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Indexing & Abstracting Of
Journal of Pharmaceutical and Biomedical Analysis
(JPBA)
YEAR 2017
Published by
LIBRARY & INFORMATION CENTRE
INDIAN PHARMACOPOEIA COMMISSION MINISTRY OF HEALTH & FAMILY WELFARE
GOVERNMENT OF INDIA
GHAZIABAD (UP)
i
INDEX
S.No. Contents Page No.
Volume 132, January 2017
1. From chemical consistency to effective consistency in precise quality discrimination of Sophora
flower-bud and Sophora flower: Discovering efficacy-associated markers by fingerprint-activity
relationship modelling 1
2. Impact of mono- and poly-ester fractions on polysorbate quantitation using mixed-mode HPLC-
CAD/ELSD and the fluorescence micelle assay 2
3. Quality assessment of marketed chamomile tea products by a validated HPTLC method
combined with multivariate analysis 3
4. Simultaneous quantification and identification of flavonoids, lignans, coumarin and amides in
leaves of Zanthoxylum armatum using UPLC-DAD-ESI-QTOF–MS/MS 3
5. Combination of HPLC–MS and QAMS as a new analytical approach for determination of
saponins in ginseng containing products 4
6. Simultaneous determination of phenolic acids and flavonoids in Chenopodium formosanum
Koidz (djulis) by HPLC-DAD-ESI–MS/MS 4
7. Design of a strong cation exchange methodology for the evaluation of charge heterogeneity in
glatiramer acetate 5
8. Development and characterization of the voriconazole loaded lipid-based nanoparticles 5
9. Selective functional activity measurement of a PEGylated protein with a modification-dependent
activity assay 6
10. Investigation of the effect of mobile phase composition on selectivity using a solvent-triangle
based approach in achiral SFC 6
11. NQO1 and CYP450 reductase decrease the systemic exposure of rifampicin-quinone and
mediate its redox cycle in rats 7
12. Study the influence of licorice and pomegranate drinks on nicotine metabolism in human urine
by LC-orbitrap MS 7
13. NMR-based metabonomics and correlation analysis reveal potential biomarkers associated with
chronic atrophic gastritis 8
14. Metabolomic profiling of doxycycline treatment in chronic obstructive pulmonary disease 8
15. Analysis of fenretinide and its metabolites in human plasma by liquid chromatography–tandem
mass spectrometry and its application to clinical pharmacokinetics 9
16. Poor and enantioselective bioavailability of naftopidil enantiomers is due to extensive and
stereoselective metabolism in rat liver 9
17. Robust HPLC–MS/MS method for levofloxacin and ciprofloxacin determination in human
prostate tissue 10
18. Pharmacokinetics, excretion of 8-cetylberberine and its main metabolites in rat urine 10
19. An UPLC–MS/MS method for the quantitation of alectinib in rat plasma 11
ii
20. Development and validation of a rapid and sensitive UPLC–MS/MS method for quantification of
kukoamine B in human plasma: Application to a clinical pharmacokinetic study 11
21. Quantitative determination of xanthorrhizol in rat plasma by HPLC–MS/MS and its application
to a pharmacokinetic study 12
22. Development and validation of a stability-indicating HPLC-UV method for the determination of
Thiocolchicoside and its degradation products 12
23. Dried blood spot analysis of gabapentin as a valid alternative for serum: a bridging study 13
24. SPR-based assays enable the full functional analysis of bispecific molecules 13
25. Angiotensin converting enzyme immobilized on magnetic beads as a tool for ligand fishing 14
26. MALDI-MS analysis and theoretical evaluation of olanzapine as a UV laser desorption
ionization (LDI) matrix 14
27. A simple and rapid UHPLC–MS/MS method for the quantitation of the dual aurora kinase A/B
inhibitor SCH-1473759 in murine plasma 15
28. Immobilized aptamer paper spray ionization source for ion mobility spectrometry 15
29. Urine metabolomics of high-fat diet induced obesity using UHPLC-Q-TOF-MS 16
Volume 133, January 2017
30. Psidium guajava L. leaves as source of proanthocyanidins: Optimization of the extraction
method by RSM and study of the degree of polymerization by NP-HPLC-FLD-ESI-MS 16
31. Identification, synthesis and structural characterization of process related and degradation
impurities of acrivastine and validation of HPLC method 17
32. Preparation of a monoclonal antibody against amantadine and rimantadine and development of
an indirect competitive enzyme-linked immunosorbent assay for detecting the same in chicken
muscle and liver 17
33. Determination of pharmaceutical residues in wastewater using high performance liquid
chromatography coupled to quadrupole-Orbitrap mass spectrometry 18
34. High-capacity hollow porous dummy molecular imprinted polymers using ionic liquid as
functional monomer for selective recognition of salicylic acid 18
35. Demonstration of the IgG antibody repertoire against the bacteria Escherichia coli in Chinese
intravenous immunoglobulins 19
36. Metabolites profiling reveals for antimicrobial compositional differences and action mechanism
in the toothbrushing stick ―miswak‖ Salvadora persica 19
37. The investigation of anti-inflammatory activity of Yi Guanjian decoction by serum
metabonomics approach 20
38. Quantitative profiling of 4'-geranyloxyferulic acid and its conjugate with l-nitroarginine methyl
ester in mononuclear cells by high-performance liquid chromatography with fluorescence
detection 20
39. A serum nuclear magnetic resonance-based metabolomic signature of antiphospholipid
syndrome 21
40. Development of a multi-matrix LC–MS/MS method for urea quantitation and its application in
human respiratory disease studies 21
iii
41. Determination of five potential genotoxic impurities in dalfampridine using liquid
chromatography 22
42. Isolation and structural characterization of novel photolytic degradation impurities of
Deflazacort using Q-TOF, 2D-NMR and FTIR 22
Volume 134, February 2017
43. LC–ESI–MS/MS evaluation of forced degradation behaviour of silodosin: In vitro anti cancer
activity evaluation of silodosin and major degradation products 23
44. Quality by Design in the development of hydrophilic interaction liquid chromatography method
with gradient elution for the analysis of olanzapine 23
45. Lipophilicity estimation and characterization of selected steroid derivatives of biomedical
importance applying RP HPLC 24
46. Comparison of the chemical consituents and immunomodulatory activity of ophiopogonis radix
from two different producing areas 24
47. Physicochemical analysis in the evaluation of reconstituted dry emulsion tablets 25
48. Cleaning verification: Exploring the effect of the cleanliness of stainless steel surface on sample
recovery 25
49. Raman spectroscopy and capillary zone electrophoresis for the analysis of degradation processes
in commercial effervescent tablets containing acetylsalicylic acid and ascorbic acid 26
50. RP-HPLC determination of dissociation constant using solely aqueous mobile phase 26
51. Quantitative determination of salbutamol sulfate impurities using achiral supercritical fluid
chromatography 27
52. Hydrogen/deuterium exchange, a unique and effective method for MS fragmentation behavior
elucidation of ginkgolides and its application to systematic research in Ginkgo biloba 27
53. Identification and characterization of a new dapoxetine impurity by NMR: Transformation of N-
oxide by Cope elimination 28
54. A new approach to the rapid separation of isomeric compounds in a Silybum marianum extract
using UHPLC core-shell column with F5 stationary phase 28
55. A comparison report of three advanced methods for drug-cyclodextrin interaction measure-
ments 29
56. Portable near-infrared instruments: Application for quality control of polymorphs in
pharmaceutical raw materials and calibration transfer 29
57. Characterization and quantitation of the polyphenolic compounds detected in methanol extracts
of Pistacia atlantica Desf fruits from the Guelmim region of Morocco 30
58. Quantification of biologically active O-prenylated and unprenylated phenylpropanoids in dill
(Anethum graveolens), anise (Pimpinella anisum), and wild celery (Angelica archangelica) 30
59. Microfluidic-based G-quadruplex ligand displacement assay for alkaloid anticancer drug
screening 31
60. Chrysin cocrystals: Characterization and evaluation 31
iv
61. Stability behaviour of antiretroviral drugs and their combinations 5: Characterization of novel
degradation products of abacavir sulfate by mass and nuclear magnetic resonance spectro-
metry 32
62. Comparison of miRNA signature versus conventional biomarkers before and after off-pump
coronary artery bypas graft 32
63. Microfluidic device for label-free quantitation and distinction of bladder cancer cells from the
blood cells using micro machined silicon based electrical approach; suitable in urinalysis
assays 33
64. Simultaneous determination of anemoside B4, phellodendrine, berberine, palmatine, obakunone,
esculin, esculetin in rat plasma by UPLC–ESI–MS/MS and its application to a comparative
pharmacokinetic study in normal and ulcerative colitis rats 33
65. Simultaneous determination and pharmacokinetic study of four phenolic acids in rat plasma
using UFLC–MS/MS after intravenous administration of salvianolic acid for injection 34
66. A rapid and sensitive UHPLC–MS/MS method for quantification of 83b1 in plasma and its
application to bioavailability study in rats 34
67. Accurate quantitation of choline and ethanolamine plasmalogen molecular species in human
plasma by liquid chromatography–tandem mass spectrometry 35
68. The profiling of the metabolites of hirsutine in rat by ultra-high performance liquid
chromatography coupled with linear ion trap Orbitrap mass spectrometry: An improved strategy
for the systematic screening and identification of metabolites in multi-samples in vivo 35
69. Metabolic fate and detectability of the new psychoactive substances 2-(4-bromo-2,5-
dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25B-NBOMe) and 2-(4-chloro-
2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25C-NBOMe) in human and
rat urine by GC–MS, LC–MSn, and LC–HR–MS/MS approaches 36
70. Liquid chromatography-tandem mass spectrometric determination of propofol in rat serum and
hair at attogram level after derivatization with 3-bromomethyl-propyphenazone 36
71. Investigation of the metabolites of the HIF stabilizer FG-4592 (roxadustat) in five different in
vitro models and in a human doping control sample using high resolution mass spectro-
metry 37
72. A quantitative LC–MS/MS method for simultaneous determination of cocaine and its
metabolites in whole blood 37
73. Use of FTIR spectroscopy and PCA-LDC analysis to identify cancerous lesions within the
human colon 38
74. Antibody-free detection of infectious bacteria using quantum dots-based barcode assay 38
75. A simple and sensitive liquid chromatography–tandem mass spectrometry method for trans-ε-
viniferin quantification in mouse plasma and its application to a pharmacokinetic study in
mice 39
76. LC–MS/MS assay for the quantitation of the tyrosine kinase inhibitor neratinib in human
plasma 39
v
77. Comparative tissue distribution and excretion study of alkaloids from Herba Ephedrae-Radix
Aconiti Lateralis extracts in rats 40
78. Deep eutectic solvents as green media for extraction of flavonoid glycosides and aglycones from
Platycladi Cacumen 40
79. Testosterone and its dimers alter tRNA morphology 41
80. Development and validation of a simple and robust HPLC method with UV detection for
quantification of the hepatitis C virus inhibitor daclatasvir in human plasma 41
81. Evaluation of ion mobility spectrometry for the detection of mitragynine in kratom pro-
ducts 42
82. A single MCR-ALS model for drug analysis in different formulations: Application on diazepam
commercial preparations 42
83. Selective recognition of cis-trans-isomers of platinum drugs and the detection of triplex DNA
based on fluorescence reversible model of quantum dots 43
84. Drug-protein binding of Danhong injection and the potential influence of drug combination with
aspirin: Insight by ultrafiltration LC–MS and molecular modelling 43
85. The importance of vial composition in HPLC analysis: An unusual case of phosphorous
pseudorotation 44
86. Supercritical fluid chromatography for separation and preparation of tautomeric 7-epimeric spiro
oxindole alkaloids from Uncaria macrophylla 44
Volume 135, February 2017
87. Renewal of an old European Pharmacopoeia method for Terazosin using modeling with mass
spectrometric peak tracking 45
88. A revisited structure for nitrosoprodenafil from NMR, mass spectrometry, X-ray and hydrolysis
data 45
89. The importance of system band broadening in modern size exclusion chromatography 46
90. Development of a multi-residue method for the determination of human and veterinary
pharmaceuticals and some of their metabolites in aqueous environmental matrices by SPE-
UHPLC–MS/MS 46
91. Development of new efficient method for isolation of phenolics from sea algae prior to their
rapid resolution liquid chromatographic–tandem mass spectrometric determination 47
92. Physico-chemical profiling of semisynthetic opioids 47
93. Interaction of anticancer drug clofarabine with human serum albumin and human α-1 acid
glycoprotein Spectroscopic and molecular docking approach 48
94. A novel method for the determination of chemical purity and assay of menaquinone-7
Comparison with the methods from the official USP monograph 48
95. Structure and pharmaceutical formulation development of a new long-acting recombinant human
insulin analog studied by NMR and MS 49
vi
96. Simultaneous HPLC assay for pretomanid (PA-824), moxifloxacin and pyrazinamide in an
inhaler formulation for drug-resistant tuberculosis 49
97. Levothyroxine sodium revisited: A wholistic structural elucidation approach of new impurities
via HPLC-HRMS/MS, on-line H/D exchange, NMR spectroscopy and chemical synthesis 50
98. Verification of the authenticity of drugs by means of NMR relaxometry—Viagra® as an
example 50
99. Metabolomics study of Populus type propolis 51
100. Hydrophilic interaction liquid chromatography method development and validation for the assay
of HEPES zwitterionic buffer 51
101. Simultaneous analysis of glucocorticosteroid fluticasone propionate and its metabolite
fluticasone propionate 17β-carboxylic acid in human plasma by UPLC–MS/MS at sub pg/mL
level 52
102. Fast Screening of Tissue Samples for Glycogen 52
103. Urinary metabolic profiling of cisplatin nephrotoxicity and nephroprotective effects of
Orthosiphon stamineus leaves elucidated by 1H NMR spectroscopy 53
104. Preparation, characterization and in vivo evaluation of a formulation of dantrolene sodium with
hydroxypropyl-β-cyclodextrin 54
105. Promotion of classic neutral bile acids synthesis pathway is responsible for cholesterol-lowing
effect of Si-miao-yong-an decoction: Application of LC–MS/MS method to determine 6 major
bile acids in rat liver and plasma 54
106. Analysis of amino acid and monoamine neurotransmitters and their metabolites in rat urine of
Alzheimer‘s disease using in situ ultrasound-assisted derivatization dispersive liquid-liquid
microextraction with UHPLC–MS/MS 55
107. Rapid determination of alkaloids in Macleaya cordata using ionic liquid extraction followed by
multiple reaction monitoring UPLC–MS/MS analysis 55
108. Volumetric absorptive microsampling (VAMS) as an alternative to conventional dried blood
spots in the quantification of miltefosine in dried blood samples 56
109. The integration of GC–MS and LC–MS to assay the metabolomics profiling in Panax ginseng
and Panax quinquefolius reveals a tissue- and species-specific connectivity of primary
metabolites and ginsenosides accumulation 56
110. Hierarchical identification of bioactive components in a medicinal herb by preparative high-
performance liquid chromatography and selective knock-out strategy 57
Volume 136, March 2017
111. Metabolomics: A potential way to know the role of vitamin D on multiple sclerosis 57
112. Impact of space environment on stability of medicines: Challenges and prospects 58
113. Evaluation of size-exclusion chromatography for the analysis of phosphorothioate
oligonucleotides 58
vii
114. Molecular insight into atypical instability behavior of fixed-dose combination containing
amlodipine besylate and losartan potassium 59
115. Qualification of HSQC methods for quantitative composition of heparin and low molecular
weight heparins 59
116. Selecting optimal columns for clarithromycin impurity analysis according to the quantitative
relationship of hydrophobic subtraction model 60
117. Efficacy of metformin in human single hair fibre by ATR-FTIR spectroscopy coupled with
statistical analysis 60
118. Primer design for SNP genotyping based on allele-specific amplification—Application to organ
transplantation pharmacogenomics 61
119. Development and validation of a liquid chromatography–mass spectrometric assay for
simultaneous determination of tacrolimus and 13-O-desmethyl tacrolimus in rat kidney
tissue 61
120. Towards interference free HPLC-SERS for the trace analysis of drug metabolites in biological
fluids 62
121. 1H NMR-based metabolomics study of liver damage induced by ginkgolic acid (15:1) in
mice 62
122. Dose-response characteristics of Clematis triterpenoid saponins and clematichinenoside AR in
rheumatoid arthritis rats by liquid chromatography/mass spectrometry-based serum and urine
metabolomics 63
123. Enhancing analysis throughput, sensitivity and specificity in LC/ESI–MS/MS assay of plasma
25-hydroxyvitamin D3 by derivatization with triplex 4-(4-dimethylaminophenyl)-1,2,4-
triazoline-3,5-dione (DAPTAD) isotopologues 63
124. A highly sensitive quantum dots-DNA nanobiosensor based on fluorescence resonance energy
transfer for rapid detection of nanomolar amounts of human papillomavirus 18 64
125. Development and validation of stability indicating HPLC methods for related substances and
assay analyses of amoxicillin and potassium clavulanate mixtures 64
126. Rapid analysis of drug dissolution by paper spray ionization mass spectrometry 65
127. Development and validation of a liquid chromatography-MS/MS method for simultaneous
quantification of tenofovir and efavirenz in biological tissues and fluids 65
128. Isolation, characterization using LC-ESI-QTOF, NMR and in vitro cytotoxicity assay of
niclosamide forced degradation products 66
129. Quantitative determinations using portable Raman spectroscopy 66
130. Screening active compounds from Corydalis yanhusuo by combining high expression VEGF
receptor HEK293 cell membrane chromatography with HPLC - ESI - IT - TOF - MSn
method 67
viii
Volume 137, April 2017
131. Powerful combination of analytical and chemometric methods for the photodegradation of 5-
Fluorouracil 67
132. Separation of antibody drug conjugate species by RPLC: A generic method development
approach 68
133. Time domain NMR as a new process monitoring method for characterization of pharmaceutical
hydrates 68
134. LC-method development for the quantification of neuromedin-like peptides Emphasis on column
choice and mobile phase composition 69
135. 2D-LC as an on-line desalting tool allowing peptide identification directly from MS unfriendly
HPLC methods 69
136. Quantification of EC-18, a synthetic monoacetyldiglyceride (1-palmitoyl-2-linoleoyl-3-acetyl-
rac-glycerol), in rat and mouse plasma by liquid-chromatography/tandem mass spect-
rometry 70
137. Compatibility study of a parenteral microdose polyethylene glycol formulation in medical
devices and identification of degradation impurity by 2D-LC/MS 70
138. Use of mixture design in drug-excipient compatibility determinations: Thymol nanoparticles
case study 71
139. Rapid analysis of Aurantii Fructus Immaturus (Zhishi) using paper spray ionization mass
spectrometry 71
140. Rapid determination of 30 bioactive constituents in XueBiJing injection using ultra high
performance liquid chromatography-high resolution hybrid quadrupole-orbitrap mass
spectrometry coupled with principal component analysis 72
141. Generic DART-MS platform for monitoring the on-demand continuous-flow production of
pharmaceuticals: Advancing the quantitative protocol for caffeates in microfluidic bioca-
talysis 72
142. Potential impurities of anxiolytic drug, clobazam: Identification, synthesis and characterization
using HPLC, LC-ESI/MSn and NMR 73
143. Simultaneous quantification of twenty-one ginsenosides and their three aglycones in rat plasma
by a developed UFLC–MS/MS assay: Application to a pharmacokinetic study of red
ginseng 73
144. Metabolic profiles of exudates from chronic leg ulcerations 74
145. Development and validation of a sensitive and fast UPLC–MS/MS method for simultaneous
determination of seven bioactive compounds in rat plasma after oral administration of Guizhi-
gancao decoction 74
146. Universal efavirenz determination in transport study, rat placenta perfusion and placenta lysate
by HPLC-UV 75
ix
147. Metabolite characterization of a novel sedative drug, remimazolam in human plasma and urine
using ultra high-performance liquid chromatography coupled with synapt high-definition mass
spectrometry 75
148. Chiral separation of new sulfonamide derivatives and evaluation of their enantioselective affinity
for human carbonic anhydrase II by microscale thermophoresis and surface plasmon
resonance 76
149. Ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS)
for determination of GHB, precursors and metabolites in different specimens: Application to
clinical and forensic cases 76
150. Impedimetric nanostructured genosensor for detection of schistosomiasis in cerebrospinal fluid
and serum samples 77
151. LC–MS/MS method for the simultaneous determination of Lys-MCC-DM1, MCC-DM1 and
DM1 as potential intracellular catabolite of the antibody-drug conjugate trastuzumab emtansine
(T-DM1) 77
152. Quantification of hydroxyurea in human plasma by HPLC–MS/MS and its application to
pharmacokinetics in patients with chronic myeloid leukaemia 78
153. Determination of rodenticides and related metabolites in rabbit liver and biological matrices by
liquid chromatography coupled to Orbitrap high resolution mass spectrometry 78
154. Comparative pharmacokinetic profiles of selected irreversible tyrosine kinase inhibitors,
neratinib and pelitinib, with apigenin in rat plasma by UPLC–MS/MS 79
155. LC–MS/MS determination of d-mannose in human serum as a potential cancer biomarker 79
156. Assessment of in vitro cardiotoxicity of extract fractions and diterpene alkaloids from Aconitum
leucostomum Worosch: A short communication 80
157. Development and validation of a liquid chromatography–tandem mass spectrometry method for
the assay of tafamidis in rat plasma: Application to a pharmacokinetic study in rats 80
158. Isolation and characterization of a novel dithio-carbodenafil analogue from a health suppl-
ement 81
159. Preparation and 68Ga-radiolabeling of porous zirconia nanoparticle platform for PET/CT-
imaging guided drug delivery 81
160. Determination of panduratin A in rat plasma by HPLC–MS/MS and its application to a
pharmacokinetic study 82
161. Simultaneous determination of creatinine and acetate by capillary electrophoresis with
contactless conductivity detector as a feasible approach for urinary tract infection
diagnosis 82
162. Analytical method development and validation for the analysis of verapamil hydrochloride and
its related substances by using ultra perfomance liquid chromatography 83
163. Evaluation of in silico pharmacokinetic properties and in vitro cytotoxic activity of selected
newly synthesized N-succinimide derivatives 83
x
164. Biopharmaceutical characterization of praziquantel cocrystals and cyclodextrin complexes
prepared by grinding 84
165. Development of an enzyme-linked immunosorbent assay for the detection of isomiroestrol, an
identical marker, in White Kwao Krua using a monoclonal antibody 84
Volume 138, May 2017
166. Fabrication of a novel hemin-based monolithic column and its application in separation of
protein from complex bio-matrix 85
167. High-throughput NIR-chemometric methods for chemical and pharmaceutical characterization of
sustained release tablets 85
168. Optimized multi-step NMR-crystallography approach for structural characterization of a stable
quercetin solvate 86
169. Study on the forced degradation behaviour of ledipasvir: Identification of major degradation
products using LC–QTOF–MS/MS and NMR 86
170. Raman scattering-based multiconformational analysis for probing the structural differences
between acetylcholine and acetylthiocholine 87
171. Characterization of metabolites in different kiwifruit varieties by NMR and fluorescence
spectroscopy 87
172. Comparison of bioactive components and pharmacological activities of ophiopogon japonicas
extracts from different geographical origins 88
173. Tiered analytics for purity assessment of macrocyclic peptides in drug discovery: Analytical
consideration and method development 88
174. Enantiomeric separation of the antiuremic drug colchicine by electrokinetic chromatography
Method development and quantitative analysis 89
175. Integrative hepatoprotective efficacy comparison of raw and vinegar-baked Radix Bupleuri using
nuclear magnetic resonance-based metabolomics 89
176. Systematic identification of flavonols, flavonol glycosides, triterpene and siraitic acid glycosides
from Siraitia grosvenorii using high-performance liquid chromatography/quadrupole-time-of-
flight mass spectrometry combined with a screening strategy 90
177. Rapid discrimination and determination of antibiotics drugs in plastic syringes using near
infrared spectroscopy with chemometric analysis: Application to amoxicillin and pen-
icillin 90
178. Identification of UV-absorbing extractables from rubber closures used in containers of injectable
powder and safety assessment of leachables in the drug 91
179. Solid state characterization of azelnidipine–oxalic acid co-crystal and co-amorphous complexes:
The effect of different azelnidipine polymorphs 91
180. Biorelevant physicochemical profiling of (E) - and (Z)-resveratrol determined from isomeric
mixtures 92
xi
181. An improved size exclusion-HPLC method for molecular size distribution analysis of
immunoglobulin G using sodium perchlorate in the eluent 92
182. Assessment of the structure of pegylated-recombinant protein therapeutics by the NMR
fingerprint assay 93
183. Prediction of the hydroxypropyl cellulose—poly (vinyl alcohol) ratio in aqueous solution
containing papaverine hydrochloride in terms of drug loaded electrospun fiber formation 93
184. Optimization of dispersive liquid-phase microextraction based on solidified floating organic drop
combined with high-performance liquid chromatography for the analysis of glucocorticoid
residues in food 94
185. A novel LCMSMS method for quantitative measurement of short-chain fatty acids in human
stool derivatized with 12C- and 13C-labelled aniline 94
186. Mixed-mode reversed phase/positively charged repulsion chromatography for intact protein
separation 95
187. Quantitative determination of five metabolites of aspirin by UHPLC–MS/MS coupled with
enzymatic reaction and its application to evaluate the effects of aspirin dosage on the metabolic
profile 95
188. HPLC–MS/MS method for the simultaneous quantification of desmethylmebeverine acid,
mebeverine acid and mebeverine alcohol in human plasma along with its application to a
pharmacokinetics study 96
189. The impact of ion-pairing reagents on the selectivity and sensitivity in the analysis of modified
oligonucleotides in serum samples by liquid chromatography coupled with tandem mass
spectrometry 96
190. A novel LC–MS/MS method for the simultaneous quantification of topiramate and its main
metabolites in human plasma 97
191. UPLC–MS/MS method for the simultaneous quantification of three new antiretroviral drugs,
dolutegravir, elvitegravir and rilpivirine, and other thirteen antiretroviral agents plus cobicistat
and ritonavir boosters in human plasma 97
192. Concentrations of antibodies against β-amyloid 40/42 monomer and oligomers in Chinese
intravenous immunoglobulins 98
193. Simultaneous quantification of estrogens, their precursors and conjugated metabolites in human
breast cancer cells by LC–HRMS without derivatization 98
194. Quantification and clinical application of carboplatin in plasma ultrafiltrate 99
195. An LC–MS/MS method for the determination of digitoxigenin in skin samples and its application
to skin permeation and metabolic stability studies 99
196. Stress-induced changes of neurosteroid profiles in rat brain and plasma under immobilized
condition 100
197. Identification, characterization and in silico ADMET prediction of Roflumilast degradation
products 100
xii
198. A rapid and robust UHPLC-DAD method for the quantification of amphotericin B in human
plasma 101
199. Quantitative analysis of mycosporine-like amino acids in marine algae by capillary
electrophoresis with diode-array detection 101
200. LC–MS/MS assay for the simultaneous quantitation of the ATM inhibitor AZ31 and the ATR
inhibitor AZD6738 in mouse plasma 102
201. The atypical excretion profile of meldonium: Comparison of urinary detection windows after
single- and multiple-dose application in healthy volunteers 102
202. Simultaneous quantitation of abiraterone, enzalutamide, N-desmethyl enzalutamide, and
bicalutamide in human plasma by LC–MS/MS 103
203. New enantioselective LC method development and validation for the assay of modafinil 103
204. Identification of a novel low-level impurity in fungicide pyraclostrobin by high-performance
liquid chromatography/tandem mass spectrometry 104
205. Paeonifiorin sulfonate as a characteristic marker for specifically inspecting Chinese patent
medicine Liu-Wei-Di-Huang-Wan contained sulfur-fumigated Moutan Cortex 104
206. Simple on-line pretreatment of column-switching coupled with ion chromatography for the
determination of lactic acid in lobaplatin 105
207. Discovery of discriminatory quality control markers for Chinese herbal medicines and related
processed products by combination of chromatographic analysis and chemometrics methods:
Radix Scutellariae as a case study 105
208. New and cost effective cell-based assay for Dialyzed Leukocyte Extract (DLE)-induced Jurkat
cells proliferation under azathioprine treatment 106
209. Spectroscopy analysis and molecular dynamics studies on the binding of penicillin V and
sulbactam to beta-lactamase II from Bacillus cereus 106
210. Variations in gut microbiota and fecal metabolic phenotype associated with depression by 16S
rRNA gene sequencing and LC/MS-based metabolomics 107
211. Chemical profiling of Fufang-Xialian-Capsule by UHPLC-Q-TOF-MS and its antioxidant
activity evaluated by in vitro method 107
Volume 139, May 2017
212. Development of an UPLC–MS/MS method for quantification of Avitinib (AC0010) and its five
metabolites in human cerebrospinal fluid: Application to a study of the blood-brain barrier
penetration rate of non-small cell lung cancer patients 108
213. The sodium salt of the enantiomers of ricobendazole: Preparation, solubility and chiroptical
properties 108
214. Application of design space optimization strategy to the development of LC methods for
simultaneous analysis of 18 antiretroviral medicines and 4 major excipients used in various
pharmaceutical formulations 109
xiii
215. Modeling and optimizing inhibitory activities of Nelumbinis folium extract on xanthine oxidase
using response surface methodology 109
216. Comparative preclinical evaluation of 68Ga-NODAGA and 68Ga-HBED-CC conjugated
procainamide in melanoma imaging 110
217. Development and validation of a HILIC-ELSD method for simultaneous analysis of non-
substituted and acetylated xylo-oligosaccharides 110
218. Enantioselective recognition of radezolid by cyclodextrin modified capillary electrokinetic
chromatography and electronic circular dichroism 111
219. Identification of leachables observed in the size exclusion chromatograms of a low concentration
product stored in prefilled syringes 111
220. Isolation and characterization of bioactive polyacetylenes Panax ginseng Meyer roots 112
221. Purity assessment of ginsenoside Rg1 using quantitative 1H nuclear magnetic resonance 112
222. Identification of forced degradation products of tedizolid phosphate by liquid
chromatography/electrospray ionization tandem mass spectrometry 113
223. Evaluation of automated Wes system as an analytical and characterization tool to support
monoclonal antibody drug product development 113
224. Pharmacokinetics and tissue distribution of 4, 5-dimethoxycanthin-6-one and its major
metabolites in rats 114
225. Development and validation of an ELISA method for the quantification of nivolumab in plasma
from non-small-cell lung cancer patients 114
226. Bioanalysis of Pseudomonas aeruginosa alkyl quinolone signalling molecules in infected mouse
tissue using LC–MS/MS; and its application to a pharmacodynamic evaluation of MvfR
inhibition 115
227. Metabolites identification of berberine in rats using ultra-high performance liquid
chromatography/quadrupole time-of-flight mass spectrometry 115
228. Quantitative chiral and achiral determination of ketamine and its metabolites by LC–MS/MS in
human serum, urine and fecal samples 116
229. Irinotecan binds to the internal cavity of beta-lactoglobulin: A multi-spectroscopic and
computational investigation 116
230. Determination of drugs in plasma samples by disposable pipette extraction with C18-BSA phase
and liquid chromatography–tandem mass spectrometry 117
231. Differentiation of protein secondary structure in clear and opaque human lenses: AFM – IR
studies 117
232. Introduction of a carbon paste electrode based on nickel carbide for investigation of interaction
between warfarin and vitamin K1 118
233. An integrated strategy using UPLC–QTOF-MSE and UPLC–QTOF-MRM (enhanced target) for
pharmacokinetics study of wine processed Schisandra Chinensis fructus in rats 118
xiv
234. Simultaneous determination of glipizide and its four hydroxylated metabolites in human urine
using LC–MS/MS and its application in urinary phenotype study 119
235. Plasma pharmacokinetics and bioavailability of verticillin A following different routes of
administration in mice using liquid chromatography tandem mass spectrometry 119
236. Robust quantitation of basic-protein higher-order aggregates using size-exclusion chromate-
graphy 120
237. A metabolomic approach shows sphingosine 1-phosphate and lysophospholipids as mediators of
the therapeutic effect of liver growth factor in emphysema 120
238. Hepatic and renal metabolism of genistein: An individual-based model to predict glucuronidation
behavior of genistein in different organs 121
239. Determination of free polysaccharide in Vi glycoconjugate vaccine against typhoid fever 121
240. HPLC–MS/MS method for quantification of paclitaxel from keratin containing samples 122
241. Development of an enzyme-linked immunosorbent assay for detection of CDCP1 shed from the
cell surface and present in colorectal cancer serum specimens 122
Volume 140, June 2017
242. Capillary blood collected on volumetric absorptive microsampling (VAMS) device for monit-
oring hydroxychloroquine in rheumatoid arthritis patients 123
243. Isolation and characterization of novel degradation products of Doxofylline using HPLC, FTIR,
LCMS and NMR 123
244. Enantiomers of triclabendazole sulfoxide: Analytical and semipreparative HPLC separation,
absolute configuration assignment, and transformation into sodium salt 124
245. Separation and characterization of unknown impurities and isomers in flomoxef sodium by LC-
IT-TOF MS and study of their negative-ion fragmentation regularities 124
246. Heparin and homogeneous model heparin oligosaccharides form distinct complexes with
protamine: Light scattering and zeta potential analysis 125
247. Quantitative analysis of binary polymorphs mixtures of fusidic acid by diffuse reflectance FTIR
spectroscopy, diffuse reflectance FT-NIR spectroscopy, Raman spectroscopy and multivariate
calibration 125
248. Mesoporous silica nanoparticles incorporated hybrid monolithic stationary phase immobilized
with pepsin for enantioseparation by capillary electrochromatography 126
249. Host-guest kinetic interactions between HP-β-cyclodextrin and drugs for prediction of bitter taste
masking 126
250. Crystal structures and physicochemical properties of amisulpride polymorphs 127
251. A UHPLC method for the rapid separation and quantification of phytosterols using tandem
UV/Charged aerosol detection – A comparison of both detection techniques 127
252. Analysis of chemical constituents in an herbal formula Jitong Ning Tablet 128
xv
253. Molecular recognition of pseudodistamine isomeric precursor trans-3(4)-aminopiperidin-4(3)-ols
by EI mass spectrometry 128
254. Development and validation of a general derivatization HPLC method for the trace analysis of
acyl chlorides in lipophilic drug substances 129
255. An isocratic hydrophilic interaction liquid chromatographic method for simultaneous determi-
nation of iodixanol and its related impurities in drug substance 129
256. Electrochemical and optical study of metallothionein interactions with prion proteins 130
257. Development of matrix effect-free MISPE-UHPLC–MS/MS method for determination of
lovastatin in Pu-erh tea, oyster mushroom, and red yeast rice 130
258. Facile preparation of fibrin coated open tubular column for characterization of monoclonal
antibody variants by capillary electrochromatography 131
259. Development and validation of a fast SFC method for the analysis of flavonoids in plant
extracts 131
260. A multi-matrix HILIC-MS/MS method for the quantitation of endogenous small molecule
neurological biomarker N-acetyl aspartic acid (NAA) 132
261. Chemotaxonomic studies of nine Paris species from China based on ultra-high performance
liquid chromatography tandem mass spectrometry and Fourier transform infrared spec-
troscopy 132
262. Rapid profiling and pharmacokinetic studies of major compounds in crude extract from
Polygonum multiflorum by UHPLC-Q-TOF-MS and UPLC–MS/MS 133
263. Metabolic profiling of nuciferine in rat urine, plasma, bile and feces after oral administration
using ultra-high performance liquid chromatography-diode array detection-quadrupole time-of-
flight mass spectrometry 133
264. Development and validation of liquid chromatography tandem mass spectrometry method
quantitative determination of polymyxin B1, polymyxin B2, polymyxin B3 and isoleucine-
polymyxin B1 in human plasma and its application in clinical studies 134
265. Human exposure to Bisphenol A and liver health status: Quantification of urinary and circulating
levels by LC–MS/MS 134
266. Determination of prodrug treosulfan and its biologically active monoepoxide in rat plasma, liver,
lungs, kidneys, muscle, and brain by HPLC–ESI–MS/MS method 135
267. Profiles of amino acids and biogenic amines in the plasma of Cri-du-Chat patients 135
268. Online microdialysis-ultra performance liquid chromatography–mass spectrometry method for
comparative pharmacokinetic investigation on iridoids from Gardenia jasminoides Ellis in rats
with different progressions of type 2 diabetic complications 136
269. Optimization of a new methodology for trace determination of elements in biological fluids:
Application for speciation of inorganic selenium in children‘s blood 136
270. Detailed analysis of cortisol, cortisone and their tetrahydro- and allo-tetrahydrometabolites in
human urine by LC–MS/MS 137
xvi
271. Urinary metabonomics study of the hepatoprotective effects of total alkaloids from Corydalis
saxicola Bunting on carbon tetrachloride-induced chronic hepatotoxicity in rats using 1H NMR
analysis 137
272. Pharmacokinetic properties of the synthetic cannabinoid JWH-018 and of its metabolites in
serum after inhalation 138
273. Charged derivatization and on-line solid phase extraction to measure extremely low cortisol and
cortisone levels in human saliva with liquid chromatography–tandem mass spectrometry 138
274. Comparability study of Rituximab originator and follow-on biopharmaceutical 139
275. Whole blood microsampling for the quantitation of estetrol without derivatization by liquid
chromatography-tandem mass spectrometry 139
276. Meropenem, levofloxacin and linezolid in human plasma of critical care patients: A fast semi-
automated micro-extraction by packed sorbent UHPLC-PDA method for their simultaneous
determination 140
277. Investigation on the combined effect of cocaine and ethanol administration through a liquid
chromatography–mass spectrometry metabolomics approach 140
278. Validation of a dried blood spot method for therapeutic drug monitoring of citalopram,
mirtazapine and risperidone and its active metabolite 9-hydroxyrisperidone using
HPLC–MS 141
279. Characterization of an unknown impurity in doxofylline using LC–MS and NMR 141
280. Determination of AB-CHMINACA and its metabolites in human hair and their deposition in hair
of abusers 142
281. Development of a new chlorogenic acid certified reference material for food and drug
analysis 142
282. Automation of plasma protein binding assay using rapid equilibrium dialysis device and Tecan
workstation 143
283. ―Ghost peaks‖ of ezetimibe: Solution degradation products of ezetimibe in acetonitrile induced
by alkaline impurities from glass HPLC vials 143
284. Metabolite quantification by NMR and LC-MS/MS reveals differences between unstimulated,
stimulated, and pure parotid saliva 144
285. Determination of AZD3759 in rat plasma and brain tissue by LC–MS/MS and its application in
pharmacokinetic and brain distribution studies 144
286. Studying the effects of natural extracts with metabolomics: A longitudinal study on the
supplementation of healthy rats with Polygonum cuspidatum Sieb et Zucc. 145
287. Identification and characterization of a thermally cleaved fragment of monoclonal antibody-A
detected by sodium dodecyl sulfate-capillary gel electrophoresis 145
288. Synchronous determination with double-wavelength by RP-HPLC-UV and optimization of
ultrasound-assisted extraction of phenolic acids from Caragana species using response surface
methodology 146
xvii
289. Untargeted metabolite analysis-based UHPLC-Q-TOF-MS reveals significant enrichment of p-
hydroxybenzyl dimers of citric acids in fresh beige-scape Gastrodia elata (Wutianma) 146
Volume 141, July 2017
290. Matrix-assisted laser-desorption/ionization mass spectrometric imaging of olanzapine in a single
hair using esculetin as a matrix 147
291. A novel liquid chromatography/tandem mass spectrometry (LC–MS/MS) based bioanalytical
method for quantification of ethyl esters of Eicosapentaenoic acid (EPA) and Docosahexaenoic
acid (DHA) and its application in pharmacokinetic study 147
292. Identification of a host cell protein impurity in therapeutic protein, P1 148
293. Simultaneous separation and determination of four uncaria alkaloids by capillary electrophoresis
using dual cyclodextrin system 148
294. CE method for the in-process control of the synthesis of active substances conjugated with gold
nanoparticles 149
295. Achievable separation performance and analysis time in current liquid chromatographic practice
for monoclonal antibody separations 149
296. Identification and characterization of process-related substances and degradation products in
apremilast: Process optimization and degradation pathway elucidation 150
297. Antiproliferative hydroxy-fatty acids from the fodder legume Stylosanthes guianensis 150
298. Development and validation of a liquid chromatographic method for the analysis of squaric acid
dibutyl ester and its impurities 151
299. Macro-Raman spectroscopy for bulk composition and homogeneity analysis of multi-component
pharmaceutical powders 151
300. Isolation, identification and characterization of potential impurities of anidulafungin 152
301. Dialkyl anionic surfactant in field-amplified sample injection and sweeping-micellar
electrokinetic chromatography for determination of eight leanness-promoting β-agonists in
animal feeds 152
302. Isolation and structural characterization of glucosylceramides from Ethiopian plants by
LC/APCI-MS/MS 153
303. Drug-protein binding mechanism of juglone for early pharmacokinetic profiling: Insights from
ultrafiltration, multi-spectroscopic and molecular docking methods 153
304. Quantification of alprenolol and propranolol in human plasma using a two-dimensional liquid
chromatography (2D-LC) 154
305. An LC–MS/MS method for quantification of AC0010, a novel mutant-selective epidermal
growth factor receptor (EGFR) inhibitor, and its metabolites in human plasma and the application
to a pharmacokinetic study 154
306. Validation and application of an ultrahigh-performance liquid chromatographic-Orbitrap mass
spectrometric method for the simultaneous detection and quantification of volatile and non-
volatile organic acids in human faecal samples 155
xviii
307. Validation of a SPE HPLC–UV method for the quantification of a new ER-specific
photosensitizer OR-141 in blood serum using total error concept 155
308. Determination of a novel anticancer AMPK activator hernandezine in rat plasma and tissues with
a validated UHPLC–MS/MS method: Application to pharmacokinetics and tissue distribution
study 156
309. LC–MS/MS determination of tranexamic acid in human plasma after phospholipid
clean-up 156
310. Metabolic profiles of neotuberostemonine and tuberostemonine in rats by high performance
liquid chromatography/quadrupole time-of-flight mass spectrometry 157
311. Comprehensive profiling and characterization of coumarins from roots, stems, leaves, branches,
and seeds of Chimonanthus nitens Oliv using ultra-performance liquid chromatography/
quadrupole-time-of-flight mass spectrometry combined with modified mass defect filter 157
312. New analytical method for determination of epimer metabolites in rat plasma after oral
administration of Paeoniflorin by UPLC-TOF-MS following picolinoyl derivatization 158
313. Application of 2D-NMR with room temperature NMR probes for the assessment of the higher
order structure of filgrastim 158
314. Influence of sulfur fumigation on the chemical profiles of Atractylodes macrocephala Koidz
evaluated by UFLC–QTOF–MS combined with multivariate statistical analysis 159
315. A sandwich immunoassay for brucellosis diagnosis based on immune magnetic beads and
quantum dots 159
316. Systematically characterize the absorbed effective substances of Wutou Decoction and their
metabolic pathways in rat plasma using UHPLC-Q-TOF-MS combined with a target network
pharmacological analysis 160
317. Nontargeted metabolomics approach for the differentiation of cultivation ages of mountain
cultivated ginseng leaves using UHPLC/QTOF-MS 160
318. Studies on the metabolites difference of psoralen/isopsoralen in human and six mammalian liver
microsomes in vitro by UHPLC–MS/MS 161
319. Al cation induces aggregation of serum proteins 161
Volume 142, August 2017
320. Fighting falsified medicines: The analytical approach 162
321. Lipophilicity estimation of statins as a decisive physicochemical parameter for their hepato-
selectivity using reversed-phase thin layer chromatography 162
322. Metabolite profiling of flavonols and in vitro antioxidant activity of young shoots of wild
Humulus lupulus L. (hop) 163
323. Development of assay for determination of eletriptan hydrobromide in loaded PLGA
nanoparticles 163
xix
324. Characterization of flavonol mono-, di-, tri- and tetra-O-glycosides by ultra-performance liquid
chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry and its
application for identification of flavonol glycosides in Viola tianschanica 164
325. Development and validation of a rapid reversed-phase HPLC method for the quantification of
monoclonal antibody bevacizumab from polyester-based nanoparticles 164
326. Quantification of active ingredients in semi-solid pharmaceutical formulations by near infrared
spectroscopy 165
327. Detection of regulated herbs and plants in plant food supplements and traditional medicines using
infrared spectroscopy 165
328. UV-induced electron transfer between triethylamine and 5-bromo-2′-deoxyuridine A puzzle
concerning the photochemical debromination of labeled DNA 166
329. Development of an in vivo-relevant drug product performance method for an amorphous solid
dispersion 166
330. Ultrasound-assisted low-density solvent dispersive liquid–liquid microextraction for the
simultaneous determination of 12 new antidepressants and 2 antipsychotics in whole blood by
gas chromatography–mass spectrometry 167
331. Application of protein A-modified capillary-channeled polymer polypropylene fibers to the
quantitation of IgG in complex matrices 167
332. Simple and rapid quantification of vancomycin in serum, urine and peritoneal/pleural effusion
via UHPLC–MS/MS applicable to personalized antibiotic dosing research 168
333. Biphenyl based stationary phases for improved selectivity in complex steroid assays 168
334. Ultra-sensitive and selective quantification of endothelin-1 in human plasma using ultra-
performance liquid chromatography coupled to tandem mass spectrometry 169
335. Serum metabolomics reveals the mechanistic role of functional foods and exercise for obesity
management in rats 169
336. Systematic screening and characterization of prototype constituents and metabolites of total
astragalosides using HPLC-ESI-IT-TOF-MSn after oral administration to rats 170
337. Development, validation and application of a novel liquid chromatography tandem mass
spectrometry assay measuring uracil, 5,6-dihydrouracil, 5-fluorouracil, 5,6-dihydro-5-
fluorouracil, α-fluoro-β-ureidopropionic acid and α-fluoro-β-alanine in human plasma 170
338. Multi-platform metabolomics and a genetic approach support the authentication of agarwood
produced by Aquilaria crassna and Aquilaria malaccensis 171
339. Apoptosis induction activity and molecular docking studies of survivin siRNA carried by Fe3O4-
PEG-LAC-chitosan-PEI nanoparticles in MCF-7 human breast cancer cells 171
340. Exploring the neuroprotective effects of ginkgolides injection in a rodent model of cerebral
ischemia–reperfusion injury by GC–MS based metabolomic profiling 172
341. Quantitative LC–HRMS determination of selected cardiovascular drugs, in dried blood spots, as
an indicator of adherence to medication 172
xx
342. Determination of thiopurine S-methyltransferase activity by hydrophilic interaction liquid
chromatography hyphenated with mass spectrometry 173
343. Workflow methodology for rat brain metabolome exploration using NMR, LC–MS and GC–MS
analytical platforms 173
344. Bioelectrical impedimetric sensor for single cell analysis based on nanoroughened quartz
substrate; suitable for cancer therapeutic purposes 174
345. Highly sensitive UHPLC–MS/MS method for the simultaneous estimation of propafenone and its
metabolites 5-hydroxypropafenone and N-depropylpropafenone on human dried blood spots
technique and application to a pharmacokinetic study 174
346. Simple determination of L-hydroxyproline in idiopathic pulmonary fibrosis lung tissues of rats
using non-extractive high-performance liquid chromatography coupled with fluorescence
detection after pre-column derivatization with novel synthetic 9-acetylimidazol-
carbazole 175
347. A simple method for assaying colistimethate sodium in pharmaceutical aerosol samples using
high performance liquid chromatography 175
348. Quantification of apigenin trimethyl ether in rat plasma by liquid chromatography–tandem mass
spectrometry: Application to a pre-clinical pharmacokinetic study 176
349. Simultaneous analysis of regorafenib and sorafenib and three of their metabolites in human
plasma using LC–MS/MS 176
350. Stability behavior of antiretroviral drugs and their combinations 7: Comparative degradation
pathways of lamivudine and emtricitabine and explanation to their differential degradation
behavior by density functional theory 177
351. An atmospheric pressure ionization source using a high voltage target compared to electrospray
ionization for the LC/MS analysis of pharmaceutical compounds 177
352. UHPLC–MS/MS method with sample dilution to test therapeutic adherence through
quantification of ten antihypertensive drugs in urine samples 178
353. A novel carbohydrate labeling method utilizing transfer hydrogenation-mediated reductive
amination 178
354. Non-volatile extractable analysis of prefilled syringes for parenteral administration of drug
products 179
355. A rapid microextraction by packed sorbent − liquid chromatography tandem mass spectrometry
method for the determination of dexamethasone disodium phosphate and dexamethasone in
aqueous humor of patients with uveitis 179
356. Isotope-coded derivatization based LC/ESI-MS/MS methods using a pair of novel reagents for
quantification of hydroxycinnamic acids and hydroxybenzoic acids in fermented brown rice
product 180
357. Rapid discovery of cyclopamine analogs from Fritillaria and Veratrum plants using LC-Q-TOF-
MS and LC-QqQ-MS 180
xxi
358. Development and validation of a HS/GC–MS method for the simultaneous analysis of diacetyl
and acetylpropionyl in electronic cigarette refills 181
359. Structural characterization and discrimination of the Paris polyphylla var. yunnanensis and Paris
vietnamensis based on metabolite profiling analysis 181
Volume 143, September 2017
360. Reliability of point-of-collection testing devices for drugs of abuse in oral fluid: A systematic
review and meta-analysis 182
361. High-throughput thermofluor-based assays for inhibitor screening of STAT SH2 domains 182
362. Trace level determination of 5-hydroxytryptamine and its related indoles in amniotic fluid by gas
chromatography–mass spectrometry 183
363. Identification of related substances in tofacitinib citrate by LC-MS techniques for synthetic
process optimization 183
364. Macro- and microstructural tracking of ageing-related changes of papaverine hydrochloride-
loaded electrospun nanofibrous buccal sheets 184
365. A simplified guide for charged aerosol detection of non-chromophoric compounds—Analytical
method development and validation for the HPLC assay of aerosol particle size distribution for
amikacin 184
366. Proton dissociation properties of arylphosphonates: Determination of accurate Hammett equation
parameters 185
367. Sub–1 min separation in sequential injection chromatography for determination of synthetic
water-soluble dyes in pharmaceutical formulation 185
368. Impurity profiling of liothyronine sodium by means of reversed phase HPLC, high resolution
mass spectrometry, on-line H/D exchange and UV/Vis absorption 186
369. A UHPLC method for the rapid separation and quantification of anthocyanins in acai berry and
dry blueberry extracts 186
370. Combined computational-experimental approach to predict blood–brain barrier (BBB)
permeation based on ―green‖ salting-out thin layer chromatography supported by simple
molecular descriptors 187
371. Development of a new extraction technique and HPLC method for the analysis of non-
psychoactive cannabinoids in fibre-type Cannabis sativa L. (hemp) 187
372. Analytical profiling of selected antioxidants and total antioxidant capacity of goji (Lycium spp.)
berries 188
373. Characterization and inhibition studies of tissue nonspecific alkaline phosphatase by
aminoalkanol derivatives of 1,7-dimethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-
3,5,10-trione, new competitive and non-competitive inhibitors, by capillary electrophoresis 188
374. A validated UHPLC-QTOF-MS method for quantification of metformin and teneligliptin in rat
plasma: Application to pharmacokinetic interaction study 189
xxii
375. Overcoming interference with the detection of a stable isotopically labeled microtracer in the
evaluation of beclabuvir absolute bioavailability using a concomitant microtracer approach 189
376. Ex-vivo measurement of scalp follicular infundibulum delivery of zinc pyrithione and climbazole
from an anti-dandruff shampoo 190
377. Pooled human liver preparations, HepaRG, or HepG2 cell lines for metabolism studies of new
psychoactive substances? A study using MDMA, MDBD, butylone, MDPPP, MDPV, MDPB, 5-
MAPB, and 5-API as examples 190
378. Evaluation of pharmacokinetics and blood-brain barrier permeability of mitragynine using in
vivo microdialysis technique 191
379. 1H NMR spectral identification of medication in cerebrospinal fluid of pediatric meningitis 191
380. Therapeutic drug monitoring of beta-lactam antibiotics – Influence of sample stability on the
analysis of piperacillin, meropenem, ceftazidime and flucloxacillin by HPLC-UV 192
381. Development and validation of an ultra-performance liquid chromatography–tandem mass
spectrometry method for quantification of SR1001, an inverse agonist of retinoid-related orphan
receptors, and its application to pharmacokinetic studies in streptozotocin-induced diabetic
mice 193
382. Quantum dot-linked immunosorbent assay (QLISA) using orientation-directed antibodies 193
383. Validation of liquid and gaseous calibration techniques for quantification of propofol in breath
with sorbent tube Thermal Desorption System GC–MS 194
384. Development and validation of a bioanalytical method based on LC–MS/MS analysis for the
quantitation of CIGB-814 peptide in plasma from Rheumatoid Arthritis patients 194
385. Application of volumetric absorptive microsampling device for quantification of tacrolimus in
human blood as a model drug of high blood cell partition 195
386. Fast and efficient zirconia-based reversed phase chromatography for selective determination of
triptans in rat plasma 195
387. Monitoring breast cancer treatment using a Fourier transform infrared spectroscopy-based
computational model 196
388. Enhancement in recovery of drugs with high protein binding efficiency from human plasma
using magnetic nanoparticles 196
389. Simultaneous quantification of endothelin receptor antagonists and phosphodiesterase
5 inhibitors currently used in pulmonary arterial hypertension 197
390. Application of 1H NMR spectroscopy to the metabolic phenotyping of rodent brain extracts: A
metabonomic study of gut microbial influence on host brain metabolism 197
391. A re-investigation of the phytochemical composition of the edible herb Amaranthus retro-
flexus L 198
392. A vote for robustness: Monitoring serum enzyme activity by thin-layer chromatography of
dabsylated bradykinin products 198
xxiii
393. Salting-out assisted liquid–liquid extraction combined with gas chromatography-mass
spectrometry for the determination of pyrethroid insecticides in high salinity and biological
samples 199
394. HPLC–MS/MS method for troventol determination in human plasma and its application to
biological samples 199
395. 1,4-Anthraquinone: A new useful pre-column reagent for the determination of N-acetylcysteine
and captopril in pharmaceuticals by high performance liquid chromatography 200
396. Development a validated highly sensitive LC–MS/MS method for simultaneous quantification of
Ledipasvir, sofosbuvir and its major metabolite GS-331007 in human plasma: Application to a
human pharmacokinetic study 200
397. Chemometrics and chromatographic fingerprints to classify plant food supplements according to
the content of regulated plants 201
398. Surrogate CD16-expressing effector cell lines for determining the bioactivity of therapeutic
monoclonal antibodies 201
399. Dual-target screening of bioactive components from traditional Chinese medicines by hollow
fiber-based ligand fishing combined with liquid chromatography–mass spectrometry 202
Volume 144, September 2017
400. Circular dichroism analysis of the calicheamicin-DNA interaction revisited 202
401. Induced circularly polarized luminescence for revealing DNA binding with fluorescent
dyes 203
402. Analysis of stereoselective drug interactions with serum proteins by high-performance affinity
chromatography: A historical perspective 203
403. An indirect stereoselective analysis of nebivolol glucuronides in plasma by LC–MS/MS:
Application to clinical pharmacokinetics 204
404. N-Decyl-S-trityl-(R)-cysteine, a new chiral selector for ―green‖ ligand-exchange chromatography
applications 204
405. The role of chirality in a set of key intermediates of pharmaceutical interest, 3-aryl-substituted-γ-
butyrolactones, evidenced by chiral HPLC separation and by chiroptical spectroscopies 205
406. Determination of the absolute configuration of a novel tetrasubstituted isoindolinone by
vibrational circular dichroism 205
407. GC–MS based Gestational Diabetes Mellitus longitudinal study: Identification of 2-and 3-
hydroxybutyrate as potential prognostic biomarkers 206
408. Development and validation of a quantification method for cucurbitacins E and I in rat plasma:
Application to population pharmacokinetic studies 206
409. Ultra-fast quantitation of voriconazole in human plasma by coated blade spray mass
spectrometry 207
410. Pharmacokinetic profile of bilberry anthocyanins in rats and the role of glucose transporters: LC–
MS/MS and computational studies 207
411. Comparative pharmacodynamic analysis of imidazoline compounds using rat model of ocular
mydriasis with a test of quantitative structure–activity relationships 208
xxiv
412. Characterization of oxycodone in vitro metabolism by human cytochromes P450 and UDP-
glucuronosyltransferases 208
413. Structural and functional integrity of human serum albumin: Analytical approaches and clinical
relevance in patients with liver cirrhosis 209
414. Targeted proteomics of cannabinoid receptor CB1 and the CB1b isoform 209
415. Application of an ESI-QTOF method for the detailed characterization of GSK-3β
inhibitors 210
416. Quantitative estimation of cholinesterase-specific drug metabolism of carbamate inhibitors
provided by the analysis of the area under the inhibition-time curve 210
417. A new method to characterize the kinetics of cholinesterases inhibited by carbamates 211
418. Cyclodextrins as inhibitors of the precipitation of riboflavin-5‘-phosphate due to presence of zinc
chloride: A NMR investigation 211
419. Monitoring drug–serum protein interactions for early ADME prediction through Surface
Plasmon Resonance technology 212
420. Capillary electrophoresis in the context of drug discovery 212
421. Simultaneous analysis of nucleobases, nucleosides and ginsenosides in ginseng extracts using
supercritical fluid chromatography coupled with single quadrupole mass spectrometry 213
422. Combined approach using capillary electrophoresis, NMR and molecular modeling for
ambrisentan related substances analysis: Investigation of intermolecular affinities, complexation
and separation mechanism 213
423. Molecularly imprinted polymer for glutathione by modified precipitation polymerization and its
application to determination of glutathione in supplements 214
424. Efficacy of a titanium dioxide nanoparticles − based indoor anti-odor product as assessed by
electronic nose and gaschromatography–mass spectrometry 214
425. Comprehensive study on the effects of sodium and potassium additives in size exclusion
chromatographic separations of protein biopharmaceuticals 215
426. Application of a rapid HILIC-UV method for synthesis optimization and stability studies of
immunogenic neo-glycoconjugates 215
427. Simple and rapid LC–MS method for the determination of circulating albumin
microheterogeneity in veal calves exposed to heat stress 216
428. Molecular fingerprinting of principal neurons in the rodent hippocampus: A neuroinformatics
approach 216
Volume 145, October 2017
429. Two-dimensional liquid chromatography in pharmaceutical analysis Instrumental aspects, trends
and applications 217
430. Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A
generic method development approach 217
431. A new platform for serological analysis based on porous 3-dimensional polyethylene sinter
bodies 218
xxv
432. Designing a calibration set in spectral space for efficient development of an NIR method for
tablet analysis 218
433. On-line prediction of the glucose concentration of CHO cell cultivations by NIR and Raman
spectroscopy: Comparative scalability test with a shake flask model system 219
434. Conventional and accelerated-solvent extractions of green tea (camellia sinensis) for
metabolomics-based chemometrics 219
435. Phase separation of in situ forming poly (lactide-co-glycolide acid) implants investigated using a
hydrogel-based subcutaneous tissue surrogate and UV–vis imaging 220
436. Mid-infrared and near-infrared spectroscopy for rapid detection of Gardeniae Fructus by a liquid-
liquid extraction process 220
437. Quantitative analysis of a biopharmaceutical protein in cell culture samples using automated
capillary electrophoresis (CE) western blot 221
438. Synchronous characterization of carbohydrates and ginsenosides yields deeper insights into the
processing chemistry of ginseng 221
439. Development and validation of an ICP-MS method for the determination of elemental impurities
in TP-6076 active pharmaceutical ingredient (API) according to USP 〈232〉/〈233〉 222
440. Comparison of SEC and CE-SDS methods for monitoring hinge fragmentation in IgG1
monoclonal antibodies 222
441. Revisiting blood-brain barrier: A chromatographic approach 223
442. Liquid chromatographic enantioseparation of carbocyclic β-amino acids possessing limonene
skeleton on macrocyclic glycopeptide-based chiral stationary phases 223
443. On-line coupling of molecularly imprinted solid phase extraction with liquid chromatography for
the fast determination of coumarins from complex samples 224
444. Metabolic profiling analysis of Siraitia grosvenorii revealed different characteristics of green
fruit and saccharified yellow fruit 224
445. Comparison of α-glucosidase inhibitory effect and bioactive constituents of Anemarrhenae
Rhizoma and Fibrous Roots 225
446. Characterization of forced degradation products of torasemide through MS tools and explanation
of unusual losses observed during mass fragmentation of drug and degradation products through
density functional theory 225
447. Metabolic profiling of the traditional Chinese medicine formulation Yu Ping Feng San for the
identification of constituents relevant for effects on expression of TNF-α, IFN-γ, IL-1β and IL-4
in U937 cells 226
448. A comprehensive stability-indicating HPLC method for determination of chloroquine in active
pharmaceutical ingredient and tablets: Identification of oxidation impurities 226
449. Identification of impurities in macrolides by liquid chromatography–mass spectrometric
detection and prediction of retention times of impurities by constructing quantitative structure–
retention relationship (QSRR) 227
xxvi
450. The impact of ZnO and TiO2 on the stability of clotrimazole under UVA irradiation:
Identification of photocatalytic degradation products and in vitro cytotoxicity assessment 227
451. Chemometrically assisted development and validation of LC–MS/MS method for the analysis of
potential genotoxic impurities in meropenem active pharmaceutical ingredient 228
452. Identification and interconversion of isomeric 4,5-functionalized 1,2,3-thiadiazoles and 1,2,3-
triazoles in conditions of electrospray ionization 228
453. Dissolution assessment of allopurinol immediate release tablets by near infrared spect-
roscopy 229
454. Enhanced and green extraction polyphenols and furanocoumarins from Fig (Ficus carica L.)
leave using deep eutectic solvents 229
455. Site- and species-specific hydrolysis rates of cocaine 230
456. Comparative stability-indicating chromatographic methods for determination of 4-
hexylresorcinol in pharmaceutical formulation and shrimps 230
457. Enantiomeric separation of seven β-agonists by NACE—Study of chiral selectivity with
diacetone-d-mannitol–boric acid complex 231
458. An optimized HPLC-UV method for quantitatively determining sesquiterpenes in Nardostachyos
Radix et Rhizoma 231
459. Photostability testing using online reactor HPLC hyphenation and mass spectrometric compound
identification illustrated by ketoprofen as model compound 232
460. Optoelectronic iron detectors for pharmaceutical flow analysis 232
461. Isoconversional approach for non-isothermal decomposition of un-irradiated and photon-
irradiated 5-fluorouracil 233
462. Characterization of impurities in sodium cromoglycate drug substance and eye drops using LC-
ESI-ion trap MS and LC-ESI-QTOF MS 233
463. Method validation and nanoparticle characterization assays for an innovative amphothericin B
formulation to reach increased stability and safety in infectious diseases 234
464. Surfactants enhance recovery of poorly soluble drugs during microdialysis sampling:
Implications for in vitro dissolution-/permeation-studies 234
465. Simultaneous identification and quantification of polymethoxyflavones, coumarin and phenolic
acids in Ageratum conyzoides by UPLC-ESI-QToF-MS and UPLC-PDA 235
466. Degradation and metabolite formation of estrogen conjugates in an agricultural soil 235
467. Chemical analysis and potential endocrine activities of aluminium coatings intended to be in
contact with cosmetic water 236
468. Identification and determination of related substances of ceftaroline fosamil in medicinal product
by high performance liquid chromatography with diode array detection and tandem mass
spectrometry 236
xxvii
469. Quantification of concentrated Chinese medicine granules by quantitative polymerase chain
reaction 237
470. Chiral separation of terbutaline and non-steroidal anti-inflammatory drugs by using a new
lysine–bridged hemispherodextrin in capillary electrophoresis 237
471. Separation and characterization of allergic polymerized impurities in cephalosporins by 2D-
HPSEC × LC-IT-TOF MS 238
472. Phytochemical analysis and anti-inflammatory evaluation of compounds from an aqueous extract
of Croton cajucara Benth 238
473. Supercritical fluid chromatography approach for a sustainable manufacture of new
stereoisomeric anticancer agent 239
474. Use of ionic liquids as headspace gas chromatography diluents for the analysis of residual
solvents in pharmaceuticals 239
475. Adduct ion-targeted qualitative and quantitative analysis of polyoxypregnanes by ultra-high
pressure liquid chromatography coupled with triple quadrupole mass spectrometry 240
476. Quantitative determination of dobutamine in newborn pig plasma samples by HPLC–
MS/MS 240
477. An integrative investigation of the toxicity of Aconiti kusnezoffii radix and the attenuation effect
of its processed drug using a UHPLC-Q-TOF based rat serum and urine metabolomics
strategy 241
478. Calibration and validation of a MCC/IMS prototype for exhaled propofol online measu-
rement 241
479. Biochemical and functional analysis of corticotropin releasing factor purified from an aqueous
extract of human placenta used as wound healer 242
480. Toxic compounds from tobacco in placenta samples analyzed by UPLC-QTOF-MS 242
481. HPLC-HG-AFS determination of arsenic species in acute promyelocytic leukemia (APL) plasma
and blood cells 243
482. Quantification of IDP-73152, a novel antibiotic, in plasma from mice, rats and humans using an
ultra-high performance liquid chromatography/tandem mass spectrometry method for use in
pharmacokinetic studies 243
483. New approach for the diagnosis of histamine intolerance based on the determination of histamine
and methylhistamine in urine 244
484. Development and validation of sensitive LC–MS/MS method for the quantification of SUVN-
502 and its metabolite and its application for first in human pharmacokinetic study 244
485. Glycosylation patterns of selected proteins in individual serum and cerebrospinal fluid
samples 245
486. MIL-101(Cr)@GO for dispersive micro-solid-phase extraction of pharmaceutical residue in
chicken breast used in microwave-assisted coupling with HPLC–MS/MS detection 245
xxviii
487. Development of a robust reporter gene assay to measure the bioactivity of anti-PD-1/anti-PD-L1
therapeutic antibodies 246
488. LC–MS/MS-ESI method for simultaneous quantification of darolutamide and its active
metabolite, ORM-15341 in mice plasma and its application to a pharmacokinetic study 246
489. Metabolite identification of AZD8055 in Sprague-Dawley rats after a single oral administration
using ultra-performance liquid chromatography and mass spectrometry 247
490. Quantification of 16 β-lactams in chicken muscle by QuEChERS extraction and UPLC-Q-
Orbitrap-MS with parallel reaction monitoring 247
491. Characterization of the phase I and phase II metabolic profile of tolvaptan by in vitro studies and
liquid chromatography–mass spectrometry profiling: Relevance to doping control
analysis 248
492. An LC–MS/MS method for simultaneous determination of nine steroidal saponins from Paris
polyphylla var. in rat plasma and its application to pharmacokinetic study 248
493. N-glucuronidation catalyzed by UGT1A4 and UGT2B10 in human liver microsomes: Assay
optimization and substrate identification 249
494. A simple high performance liquid chromatography–mass spectrometry method for Therapeutic
Drug Monitoring of isavuconazole and four other antifungal drugs in human plasma
samples 249
495. Metabolic profiling of dehydrodiisoeugenol using xenobiotic metabolomics 250
496. Development and validation of a reliable method for thiopurine methyltransferase (TPMT)
enzyme activity in human whole blood by LC–MS/MS: An application for phenotypic and
genotypic correlations 250
497. Development and validation of a GC–MS method for the determination of hydroxyzine and its
active metabolite, cetirizine, in whole blood 251
498. An on-spot internal standard addition approach for accurately determining colistin A and colistin
B in dried blood spots using ultra high-performance liquid chromatography–tandem mass
spectrometry 251
499. Comparative study of single/combination use of Huang-Lian-Jie-Du decoction and berberine on
their protection on sepsis induced acute liver injury by NMR metabolic profiling 252
500. Screening and confirmation of steroids and nitroimidazoles in urine, blood, and food matrices:
Sample preparation methods and liquid chromatography tandem mass spectrometric
separations 252
501. Validation of an HPLC-UV method for analysis of Kaempferol-loaded nanoemulsion and its
application to in vitro and in vivo tests 253
502. Development of direct assays for Toxoplasma gondii and its use in genomic DNA
sample 253
503. Investigation and structural elucidation of a new impurity in bulk drug of cilostazol by
LC/MS/MS, FT-IR and NMR 254
xxix
504. Selective screening of glutaric acid acidurias by capillary electrophoresis-mass spect-
rometry 254
505. Online turbulent flow extraction coupled with liquid chromatography–tandem mass spectrometry
for high throughput screening of anabolic steroids in horse urine 255
506. Development of a mixed-mode chromatography with tandem mass spectrometry method for the
quantitative analysis of 23 underivatized amino acids in human serum 255
507. Inhibitory assay for degradation of collagen IV by cathepsin B with a surface plasmon resonance
sensor 256
508. Liquid chromatography-tandem mass spectrometry assay to quantify plitidepsin in human
plasma, whole blood and urine 256
509. Rapid analysis of benzalkonium chloride using paper spray mass spectrometry 257
510. Rapid screening of non-steroidal anti-inflammatory drugs illegally added in anti-rheumatic
herbal supplements and herbal remedies by portable ion mobility spectrometry 257
511. Simultaneous quantitative analysis of polyethylene glycol (PEG), PEGylated paclitaxel and
paclitaxel in rats by MS/MSALL technique with hybrid quadrupole time-of-flight mass
spectrometry 258
512. Lyophilic matrix method for dissolution and release studies of nanoscale particles 258
513. Incorporation of 14C-cholesterol in human adrenal corticocarcinoma H295R cell line and online-
radiodetection of produced 14C-steroid hormone metabolites 259
514. Verification of the effectiveness of the Fourier transform infrared spectroscopy computational
model for colorectal cancer 259
515. Quantification of paracetamol and 5-oxoproline in serum by capillary electrophoresis:
Implication for clinical toxicology 260
516. Quantification of cyclocreatine in mouse and rat plasma using hydrophilic-interaction ultra-
performance liquid chromatography-tandem mass spectrometry 260
517. Volumetric adsorptive microsampling-liquid chromatography tandem mass spectrometry assay
for the simultaneous quantification of four antibiotics in human blood: Method development,
validation and comparison with dried blood spot 261
518. PLGA Ethionamide Nanoparticles for Pulmonary Delivery: Development and in vivo evaluation
of dry powder inhaler 261
519. Simultaneous determination and pharmacokinetics of danshensu, protocatechuic aldehyde, 4-
hydroxy-3-methyloxyphenyl lactic acid and protocatechuic acid in human plasma by LC–
MS/MS after oral administration of Compound Danshen Dripping Pills 262
520. LC–MS bioanalysis of Trastuzumab and released emtansine using nano-surface and molecular-
orientation limited (nSMOL) proteolysis and liquid–liquid partition in plasma of Trastuzumab
emtansine-treated breast cancer patients 262
521. Separation of furostanol saponins by supercritical fluid chromatography 263
xxx
522. Macroporous monoliths for biodegradation study of polymer particles considered as drug
delivery systems 263
523. A novel enantioseparation approach based on liposome electrokinetic capillary chroma-
tography 264
524. Comparative study on the anticancer activities and binding properties of a hetero metal binuclear
complex [Co(dipic)2Ni(OH2)5]·2H2O (dipic = dipicolinate) with two carrier proteins 264
525. Characterization and quantitative analysis of phenolic derivatives in Longxuetongluo Capsule by
HPLC-DAD-IT-TOF-MS 265
Volume 145, October 2017
526. Two-dimensional liquid chromatography in pharmaceutical analysis Instrumental aspects, trends
and applications 265
527. Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A
generic method development approach 266
528. A new platform for serological analysis based on porous 3-dimensional polyethylene sinter
bodies 266
529. Designing a calibration set in spectral space for efficient development of an NIR method for
tablet analysis 267
530. On-line prediction of the glucose concentration of CHO cell cultivations by NIR and Raman
spectroscopy: Comparative scalability test with a shake flask model system 267
531. Conventional and accelerated-solvent extractions of green tea (camellia sinensis) for
metabolomics-based chemometrics 268
532. Phase separation of in situ forming poly (lactide-co-glycolide acid) implants investigated using a
hydrogel-based subcutaneous tissue surrogate and UV–vis imaging 268
533. Mid-infrared and near-infrared spectroscopy for rapid detection of Gardeniae Fructus by a liquid-
liquid extraction process 269
534. Quantitative analysis of a biopharmaceutical protein in cell culture samples using automated
capillary electrophoresis (CE) western blot 269
535. Synchronous characterization of carbohydrates and ginsenosides yields deeper insights into the
processing chemistry of ginseng 270
536. Development and validation of an ICP-MS method for the determination of elemental impurities
in TP-6076 active pharmaceutical ingredient (API) according to USP 〈232〉/〈233〉 270
537. Comparison of SEC and CE-SDS methods for monitoring hinge fragmentation in IgG1
monoclonal antibodies 271
538. Revisiting blood-brain barrier: A chromatographic approach 271
539. Liquid chromatographic enantioseparation of carbocyclic β-amino acids possessing limonene
skeleton on macrocyclic glycopeptide-based chiral stationary phases 272
xxxi
540. On-line coupling of molecularly imprinted solid phase extraction with liquid chromatography for
the fast determination of coumarins from complex samples 272
541. Metabolic profiling analysis of Siraitia grosvenorii revealed different characteristics of green
fruit and saccharified yellow fruit 273
542. Comparison of α-glucosidase inhibitory effect and bioactive constituents of Anemarrhenae
Rhizoma and Fibrous Roots 273
543. Characterization of forced degradation products of torasemide through MS tools and explanation
of unusual losses observed during mass fragmentation of drug and degradation products through
density functional theory 274
544. Metabolic profiling of the traditional Chinese medicine formulation Yu Ping Feng San for the
identification of constituents relevant for effects on expression of TNF-α, IFN-γ, IL-1β and IL-4
in U937 cells 274
545. A comprehensive stability-indicating HPLC method for determination of chloroquine in active
pharmaceutical ingredient and tablets: Identification of oxidation impurities 275
546. Identification of impurities in macrolides by liquid chromatography–mass spectrometric
detection and prediction of retention times of impurities by constructing quantitative structure–
retention relationship (QSRR) 275
547. The impact of ZnO and TiO2 on the stability of clotrimazole under UVA irradiation:
Identification of photocatalytic degradation products and in vitro cytotoxicity assessment 276
548. Chemometrically assisted development and validation of LC–MS/MS method for the analysis of
potential genotoxic impurities in meropenem active pharmaceutical ingredient 276
549. Identification and interconversion of isomeric 4,5-functionalized 1,2,3-thiadiazoles and 1,2,3-
triazoles in conditions of electrospray ionization 277
550. Dissolution assessment of allopurinol immediate release tablets by near infrared spectr-
oscopy 277
551. Enhanced and green extraction polyphenols and furanocoumarins from Fig (Ficus carica L.)
leave using deep eutectic solvents 278
552. Site- and species-specific hydrolysis rates of cocaine 278
553. Comparative stability-indicating chromatographic methods for determination of 4-hexylre-
sorcinol in pharmaceutical formulation and shrimps 279
554. Enantiomeric separation of seven β-agonists by NACE—Study of chiral selectivity with
diacetone-d-mannitol–boric acid complex 279
555. An optimized HPLC-UV method for quantitatively determining sesquiterpenes in Nardostachyos
Radix et Rhizoma 280
556. Photostability testing using online reactor HPLC hyphenation and mass spectrometric compound
identification illustrated by ketoprofen as model compound 280
557. Optoelectronic iron detectors for pharmaceutical flow analysis 281
xxxii
558. Isoconversional approach for non-isothermal decomposition of un-irradiated and photon-
irradiated 5-fluorouracil 281
559. Characterization of impurities in sodium cromoglycate drug substance and eye drops using LC-
ESI-ion trap MS and LC-ESI-QTOF MS 282
560. Method validation and nanoparticle characterization assays for an innovative amphothericin B
formulation to reach increased stability and safety in infectious diseases 282
561. Surfactants enhance recovery of poorly soluble drugs during microdialysis sampling:
Implications for in vitro dissolution-/permeation-studies 283
562. Simultaneous identification and quantification of polymethoxyflavones, coumarin and phenolic
acids in Ageratum conyzoides by UPLC-ESI-QToF-MS and UPLC-PDA 283
563. Degradation and metabolite formation of estrogen conjugates in an agricultural soil 284
564. Chemical analysis and potential endocrine activities of aluminium coatings intended to be in
contact with cosmetic water 284
565. Identification and determination of related substances of ceftaroline fosamil in medicinal product
by high performance liquid chromatography with diode array detection and tandem mass
spectrometry 285
566. Quantification of concentrated Chinese medicine granules by quantitative polymerase chain
reaction 285
567. Chiral separation of terbutaline and non-steroidal anti-inflammatory drugs by using a new
lysine–bridged hemispherodextrin in capillary electrophoresis 286
568. Separation and characterization of allergic polymerized impurities in cephalosporins by 2D-
HPSEC × LC-IT-TOF MS 286
569. Phytochemical analysis and anti-inflammatory evaluation of compounds from an aqueous extract
of Croton cajucara Benth 287
570. Supercritical fluid chromatography approach for a sustainable manufacture of new
stereoisomeric anticancer agent 287
571. Use of ionic liquids as headspace gas chromatography diluents for the analysis of residual
solvents in pharmaceuticals 288
572. Adduct ion-targeted qualitative and quantitative analysis of polyoxypregnanes by ultra-high
pressure liquid chromatography coupled with triple quadrupole mass spectrometry 288
573. Quantitative determination of dobutamine in newborn pig plasma samples by HPLC–
MS/MS 289
574. An integrative investigation of the toxicity of Aconiti kusnezoffii radix and the attenuation effect
of its processed drug using a UHPLC-Q-TOF based rat serum and urine metabolomics
strategy 289
575. Calibration and validation of a MCC/IMS prototype for exhaled propofol online measu-
rement 290
xxxiii
576. Biochemical and functional analysis of corticotropin releasing factor purified from an aqueous
extract of human placenta used as wound healer 290
577. Toxic compounds from tobacco in placenta samples analyzed by UPLC-QTOF-MS 291
578. HPLC-HG-AFS determination of arsenic species in acute promyelocytic leukemia (APL) plasma
and blood cells 291
579. Quantification of IDP-73152, a novel antibiotic, in plasma from mice, rats and humans using an
ultra-high performance liquid chromatography/tandem mass spectrometry method for use in
pharmacokinetic studies 292
580. New approach for the diagnosis of histamine intolerance based on the determination of histamine
and methylhistamine in urine 292
581. Development and validation of sensitive LC–MS/MS method for the quantification of SUVN-
502 and its metabolite and its application for first in human pharmacokinetic study 293
582. Glycosylation patterns of selected proteins in individual serum and cerebrospinal fluid
samples 293
583. MIL-101(Cr)@GO for dispersive micro-solid-phase extraction of pharmaceutical residue in
chicken breast used in microwave-assisted coupling with HPLC–MS/MS detection 294
584. Development of a robust reporter gene assay to measure the bioactivity of anti-PD-1/anti-PD-L1
therapeutic antibodies 294
585. LC–MS/MS-ESI method for simultaneous quantification of darolutamide and its active
metabolite, ORM-15341 in mice plasma and its application to a pharmacokinetic study 295
586. Metabolite identification of AZD8055 in Sprague-Dawley rats after a single oral administration
using ultra-performance liquid chromatography and mass spectrometry 295
587. Confirmation of metabolites of the neuroleptic drug prothipendyl using human liver microsomes,
specific CYP enzymes and authentic forensic samples—Benefits for routine drug testing 296
588. Quantification of 16 β-lactams in chicken muscle by QuEChERS extraction and UPLC-Q-
Orbitrap-MS with parallel reaction monitoring 296
589. Characterization of the phase I and phase II metabolic profile of tolvaptan by in vitro studies and
liquid chromatography–mass spectrometry profiling: Relevance to doping control
analysis 297
590. An LC–MS/MS method for simultaneous determination of nine steroidal saponins from Paris
polyphylla var. in rat plasma and its application to pharmacokinetic study 297
591. N-glucuronidation catalyzed by UGT1A4 and UGT2B10 in human liver microsomes: Assay
optimization and substrate identification 298
592. A simple high performance liquid chromatography–mass spectrometry method for Therapeutic
Drug Monitoring of isavuconazole and four other antifungal drugs in human plasma
samples 298
593. Metabolic profiling of dehydrodiisoeugenol using xenobiotic metabolomics 299
xxxiv
594. Development and validation of a reliable method for thiopurine methyltransferase (TPMT)
enzyme activity in human whole blood by LC–MS/MS: An application for phenotypic and
genotypic correlations 299
595. Development and validation of a GC–MS method for the determination of hydroxyzine and its
active metabolite, cetirizine, in whole blood 300
596. An on-spot internal standard addition approach for accurately determining colistin A and colistin
B in dried blood spots using ultra high-performance liquid chromatography–tandem mass
spectrometry 300
597. Comparative study of single/combination use of Huang-Lian-Jie-Du decoction and berberine on
their protection on sepsis induced acute liver injury by NMR metabolic profiling 301
598. Screening and confirmation of steroids and nitroimidazoles in urine, blood, and food matrices:
Sample preparation methods and liquid chromatography tandem mass spectrometric
separations 301
599. Validation of an HPLC-UV method for analysis of Kaempferol-loaded nanoemulsion and its
application to in vitro and in vivo tests 302
600. Development of direct assays for Toxoplasma gondii and its use in genomic DNA
sample 302
601. Investigation and structural elucidation of a new impurity in bulk drug of cilostazol by
LC/MS/MS, FT-IR and NMR 303
602. Selective screening of glutaric acid acidurias by capillary electrophoresis-mass spect-
rometry 303
603. Online turbulent flow extraction coupled with liquid chromatography–tandem mass spectrometry
for high throughput screening of anabolic steroids in horse urine 304
604. Development of a mixed-mode chromatography with tandem mass spectrometry method for the
quantitative analysis of 23 underivatized amino acids in human serum 304
605. Inhibitory assay for degradation of collagen IV by cathepsin B with a surface plasmon resonance
sensor 305
606. Liquid chromatography-tandem mass spectrometry assay to quantify plitidepsin in human
plasma, whole blood and urine 305
607. Rapid analysis of benzalkonium chloride using paper spray mass spectrometry 306
608. Rapid screening of non-steroidal anti-inflammatory drugs illegally added in anti-rheumatic
herbal supplements and herbal remedies by portable ion mobility spectrometry 306
609. Simultaneous quantitative analysis of polyethylene glycol (PEG), PEGylated paclitaxel and
paclitaxel in rats by MS/MSALL technique with hybrid quadrupole time-of-flight mass
spectrometry 307
610. Lyophilic matrix method for dissolution and release studies of nanoscale particles 307
611. Incorporation of 14C-cholesterol in human adrenal corticocarcinoma H295R cell line and online-
radiodetection of produced 14C-steroid hormone metabolites 308
xxxv
612. Verification of the effectiveness of the Fourier transform infrared spectroscopy computational
model for colorectal cancer 308
613. Quantification of paracetamol and 5-oxoproline in serum by capillary electrophoresis:
Implication for clinical toxicology 309
614. Quantification of cyclocreatine in mouse and rat plasma using hydrophilic-interaction ultra-
performance liquid chromatography-tandem mass spectrometry 309
615. Volumetric adsorptive microsampling-liquid chromatography tandem mass spectrometry assay
for the simultaneous quantification of four antibiotics in human blood: Method development,
validation and comparison with dried blood spot 310
616. PLGA Ethionamide Nanoparticles for Pulmonary Delivery: Development and in vivo evaluation
of dry powder inhaler 310
617. Simultaneous determination and pharmacokinetics of danshensu, protocatechuic aldehyde, 4-
hydroxy-3-methyloxyphenyl lactic acid and protocatechuic acid in human plasma by LC–
MS/MS after oral administration of Compound Danshen Dripping Pills 311
618. LC–MS bioanalysis of Trastuzumab and released emtansine using nano-surface and molecular-
orientation limited (nSMOL) proteolysis and liquid–liquid partition in plasma of Trastuzumab
emtansine-treated breast cancer patients 311
619. Separation of furostanol saponins by supercritical fluid chromatography 312
620. Macroporous monoliths for biodegradation study of polymer particles considered as drug
delivery systems 312
621. A novel enantioseparation approach based on liposome electrokinetic capillary chroma-
tography 313
622. Comparative study on the anticancer activities and binding properties of a hetero metal binuclear
complex [Co(dipic)2Ni(OH2)5]·2H2O (dipic = dipicolinate) with two carrier proteins 313
623. Characterization and quantitative analysis of phenolic derivatives in Longxuetongluo Capsule by
HPLC-DAD-IT-TOF-MS 314
Volume 146, November 2017
624. Analytical characterization of human milk oligosaccharides – potential applications in
pharmaceutical analysis 314
625. Analysis of macrolide antibiotics in water by magnetic solid-phase extraction and liquid
chromatography–tandem mass spectrometry 315
626. Second harmonic generation microscopy as a tool for the early detection of crystallization in
spray dried dispersions 315
627. Sensitive analysis of bioactive secondary metabolites in lichen species using liquid
chromatography–mass spectrometry 316
628. Semiautomated determination of neonicotinoids and characteristic metabolite in urine samples
using TurboFlow™ coupled to ultra high performance liquid chromatography coupled to
Orbitrap analyzer 316
xxxvi
629. A new polymorph of ciprofloxacin saccharinate: Structural characterization and pharmaceutical
profile 317
630. Qualitative and quantitative measurement of cannabinoids in cannabis using modified
HPLC/DAD method 317
631. Quantification of gabapentin polymorphs in gabapentin/excipient mixtures using solid state 13C
NMR spectroscopy and X-ray powder diffraction 318
632. Accurate recognition and feature qualify for flavonoid extracts from Liang-wai Gan Cao by
liquid chromatography-high resolution-mass spectrometry and computational MS/MS
fragmentation 318
633. Sample preparation composite and replicate strategy case studies for assay of solid oral drug
products 319
634. Separation and characterization of chemical constituents in Ginkgo biloba extract by off-line
hydrophilic interaction × reversed-phase two-dimensional liquid chromatography coupled with
quadrupole-time of flight mass spectrometry 319
635. Double-Track Electrochemical Green Approach for Simultaneous Dissolution Profiling of
Naproxen Sodium and Diphenhydramine Hydrochloride 320
636. A workflow for column interchangeability in liquid chromatography using modeling software
and quality-by-design principles 320
637. Absolute quantification of poly(dl-lactide-co-glycolide) in microspheres using quantitative 1H
NMR spectroscopy 321
638. A novel LC-MS/MS method for the quantitative measurement of the acetate content in
pharmaceutical peptides 321
639. A quality by design-based approach to a capillary electrokinetic assay for the determination of
dextromepromazine and levomepromazine sulfoxide as impurities of levomepromazine 322
640. An HPLC–MS/MS method for quantitation of Gly-MCA in mouse plasma: Application to a
pharmacokinetic study 322
641. Development and validation of a sensitive LC–MS/MS method without derivatization/ion-pairing
agents for etimicin quantification in rat plasma, internal ear and kidney 323
642. Bioanalysis of monomethyl fumarate in human plasma by a sensitive and rapid LC–MS/MS
method and its pharmacokinetic application 323
643. Simultaneous determination of tenofovir alafenamide and its active metabolites tenofovir and
tenofovir diphosphate in HBV-infected hepatocyte with a sensitive LC–MS/MS method 324
644. LC–MS/MS method for preclinical pharmacokinetic study of QX-OH, a novel long-acting local
anesthetic, in sciatic nerve blockade in rats 324
645. Capillary electrophoresis study on the base-catalyzed formation of bioactive oxidized metabolites
of 20-hydroxyecdysone 325
646. Quantification of the antimalarial drug pyronaridine in whole blood using LC–MS/MS —
Increased sensitivity resulting from reduced non-specific binding 325
647. Simultaneous extraction of propofol and propofol glucuronide from hair followed by validated
LC–MS/MS analyses 326
648. 11-nor-9-carboxy-Δ9-tetrahydrocannabinol glucuronide exhibits acyl-migration isomers 326
xxxvii
649. Simple and rapid LC-MS/MS method for the absolute determination of cetuximab in human
serum using an immobilized trypsin 327
650. Development of quantitative immunochromatographic assay for rapid and sensitive detection of
carbohydrate antigen 19-9 (CA 19-9) in human plasma 327
651. Simultaneous determination of pentoxifylline, metabolites M1 (lisofylline), M4 and M5, and
caffeine in plasma and dried blood spots for pharmacokinetic studies in preterm infants and
neonates 328
652. Pharmacokinetics and tissue distribution of five major triterpenoids after oral administration of
Rhizoma Alismatis extract to rats using ultra high-performance liquid chromatography–tandem
mass spectrometry 328
653. Solid-phase microextraction coupled to gas chromatography–mass spectrometry followed by
multivariate data analysis for the identification of volatile organic compounds as possible
biomarkers in lung cancer tissues 329
654. UPLC–MS/MS assay of riluzole in human plasma and cerebrospinal fluid (CSF): Application in
samples from spinal cord injured patients 329
655. Simultaneous determination and pharmacokinetic study of twelve bioactive compounds in rat
plasma after intravenous administration of Xuebijing injection by UHPLC-Q-Orbitrap
HRMS 330
656. A surrogate analyte-based liquid chromatography-tandem mass spectrometry method for the
determination of endogenous cyclic nucleotides in rat brain 330
657. Determination of ketamine and its main metabolites by liquid chromatography coupled to tandem
mass spectrometry in pig plasma: Comparison of extraction methods 331
658. Ketamine metabolites with antidepressant effects: Fast, economical, and eco-friendly
enantioselective separation based on supercritical-fluid chromatography (SFC) and single
quadrupole MS detection 331
659. A new method based on supercritical fluid extraction for polyacetylenes and polyenes from
Echinacea pallida (Nutt.) Nutt. Roots 332
660. Validated LC–MS/MS method for the simultaneous determination of rotigotine and its prodrug
in rat plasma and an application to pharmacokinetics and biological conversion in vitro 332
661. Determination of higenamine in dietary supplements by UHPLC/MS/MS method 333
662. Determination of genotoxic epoxide at trace level in drug substance by direct injection
GC/MS 333
663. LC–MS/MS assay for the quantitation of the ribonucleotide reductase inhibitor triapine in human
plasma 334
664. Development and validation of a liquid chromatography-tandem mass spectrometry method for
pharmacokinetic study of TM-53, a novel transcriptional coactivator with PDZ-binding motif
(TAZ) modulator 334
665. Development of a sensitive LC–MS/MS method for quantification of coniferyl ferulate and its
metabolite coniferyl alcohol in rat plasma: Application to a pharmacokinetic study 335
666. LC–MS/MS assay for the quantitation of the ATR kinase inhibitor VX-970 in human
plasma 335
xxxviii
667. Quantitative determination of carfilzomib in mouse plasma by liquid chromatography–tandem
mass spectrometry and its application to a pharmacokinetic study 336
668. Development and implementation of a pass/fail field-friendly method for detecting sildenafil in
suspect pharmaceutical tablets using a handheld Raman spectrometer and silver colloids 336
669. Multi-residue determination of 47 organic compounds in water, soil, sediment and fish—Turia
River as case study 337
670. Separation of bioactive chamazulene from chamomile extract using metal-organic frame-
work 337
671. 1H NMR based pharmacometabolomics analysis of urine identifies metabolic phenotype of
clopidogrel high on treatment platelets reactivity in coronary artery disease patients 338
672. Permeability through the Caco-2 cell monolayer of 42 bioactive compounds in the TCM formula
Gegen-Qinlian Decoction by liquid chromatography tandem mass spectrometry analysis 338
673. Simultaneous quantification of arginine, alanine, methionine and cysteine amino acids in
supplements using a novel bioelectro-nanosensor based on CdSe quantum dot/modified carbon
nanotube hollow fiber pencil graphite electrode via Taguchi method 339
674. Simultaneous optimization of pH and binary organic composition by grid form modeling of the
retention behavior in reversed-phase ultra high-performance liquid chromatography 339
675. On-Line two dimensional liquid chromatography based on skeleton type molecularly imprinted
column for selective determination of sulfonylurea additive in Chinese patent medicines or
functional foods 340
676. Impact of chemotherapy on metabolic reprogramming: Characterization of the metabolic profile
of breast cancer MDA-MB-231 cells using 1H HR-MAS NMR spectroscopy 340
677. An integrated strategy for rapid discovery and identification of the sequential piperine
metabolites in rats using ultra high-performance liquid chromatography/high resolution mass
spectrometery 341
***
1
Volume 132, January 2017
From chemical consistency to effective consistency in precise quality discrimination of Sophora
flower-bud and Sophora flower: Discovering efficacy-associated markers by fingerprint-activity
relationship modeling
Fei Wang, Zi-Yue Xiong, Ping Li, Hua Yang, Hui-Jun Li
ABSTRACT
Chromatographic fingerprint has been extensively used as a comprehensive approach for quality
evaluation of herbal medicines (HMs). However, similar chemical profiles do not always mean similar
efficacies. The present work, taking Sophora flower-bud and Sophora flower as a typical case,
attempts to develop a rational strategy based on fingerprint-activity relationship modeling to realize
quality evaluation from chemical consistency to effective consistency. A total of 57 batches of
Sophora samples were collected and their antioxidant and hyaluronidase inhibitory activities were
measured. Chemical fingerprints were established by high performance liquid chromatography
(HPLC) coupled with photodiode array (PDA) detector and quadrupole time-of-flight mass
spectrometry (Q-TOF MS), and similarity analyses were calculated based on eight common
characteristic peaks. Subsequently, three principal bioactive markers were discovered by correlating
biological effects with chemical fingerprints via partial least squares regression (PLSR) and back
propagation-artificial neural network modeling (BP-ANN). The selected markers were quantified by
the ‗single standard to determine multi-components‘ method, and then the quantitative data as well as
their bioactive properties were subjected to principal component analysis to generate two clear-cut
groups. This study not only demonstrates the necessity of effective consistency besides chemical
consistency in the quality evaluation of HMs, but also provides an applicable strategy to screen out
efficacy-associated markers by fingerprint-activity relationship modeling.
2
Impact of mono- and poly-ester fractions on polysorbate quantitation using mixed-mode HPLC-
CAD/ELSD and the fluorescence micelle assay
Steffen Lippold, Stijn H.S. Koshari, Robert Kopf, Rudolf Schuller, Henning Koehn
ABSTRACT
Determination of excipient content in drug formulation is an important aspect of pharmaceutical
formulation development and for analytical testing of the formulation. In this study, the influence of
polysorbate subspecies, in particular mono- and poly-esters, for determining polysorbate (PS) content
were investigated by comparing three of the most widely used PS quantitation approaches, the
Fluorescence Micelle Assay (FMA) and Mixed-Mode High Performance Liquid Chromatography
coupled with Charged Aerosol Detection (MM-CAD) or Evaporative Light Scattering Detection (MM-
ELSD). FMA and MM-CAD were employed to investigate the quantitation behavior of PS20 and
PS80 subspecies and corresponding degradation products in placebo formulations using forced
degradation conditions at 40 °C for up to 12 weeks. While both methods allowed accurate and
comparable quantification of neat PS at the beginning of stress studies, pronounced differences in
content determination between the methods were observed at later time points, which were attributable
to substantial differences in the contribution of individual mono- and poly-esters to the overall
quantitation results. It was particularly surprising to find that the main component of PS20,
polyoxyethylene sorbitan monolaurate, did not show a signal at the studied concentration using FMA.
Moreover, the degradation of polysorbate poly-esters, was reflected much stronger in FMA than MM-
CAD results. Additional experiments employing chemical oxidation and base hydrolysis to degrade
PS20, quantified by FMA and MM-ELSD, also show preferential reduction in certain subspecies
depending on the degradation pathway involved. For PS20 degraded by chemical oxidation,
quantitation results were lower for FMA than MM-ELSD, while the opposite trend was observed with
base hydrolysis.
3
Quality assessment of marketed chamomile tea products by a validated HPTLC method
combined with multivariate analysis
Etil Guzelmeric, Petar Ristivojević, Irena Vovk, Duńanka Milojković-Opsenica, Erdem Yesilada
ABSTRACT
Chamomile tea composed of dried flower heads of Matricaria recutita L. (Asteraceae) is one of the
most popular single ingredient herbal teas. Tea industries, spice shops or public bazaars are mostly
supplied chamomile as a raw material via cultivation or through nature-picking. However, one of the
drawbacks of nature-picking is adulteration. This could be either due to false authentication of the
plant materials by ingenuous pickers or intentional/unintentional substitution with other flowers
resembling to chamomile in appearance during harvesting. Therefore, quality control of raw
chamomile materials before marketing should be carefully considered not only by quantification of
apigenin 7-O-glucoside (active marker) but also by fingerprinting of chemical composition. This work
presents both quantification of apigenin 7-O-glucoside and chemical fingerprinting of commercial
chamomile tea products obtained from different food stores and spice shops by a validated HPTLC
method. In addition, HPTLC profiles of investigated chamomile tea samples were compared with
HPLC method stated in the European Pharmacopoeia and it was found that HPTLC method was
superior to HPLC method in the field of adulteration confirmation. Therefore, fingerprint profiles
performed on the silica gel 60 NH2 F254s HPTLC plates combined with pattern recognition
techniques of these marketed products were comparatively evaluated with wild and cultivar chamomile
samples and also chamomile-like species from Asteraceae. Consequently, not chamomile tea bags but
crude flowers sold on market were found to be adulterated with other plant materials.
Simultaneous quantification and identification of flavonoids, lignans, coumarin and amides in
leaves of Zanthoxylum armatum using UPLC-DAD-ESI-QTOF–MS/MS
Vinod Bhatt, Sushila Sharma, Neeraj Kumar, Upendra Sharma, Bikram Singh
ABSTRACT
The current study presents isolation and characterization of twelve compounds including catechin (1),
isovitexin (2), hesperidin (3), psoralin (4), eudesmin (5), kobusin (6), fargesin (7), sesamin (8),
asarinin (9), planispine-A (10), α-sanshool (11) and vitexin (12), from the leaves of Zanthoxylum
armatum. Further, two rapid and simple ultra performance liquid chromatography-diode array
detection (UPLC-DAD) methods were developed for the simultaneous quantitative determination of
isolated compounds from Z. armatum leaves. These analytical methods were validated for linearity,
precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ). The LOD and LOQ
were in the range of 0.06–0.21 μg/mL and 0.19–0.69 μg/mL, respectively. The validated method was
linear (R2 ≥ 0.9906), precise in terms of peak area (intra-day RSDs <3.8% and inter-day RSDs
<2.7%), and accurate (109.6–92.5%). This is the first report on the isolation and quantification of 1, 2,
4 and 12 in Z. armatum and 3 in Zanthoxylum genus. The methods: were successfully applied to assess
the quality of samples collected from different locations of Himachal Pradesh during summer and
winter season. The results demonstrated that flavonoids and furofuran lignans were the major
constituents in Z. armatum leaves. The developed methods: were further applied for tandem
electrospray ionization-mass spectrometry (UPLC-DAD-ESI-MS/MS) and total eighteen compounds
were identified including phenolic acid, flavonoids, furofuran lignans, coumarin and isobutyl amides.
4
Combination of HPLC–MS and QAMS as a new analytical approach for determination of
saponins in ginseng containing products
Andrey Stavrianidi, Elena Stekolshchikova, Anna Porotova, Igor Rodin, Oleg Shpigun
ABSTRACT
Conventional liquid chromatographic methods coupled with ultraviolet detection with low-wavelength
range are lacking selectivity and sensitivity to determine both polar and less polar ginsenosides. Also
the lack of standard substances for such quality control methods is leading to development of the
approaches using single standard for quantitative analysis of multi-component system (QAMS). The
objective of present study was to establish and compare for the first time liquid chromatography–
ultraviolet detection and liquid chromatography–mass spectrometry QAMS methods for the
simultaneous determination of protopanaxatriol-type and protopanaxadiol-type ginsenosides in a
variety of ginseng products. Sixteen polar and less polar ginsenosides were separated on a reversed-
phase C18-column (150mm × 2.0 mm, 2.2 μm) with a mobile phase consisting of 0.1% formic acid in
water and acetonitrile. Components were then detected by means of ultraviolet and mass spectrometry
detection. Characteristic sapogenin fragmentation signals with m/z 423 and 425 for two major groups
of ginseng saponins allowed their simultaneous determination in a single chromatographic run, while
the use of ultraviolet detection tends to give overvalued results. Structural correlation between the
relative response factors and saponin structure was demonstrated. The method was linear (R2 >0.999)
and sensitive (LODs, 0.01–0.03 mg/mL) within the concentration range tested. Concentrations of
individual ginsenosides and several quality control parameters were determined in ginseng root
extracts and commercial ginseng products of different types (root slices, tablets and tea samples), and
results showed that ginsenoside content can be successfully measured by means of QAMS approach.
Simultaneous determination of phenolic acids and flavonoids in Chenopodium formosanum
Koidz (djulis) by HPLC-DAD-ESI–MS/MS
B.Y. Hsu, S.W. Lin, B. Stephen Inbaraj, B.H. Chen
ABSTRACT
A high performance liquid chromatography-diode array detection-tandem mass spectrometry method
(HPLC-DAD-MS/MS) was developed for simultaneous determination of phenolic acids and
flavonoids in djulis (Chenopodium formosanum Koidz.), a traditional Chinese herb reported to possess
vital biological activities. A high yield of phenolic acids and flavonoids was attained by employing
50% ethanol in water as the extraction solvent and shaking in a 60 °C water bath for 3 h. A total of 8
phenolic acids and 14 flavonoids were separated and identified within 55 min by using a Poroshell 120
EC-C18 column with detection at 280 nm, flow rate at 0.8 mL/min, column temperature at 35 °C, and
a gradient solvent system of 0.1% formic acid in water and acetonitrile. Two internal standards caffeic
acid and kaempferol-3-O-rutinoside were used for quantitation of phenolic acids and flavonoids in
djulis respectively. The amounts of phenolic acids ranged from 11.5 ± 0.8 μg/g (caffeoyl-putrescine-
derivative (2)) to 1855.3 ± 16.9 μg/g (hydroxylphenylacetic acid pentoside), while the flavonoids
ranged from 19.93 ± 2.29 μg/g (quercetin-3-O-(coumaryl)-rutinoside-pentoside (1)) to 257.3 ± 2.05
μg/g (rutin-O-pentoside (2)). A high recovery (89.68–97.20%) and high reproducibility was obtained
for both phenolic acids and flavonoids with the relative standard deviation (RSD) for the latter ranging
from 0.09–8.22% (intra-day variability) and 0.80–8.48% (inter-day variability). This method may be
applied to determination of both phenolic acids and flavonoids in food products and Chinese herbs.
5
Design of a strong cation exchange methodology for the evaluation of charge heterogeneity in
glatiramer acetate
Víctor R. Campos-García, Carlos A. López-Morales, Eleuterio Benites-Zaragoza, Armando Jiménez-
Miranda, E. Medina-Rivero
ABSTRACT
Complex pharmaceuticals are in demand of competent analytical methods able to analyze charge
heterogeneity as a critical quality attribute (CQA), in compliance with current regulatory expectations.
A notorious example is glatiramer acetate (GA), a complex polypeptide mixture useful for the
treatment of relapsing-remitting multiple sclerosis. This pharmaceutical challenges the current state of
analytical technology in terms of the capacity to study their constituent species. Thus, a strong cation
exchange methodology was designed under the lifecycle approach to support the establishment of GA
identity, trough the evaluation of its chromatographic profile, which acts as a charge heterogeneity
fingerprint. In this regard, a maximum relative margin of error of 5% for relative retention time and
symmetry factor was proposed for the analytical target profile. The methodology met the proposed
requirements after precision and specificity tests results, the former comprised of sensitivity and
selectivity. Subsequently, method validation was conducted and showed that the method is able to
differentiate between intact GA and heterogeneity profiles coming from stressed, fractioned or
process-modified samples. In summary, these results provide evidence that the method is adequate to
assess charge heterogeneity as a CQA of this complex pharmaceutical.
Development and characterization of the voriconazole loaded lipid-based nanoparticles
Petra Füredi, Zsófia Edit Pápay, Kristóf Kovács, Borbála Dalmadi Kiss, Imre Klebovich
ABSTRACT
The number of topical fungal infections is growing, mostly owing to immunosuppressive therapy.
Several topical fungal infections, such as eye mycoses, can be treated by local administration of
antimycotic drugs. One major group of the antifungal agents is triazole, such as voriconazole (VCZ),
which is used as the first line treatment of aspergillosis. A disadvantage of VCZ is its low water
solubility making the drug difficult to administer in a liquid preparation. The lipid-based nanoparticles
(LNP) have attracted increasing attention due to their advantageous properties. Contrarily to the
conventional carrier systems, LNP can improve the poor solubility of topically used drugs, such as
VCZ. Therefore, LNP represents promising alternatives to traditional carrier systems. The aim of the
study was to formulate VCZ loaded lipid-based nanoparticles (VCZ-LNP) by high pressure
homogenization (HPH). The developed LNPs were characterized by particle size analysis, IR
spectroscopy, differential scanning calorimetry, dialysis test and antifungal efficacy studies. The
particle size of the optimized nanoparticles from the selected lipid base, Witepsol® W35, was 182 ±
4.1 nm after five cycles of homogenization at 600 bars. The antifungal study confirmed that the
optimized VCZ-LNP inhibited the fungus reproduction.
6
Selective functional activity measurement of a PEGylated protein with a modification-dependent
activity assay
Alfred Weber, Andrea Engelmaier, Gabriele Mohr, Sonja Haindl, Peter L. Turecek
ABSTRACT
BAX 855 (ADYNOVATE) is a PEGylated recombinant factor VIII (rFVIII) that showed prolonged
circulatory half-life compared to unmodified rFVIII in hemophilic patients. Here, the development and
validation of a novel assay is described that selectively measures the activity of BAX 855 as cofactor
for the serine protease factor IX, which actives factor X. This method type, termed modification-
dependent activity assay, is based on PEG-specific capture of BAX 855 by an anti-PEG IgG
preparation, followed by a chromogenic FVIII activity assay. The assay principle enabled sensitive
measurement of the FVIII cofactor activity of BAX 855 down to the pM-range without interference by
non-PEGylated FVIII. The selectivity of the capture step, shown by competition studies to primarily
target the terminal methoxy group of PEG, also allowed assessment of the intactness of the attached
PEG chains. Altogether, the modification-dependent activity not only enriches, but complements the
group of methods to selectively, accurately, and precisely measure a PEGylated drug in complex
biological matrices. In contrast to all other methods described so far, it allows measurement of the
biological activity of the PEGylated protein. Data obtained demonstrate that this new method principle
can be extended to protein modifications other than PEGylation and to a variety of functional activity
assays.
Investigation of the effect of mobile phase composition on selectivity using a solvent-triangle
based approach in achiral SFC
Charlene Muscat Galea, Debby Mangelings, Yvan Vander Heyden
ABSTRACT
Defining a method development methodology for achiral drug impurity profiling in SFC requires a
number of steps. Initially, diverse stationary phases are characterized and a small number of
orthogonal or dissimilar phases are selected for further method development. In this paper, we focus
on a next step which is the investigation of the modifier composition on chromatographic selectivity. A
solvent-triangle based approach is used in which blends of organic solvents, mainly ethanol (EtOH),
propanol (PrOH), acetonitrile (ACN) and tetrahydrofuran (THF) mixed with methanol (MeOH) are
tested as modifiers on six dissimilar stationary phases. The tested modifier blends were composed to
have equal eluotropic strengths as calculated on bare silica. The modifier leads to minor changes in
terms of elution order, retention and mixture resolution. However, varying only the modifier
composition on a given stationary phase does not lead to the creation of dissimilar systems. Therefore
the modifier composition is an optimization parameter, with the stationary phase being the factor
determining most the selectivity of a given mixture in achiral SFC.
7
NQO1 and CYP450 reductase decrease the systemic exposure of rifampicin-quinone and
mediate its redox cycle in rats
Fuguo Shi, Xiaobing Li, Hong Pan, Li Ding
ABSTRACT
Rifampicin (RIF) is used in regimens for infections caused by Mycobacteria accompanied by serious
adverse reactions. Rifampicin-quinone (RIF-Q) is a major autoxidation product of RIF. It is not clear
whether RIF-Q plays a role in RIF induced adverse reactions. Investigation of the systemic exposure of
RIF-Q is helpful to better understand the role of RIF-Q in RIF induced adverse reactions. In this study,
a simple and reproducible high performance liquid chromatography-mass spectrometry (LC–MS)
method involving a procedure to prevent the RIF from oxidation for simultaneous quantification of
RIF and RIF-Q in rat plasma has been developed and validated, and applied to elucidate the systemic
exposure of RIF-Q in rats. The pharmacokinetics data showed that the systemic exposure of RIF-Q
was very low (0.67% of RIF, AUC0-24) in rats after oral administration of RIF. However, RIF-Q may
undergo the redox cycle in vivo by the evidence that the majority of RIF-Q was reduced to RIF after
an oral dose of RIF-Q. Pretreatment with the NAD (P) H: quinone oxidoreductase 1 (NQO1) specific
inhibitor dicoumarol and/or cytochrome P450 reductase (CPR) inhibitor diphenyleneiodonium
suppressed the redox cycle and significantly increased the systemic exposure of RIF-Q. The inhibitors
also attenuated the redox cycle induced reactive oxygen species formation and cytotoxicity in RIF-Q-
treated HepG2 cells. These results indicate that NQO1 and CPR play an important role in redox cycle
of RIF-Q and may thus contribute to RIF-induced adverse reactions.
Study the influence of licorice and pomegranate drinks on nicotine metabolism in human urine
by LC-orbitrap MS
Ahmad Abu-awwad, Tawfiq Arafat, Oliver J. Schmitz
ABSTRACT
Nicotine-diet interactions have a particular importance on human health. Some food substances are
subject to change hepatic CYP2A6 metabolism rate for nicotine and its levels in smokers
consequently. This study investigates the effect of pomegranate and licorice drinks on nicotine
metabolism, by a new developed and validated method for simultaneous determination of nicotine with
its major metabolites (cotinine and nicotine N-oxide) in human urine, utilizing LC ESI-orbitrap-MS.
Twenty-four Jordanian healthy and smoker volunteers were participated in two equal groups,
crossover design for each of pomegranate and licorice test drink. In the study periods each group
assigned either to drink test juice three times a day or to be avoided from test drink for 7 successive
days, and then both groups switched their drink treatment in subsequent period. Early morning urine
samples were collected from all volunteers after each period. Nicotine metabolism rate was evaluated
from nicotine/cotinine and nicotine/nicotine N-oxide ratios in urine. A consistent trend of increase in
metabolism rate for nicotine was observed from urine analysis under pomegranate or licorice drink
conditions compared to control conditions. Pomegranate and licorice drinks are increasing the
metabolism rate for nicotine in terms of induction effect for hepatic cytochrome p450 enzymes.
8
NMR-based metabonomics and correlation analysis reveal potential biomarkers associated with
chronic atrophic gastritis
Jiajia Cui, Yuetao Liu, Yinghuan Hu, Jiayu Tong, Guanhua Du
ABSTRACT
Chronic atrophic gastritis (CAG) is one of the most important pre-cancerous states with a high
prevalence. Exploring of the underlying mechanism and potential biomarkers is of significant
importance for CAG. In the present work, 1H NMR-based metabonomics with correlative analysis was
performed to analyze the metabolic features of CAG. 19 plasma metabolites and 18 urine metabolites
were enrolled to construct the circulatory and excretory metabolome of CAG, which was in response
to alterations of energy metabolism, inflammation, immune dysfunction, as well as oxidative stress. 7
plasma biomarkers and 7 urine biomarkers were screened to elucidate the pathogenesis of CAG based
on the further correlation analysis with biochemical indexes. Finally, 3 plasma biomarkers (arginine,
succinate and 3-hydroxybutyrate) and 2 urine biomarkers (α-ketoglutarate and valine) highlighted the
potential to indicate risks of CAG in virtue of correlation with pepsin activity and ROC analysis. Here,
our results paved a way for elucidating the underlying mechanisms in the development of CAG, and
provided new avenues for the diagnosis of CAG and presented potential drug targets for treatment of
CAG.
Metabolomic profiling of doxycycline treatment in chronic obstructive pulmonary disease
Brajesh Singh, Saikat K. Jana, Nilanjana Ghosh, Soumen K. Das, Koel Chaudhury
ABSTRACT
Serum metabolic profiling can identify the metabolites responsible for discrimination between
doxycycline treated and untreated chronic obstructive pulmonary disease (COPD) and explain the
possible effect of doxycycline in improving the disease conditions. 1H nuclear magnetic resonance
(NMR)-based metabolomics was used to obtain serum metabolic profiles of 60 add-on doxycycline
treated COPD patients and 40 patients receiving standard therapy. The acquired data were analyzed
using multivariate principal component analysis (PCA), partial least-squares-discriminant analysis
(PLS-DA), and orthogonal projection to latent structure with discriminant analysis (OPLS-DA). A
clear metabolic differentiation was apparent between the pre and post doxycycline treated group. The
distinguishing metabolites lactate and fatty acids were significantly down-regulated and formate,
citrate, imidazole and l-arginine upregulated. Lactate and folate are further validated biochemically.
Metabolic changes, such as decreased lactate level, inhibited arginase activity and lowered fatty acid
level observed in COPD patients in response to add-on doxycycline treatment, reflect the anti-
inflammatory action of the drug. Doxycycline as a possible therapeutic option for COPD seems
promising.
9
Analysis of fenretinide and its metabolites in human plasma by liquid chromatography–tandem
mass spectrometry and its application to clinical pharmacokinetics
Hwang Eui Cho, H. Kang Min
ABSTRACT
A simple and accurate high-performance liquid chromatography–tandem mass spectrometry (LC–
MS/MS) method was developed for the determination of N-(4-hydroxyphenyl) retinamide (fenretinide,
4-HPR) and its metabolites, 4-oxo-N-(4-hydroxyphenyl) retinamide (4-oxo-4-HPR) and N-(4-
methoxyphenyl) retinamide (4-MPR), in human plasma. Plasma samples were prepared using protein
precipitation with ethanol. Chromatographic separation of the three analytes and N-(4-ethoxyphenyl)
retinamide (4-EPR), an internal standard, was achieved on a Zorbax SB-C18 column (3.5 μm, 50 × 2.1
mm) using gradient elution with the mobile phase of 0.1% formic acid in water and acetonitrile (pH*
2.4) at a flow rate of 0.5 mL/min. Electrospray ionization (ESI) mass spectrometry was operated in the
positive ion mode with multiple reaction monitoring (MRM). The calibration curves obtained were
linear over the concentration range of 0.2–50 ng/mL with a lower limit of quantification of 0.2 ng/mL.
The relative standard deviation of intra-day and inter-day precision was below 7.64%, and the
accuracy ranged from 94.92 to 105.43%. The extraction recoveries were found to be higher than
90.39% and no matrix effect was observed. The analytes were stable for the durations of the stability
studies. The validated method was successfully applied to the analyses of the pharmacokinetic study
for patients treated with 4-HPR in a clinical trial.
Poor and enantioselective bioavailability of naftopidil enantiomers is due to extensive and
stereoselective metabolism in rat liver
Xiawen Liu, Lijun Zhu, Biyun Huang, Junjun Huang, Mu Yuan
ABSTRACT
Racemic naftopidil (NAF) is used to treat benign prostatic hyperplasia (BPH) and prostatic cancer
(PCa). It exhibits greater efficacy but requires higher dose than other ɑ1-adrenoceptor blockers
because of its poor bioavailability. It was previously shown that bioavailability of S (−)-NAF (14.5%)
was twice that of R (+)-NAF (6.8%). The present study aimed to elucidate the major factors
contributing to the poor and enantioselective bioavailability of NAF. First, absorption of NAF
enantiomers was examined using a perfusated intestinal model. NAF enantiomers were found to be
equally and highly permeable in all segments of the intestine. Second, the metabolites formed in
different parts of the intestine and in bile were investigated. Glucuronidation of NAF enantiomers was
found to occur primarily in the liver. Third, a new method consisting of ultra performance liquid
chromatography coupled with triple-quadruple mass spectrometry (UPLC–MS/MS) was employed to
quantify and calculate the pharmacokinetic parameters of NAF enantiomers and their glucuronides
after the enantiomers were intravenously injected into rats. The amounts of R(+)-NAF glucuronide
(R(+)-NAF-G) and S(−)-NAF glucuronide (S(−)-NAF-G) were six-fold higher than that of R(+)-NAF,
and three-fold higher than that of S(−)-NAF. Glucuronidation of S(−)-NAF was faster than that of
R(+)-NAF, but the conjugated amount was half of that of R(+)-NAF. Thus, bioavailability of S(−)-
NAF was twice that of R(+)-NAF. In conclusion, extensive phase II metabolism in the liver
significantly contributes to the low bioavailability of NAF enantiomers. Glucuronidation is the most
important metabolic pathway for NAF enantiomers. Glucuronidation of S(−)-NAF is faster but occurs
to a lesser extent than that of R(+)-NAF.
10
Robust HPLC–MS/MS method for levofloxacin and ciprofloxacin determination in human
prostate tissue
O. Szerkus, J. Jacyna, A. Gibas, M. Sieczkowski, M.J. Markuszewski
ABSTRACT
Fluoroquinolones are the drugs of choice in the prevention of bacterial infections after transrectal
ultrasound guided prostate biopsy. In order to improve assessment of antibacterial efficacy in the target
tissue a simple, selective, rapid and robust HPLC-ESI–MS/MS method for the determination of
levofloxacin and ciprofloxacin concentrations in human prostate bioptates was developed and
validated. Preparation procedure for prostate samples (10 mg) was carried out using homogenization
and filtration steps. Analyses were performed within 3.5 min using RP column in the isocratic elution
mode with mobile phase composed of a mixture of 0.1% formic acid aqueous solution and 0.1%
formic acid methanol solution (v/v; 79:21). The method was linear between 0.3 μg/g and 15 μg/g for
levofloxacin and ciprofloxacin with coefficient of correlation (r) ≥0.999. The limit of detection and the
limit of quantification for levofloxacin were 0.06 μg/g and 0.2 μg/g and for ciprofloxacin were 0.04
μg/g and 0.13 μg/g, respectively. Average concentrations (±SD) of levofloxacin and ciprofloxacin
obtained from patient tissue were 5.4 ± 2.2 μg/g and 3.9 ± 1.5 μg/g, respectively. Additionally, during
validation procedure a novel, experimental design approach was applied for the robustness study. For
evaluation of analytical method robustness, Plackett-Burman design was employed and for sample
preparation method robustness Fractional Factorial design was used. The developed and validated
method was successfully applied to examine prostate tissue samples obtained from patients enrolled
into a clinical study. Up to now, there has been no other HPLC-ESI-MS/MS method reported for the
simultaneous determination of levofloxacin and ciprofloxacin in human prostatic tissue.
Pharmacokinetics, excretion of 8-cetylberberine and its main metabolites in rat urine
Yuli Hu, Shoujun Fan, Xiaobing Liao, Chao Chen, Xuegang Li
ABSTRACT
The Berberine (BBR) is a bioactive plant ingredient derived from the roots and barks of Berberis
aristata and Coptis chinensis and has a wide variety of pharmacological effects. 8-cetylberberine (8-
BBR-C16) is the berberine (BBR) derivative reconstructed from adding octadecyl at C-8 of BBR to
enhance its activity. This study presents a reliable method for the determination of BBR and 8-BBR-
C16 in rat plasma, urine and feces. BBR and 8-BBR-C16 were determined by HPLC-UV after liquid-
liquid extraction for plasma samples, and solid-phase extraction for urinary and fecal samples. The
method was linear over the concentration range of 10-300 ng·ml−1 for the plasma samples, 25-2000
ng·ml−1 for the urinary samples, and 100-2000 ng·g−1 for the fecal samples. Furthermore, a metabolic
investigation on urine was performed by LC/MS/MS analysis to identify the structures of 8-BBR-C16
metabolites by full scan and product ion scan. Adult Sprague-Dawley rats were divided into two
groups. In the control group, rats received 80 mg·kg−1 BBR, and in the drug-treated group, rats
received 80 mg·kg−1 8-BBR-C16. The results indicate that there were significant differences in the
pharmacokinetic parameters and in the accumulated excretion levels between the control group and the
drug-treated group. The Cmax and AUC0-t of 8-BBR-C16 were 2.8 and 12.9 times higher than those
of BBR, and the relative bioavailability of BBR to 8-BBR-C16 was 7.7%. The total excretion amount
through the urine and feces of 8-BBR-C16 was 76.9%, but that of BBR was only 20.5%. Additionally,
8-BBR-C16 was metabolized in rat urine with phase I demethylation and phase II glucuronidation or
sulfation.
11
An UPLC–MS/MS method for the quantitation of alectinib in rat plasma
Xiang-xin Huang, Yun-xuan Li, Xiang-yu Li, Xiao-xia Hu, Guo-xin Hu
ABSTRACT
Currently, crizotinib is the first generation drug, which has been used in the treatment of ALK-
rearranged non-small cell lung cancer (NSCLC). However, more and more patients are found in
crizotinib-resistance. In the last year, alectinib has been approved for treatment of patients with
crizotinib-resistance. In this study, we aim to develop and validate a simple, rapid and sensitive tandem
mass spectrometry (UHPLC–MS/MS) method for determination of alectinib in rat plasma. Diazepam
was chosen as an internal standard (IS). Protein precipitation by acetonitrile was utilized to prepare
plasma samples. Chromatographic separation was achieved on a RRHD Eclipse plus C18 (2.1 × 50
mm, 1.8 μ) column with a gradient mobile phase consisting of acetonitrile and water (containing 0.1%
formic acid). The analytes were detected by an electrospray ionization (ESI) source in positive mode.
A dynamic multiple reactions monitoring (MRM) method was developed to detect specific precursor
and product ions. The target fragment ions were m/z 483.2 → 396.1 for alectinib and m/z 285.0 →
192.9 for diazepam (IS). Linear calibration plots were achieved in the range of 1–500 ng/ml for
alectinib (R2 = 0.997) in rat plasma. Mean recoveries of alectinib in rat plasma ranged from 84.2% to
92.2%. The intra- and inter-day precision was below 9.3% and accuracy was from −1.4% to 12.1%. No
obvious matrix effect was found. This method shows a good performance: accuracy, precision and
stability. It has been fully validated and successfully applied to pharmacokinetic study of alectinib.
Development and validation of a rapid and sensitive UPLC–MS/MS method for quantification of
kukoamine B in human plasma: Application to a clinical pharmacokinetic study
Zhenlei Wang, Qian Zhao, Lili Li, Pei Hu, Ji Jiang
ABSTRACT
A rapid, accurate and robust method was firstly developed using ultra-performance liquid
chromatography tandem mass spectrometry (UPLC–MS/MS) assay to quantify kukoamine B, which is
a novel drug under clinical development for the treatment of sepsis, in human plasma. Solid-phase
extraction (SPE) was used to extract kukoamine B from human plasma. The extracts were separated on
a Waters Acquity HSS T3 column (2.1 × 50 mm i.d., 1.8 μm) with a gradient elution method, using
mobile phases of A (formic acid-water (1:1000, v/v)) and B(formic acid-methanol (1:1000, v/v)).
Kukoamine B and internal standard (5-deuterated isotope kukoamine B) were detected under the
multiple-reaction monitoring mode by an API 5500 triple quadrupole mass spectrometer with
electrospray ionization. The method showed good linearity from 0.100 to 50.0 ng/mL according to
1/x2 weighted linear regression analysis. Inter- and intra-batch precision of kukoamine B were less
than 15% and the accuracy was within 85–115%. The extraction recoveries and matrix effect of
kukoamine B at three concentration levels were consistent. The sensitivity, specificity and stabilities
under various conditions were validated. In conclusion, the validation results showed that this method
was rapid, accurate, robusts and can successfully fulfill the requirement of clinical pharmacokinetic
study of kukoamine B mesylate in Chinese healthy subjects.
12
Quantitative determination of xanthorrhizol in rat plasma by HPLC–MS/MS and its application
to a pharmacokinetic study
Seungmok Choi, Minsoo Kim, Changhee Kim, Jae-Kwan Hwang, Wonku Kang
ABSTRACT
Although xanthorrhizol, a sesquiterpenoid oil isolated from the rhizoma of Curcuma xanthorrhiza
Roxb. Known as Java turmeric, represents a variety of pharmacological activities, to date, there have
been no validated determination methods of xanthorrhizol in biological samples. Thus, we developed a
liquid chromatographic method using a tandem mass spectrometry for the determination of
xanthorrhizol in rat plasma. After simple protein precipitation with acetonitrile including diclofenac
(internal standard, IS), the analytes were chromatographed on a reversed-phased column with a mobile
phase of 20 mM ammonium acetate aqueous solution and acetonitrile (20:80, v/v). The ion transitions
of the precursor to the product ion were principally deprotonated ions [M−H]− at m/z 216.9 → 132.8
for xanthorrhizol and 296.1 → 251.7 for the IS. The accuracy and precision of the assay were in
accordance with FDA regulations for the validation of bioanalytical methods. This analytical method
was successfully applied to monitor plasma concentrations of xanthorrhizol over time following
intravenous administration in rats.
Development and validation of a stability-indicating HPLC-UV method for the determination of
Thiocolchicoside and its degradation products
Silvio Aprile, Rossana Canavesi, Michele Bianchi, Giorgio Grosa, Erika Del Grosso
ABSTRACT
A stability indicating high performance liquid chromatography method has been developed for the
determination of thiocolchicoside (TCC) and its main degradation products thiocolchicoside S-oxide
(D1SO) and 3-O-demethylthiocolchicine (D3) in liquid and solid formulations. The method was
developed based on a previous forced degradation study showing that TCC underwent chemical
degradation by acid/base catalyzed hydrolysis and oxidation being the main degradation products D3
and D1SO respectively. The analytes separation and quantification were achieved on a Synergi™ 4 μm
Polar-RP 80 Å, column 150 × 4.6 mm (Phenomenex) using the mobile phase constituted (flow rate 1
mL min−1) of eluant A: 20 mM sodium acetate buffer (pH 5.0) and eluant B: MeOH:CH3CN (20:80);
the elution was performed in gradient mode detecting the analytes at 254 nm. The method showed
linearity for TCC assay in the 5–15 μg mL−1, range and for unknown (TCCfu) and known (D1SO and
D3) degradation products assay, in the 0.5–10 μg mL−1 range: all the square of the correlation
coefficients were greater than 0.999. The precision, determined in terms of intra-day and inter-day
were expressed as RSDs and resulted to be 1.19, 1.10, 1.37 and 1.04% and 0.95, 0.83, 1.30 and 0.72
for TCC, TCCfu, D1SO and D3, respectively. The method demonstrated also to be accurate; indeed,
the average recoveries were 102.1/102.0% for TCC (ampoules and hard capsules respectively),
101.3/100.3% for TCCfu, 101.7/100.2% for D1SO, and 101.4/101.4% for D3. The robustness was also
evaluated by variations of mobile phase composition and pH. Finally, the applicability of the method
was evaluated by analysis of commercial liquid and solid dosage forms.
13
Dried blood spot analysis of gabapentin as a valid alternative for serum: a bridging study
Nele Sadones, Elien Van Bever, Luc Van Bortel, Willy E. Lambert, Christophe P. Stove
ABSTRACT
We evaluated the applicability of a validated GC–MS method for the determination of gabapentin in
dried blood spots (DBS). Important for the acceptance of DBS sampling as an alternative sampling
strategy is the possibility to base solid conclusions on the quantification. Therefore, bridging studies –
studies in which the correlation between both DBS and a reference matrix (e.g. serum) is evaluated
statistically- need to be conducted. To this end, a comparative study was set up to quantify gabapentin
in both blood (DBS) and serum samples. Statistically significant differences between DBS and serum
concentrations were found (p < 0.001). A mean blood-to-serum ratio of 0.85 was observed, which is in
line with expectations. Calculated serum concentrations (obtained by dividing the DBS concentrations
by 0.85) demonstrated a good correlation with measured serum concentrations, with 87% of samples
fulfilling the criterion for incurred sample reanalysis. Furthermore, our data indicate a good correlation
between capillary and venous concentrations. Conclusively, this study demonstrated that DBS are a
valid alternative to serum for the determination of gabapentin.
SPR-based assays enable the full functional analysis of bispecific molecules
W. Meschendoerfer, C. Gassner, F. Lipsmeier, J.T. Regula, J. Moelleken
ABSTRACT
The increasing complexity of novel biotherapeutics such as bispecific antibodies or fusion proteins
raises new challenges for functional characterization. When compared to standard antibodies, two
individual interactions and the inter-dependency of binding events need to be considered for bispecific
antibodies. We have previously described an SPR-based assay setup, which enables us to assess the
binding activity of a bivalent-bispecific molecule to both targets simultaneously and − in addition to
one individual target − in a single setup. However, there might be some pitfalls when applying the
bridging assay, e.g. change of antigen activity upon immobilization. Therefore, we have developed an
alternative SPR-based assay principle, which allows the individual assessment of both targets in
solution. Comparison of data between the assays showed that simultaneous binding can be calculated
based on both individual readouts, and revealed a good correlation. Hence, both SPR-based assay
principles allow a ―full‖ functional analysis of a bispecific CrossMab in only one assay. The assay
principles can be qualified and enable an efficient drug development.
14
Angiotensin converting enzyme immobilized on magnetic beads as a tool for ligand fishing
Fernando G. de Almeida, Kenia L. Vanzolini, Quezia B. Cass
ABSTRACT
Angiotensin converting enzyme (ACE) presents an important role in blood pressure regulation, since
that converts angiotensin I to the vasoconstrictor angiotensin II. Some commercially available ACE
inhibitors are captopril, lisinopril and enalapril; due to their side effects, naturally occurring inhibitors
have been prospected. In order to endorse this research field we have developed a new tool for ACE
ligand screening. To this end, ACE was extracted from bovine lung, purified and chemically
immobilized in modified ferrite magnetic beads (ACE-MBs). The ACE-MBs have shown a Michaelian
kinetic behavior towards hippuryl-histidyl-leucine. Moreover, as proof of concept, the ACE-MBs were
inhibited by lisinopril with a half maximal inhibitory concentration (IC50) of 10 nM. At the fishing
assay, ACE-MBs were able not only to fish out the reference inhibitor, but also one peptide from a
pool of tryptic digested BSA. In conclusion, ACE-MBs emerge as new straightforward tool for ACE
kinetics determination, inhibition and binder screening.
MALDI-MS analysis and theoretical evaluation of olanzapine as a UV laser desorption
ionization (LDI) matrix
Syed Ghulam Musharraf, Mariam Ameer, Arslan Ali
ABSTRACT
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) being soft ionization
technique, has become a method of choice for high-throughput analysis of proteins and peptides. In
this study, we have explored the potential of atypical anti-psychotic drug olanzapine (OLZ) as a matrix
for MALDI-MS analysis of peptides aided with the theoretical studies. Seven small peptides were
employed as target analytes to check performance of olanzapine and compared with conventional
MALDI matrix α-cyano-4-hydroxycinnamic acid (HCCA). All peptides were successfully detected
when olanzapine was used as a matrix. Moreover, peptides angiotensin Ι and angiotensin ΙΙ were
detected with better S/N ratio and resolution with this method as compared to their analysis by HCCA.
Computational studies were performed to determine the thermochemical properties of olanzapine in
order to further evaluate its similarity to MALDI matrices which were found in good agreement with
the data of existing MALDI matrices.
15
A simple and rapid UHPLC–MS/MS method for the quantitation of the dual aurora kinase A/B
inhibitor SCH-1473759 in murine plasma
Marco A. Ferraz Nogueira Filho, Cody J. Peer, Jeffers Nguyen, Amy McCalla, William D. Figg
ABSTRACT
The Aurora kinase family facilitates cell division through various processes and is overexpressed in a
wide variety of human cancers, leading to aneuploidy. For that reason, these enzymes are currently
targets of a rising class of anticancer drugs, with some molecules already in therapeutic use. In this
study, a new UHPLC–MS/MS method was developed and validated to quantitate a new pan Aurora
kinase inhibitor still in preclinical development, SCH-1473759. This bioanalytical method employed a
liquid–liquid extraction from plasma using ethyl acetate before evaporation. Calibration range
encompassed 0.5–2500 ng/mL. The inter- and intra-day accuracy and precision were assessed over
five quality control levels; all within limits required by the FDA guidelines. Assay applicability was
demonstrated in a first-in-animals study with oral administration, where the maximum plasma
concentration (34 ng/mL) occurred at 1 h, the half-life (1 h) was consistent with a previous IV study,
and oral bioavailability was poor (F = 0.002).
Immobilized aptamer paper spray ionization source for ion mobility spectrometry
Tahereh Zargar, Taghi Khayamian, Mohammad T. Jafari
ABSTRACT
A selective thin-film microextraction based on aptamer immobilized on cellulose paper was used as a
paper spray ionization source for ion mobility spectrometry (PSI-IMS), for the first time. In this
method, the paper is not only used as an ionization source but also it is utilized for the selective
extraction of analyte, based on immobilized aptamer. This combination integrates both sample
preparation and analyte ionization in a Whatman paper. To that end, an appropriate sample
introduction system with a novel design was constructed for the paper spray ionization source. Using
this system, a continuous solvent flow works as an elution and spray solvent simultaneously. In this
method, analyte is adsorbed on a triangular paper with immobilized aptamer and then it is desorbed
and ionized by elution solvent and applied high voltage on paper, respectively. The effects of different
experimental parameters such as applied voltage, angle of paper tip, distance between paper tip and
counter electrode, elution solvent type, and solvent flow rate were optimized. The proposed method
was exhaustively validated in terms of sensitivity and reproducibility by analyzing the standard
solutions of codeine and acetamiprid. The analytical results obtained are promising enough to ensure
the use of immobilized aptamer paper-spray as both the extraction and ionization techniques in IMS
for direct analysis of biomedicine.
16
Urine metabolomics of high-fat diet induced obesity using UHPLC-Q-TOF-MS
Lihui Men, Zifeng Pi, Yuan Zhou, Mengying Wei, Zhongying Liu
ABSTRACT
Obesity has become a global epidemic and public health challenge which associates with serious
health issues including diabetes, cardiovascular disease, stroke, arthritis, and some types of cancer. To
better understand obesity and obesity-related dysfunction, a high-fat diet (HFD) induced obese model
was developed on Sprague-Dawley rats. Metabolomics based on ultra high-performance liquid
chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was
untilized to identify and analyze obesity related metabolites in rat urine samples. Multivariate analyses
were applied to differentiate metabolite patterns between HFD group and normal group. The study
successfully identified 20 altered urine metabolites that correlated with obesity. These metabolites are
mainly involved in tryptophan metabolism, phenylalanine and tyrosine metabolism, gut microbiota
metabolism and insulin resistance related metabolism. They could serve as potential biomarkers to
diagnose the development of obesity.
Volume 133, January 2017
Psidium guajava L. leaves as source of proanthocyanidins: Optimization of the extraction
method by RSM and study of the degree of polymerization by NP-HPLC-FLD-ESI-MS
Elixabet Díaz-de-Cerio, Federica Pasini, Vito Verardo, Alberto Fernández-Gutiérrez, Maria Fiorenza
Caboni
ABSTRACT
Due to the importance of the proanthocyanidins (PAs) bioactivity and its relationship with the PAs
degree of polymerization (DP), an experimental design was carried out to establish the best extraction
conditions in order to evaluate the proanthocyanidins content and their degree of polymerization in
Psidium guajava leaves at different oxidation state. Optimal conditions achieved by response surface
methodology were 50% acetone/water (v/v), 48 °C, 30 min, and 0% acetic acid (v/v). The highest DP
has been found in the low oxidized state (DP 13 plus the polymers). Medium and high oxidized state
leaves reported a DP 11 plus the polymers. The total amounts of proanthocyanidins (sum of PAs by
HPLC-FLD-ESI-MS) decreased when oxidation state of leaves increased (15.8 ± 0.4, 12.6 ± 0.4, and
10.5 ± 0.3 mg/g leaf dry weight (d.w.) in low, medium and high oxidized state leaves, respectively).
Guava leaves present an interesting source of low DP-PAs.
17
Identification, synthesis and structural characterization of process related and degradation
impurities of acrivastine and validation of HPLC method
Ajay Kumar, Subba Rao Devineni, Shailender Kumar Dubey, Pradeep Kumar, Pramod Kumar
ABSTRACT
Four impurities (Imp-I–IV) were detected using gradient HPLC method in few laboratory batches of
acrivastine in the level of 0.03–0.12% and three impurities (Imp-I–III) were found to be known and
one (Imp-IV) was unknown. In forced degradation study, the drug is degraded into four degradation
products under oxidation and photolytic conditions. Two impurities (Imp-III and -IV) were concurred
with process related impurities whereas Imp-V and -VI were identified as new degradation impurities.
Based on LC-ESI/MSn study, the chemical structures of new impurities were presumed as 1-[(2E)-3-
(4-methylphenyl)-3-{6-[(1E)-3-oxobut-1-en-1-yl]pyridin-2-yl}prop-2-en-1-yl]pyrrolidin-1-ium-1-olate
(Imp-IV),1-{[3-(4-methylphenyl)-3-{6-[(1E)-3-oxobut-1-en-1-yl]pyridin-2-yl}oxiran-2-yl]methyl} py
rrolidin-1-ium-1-olate (Imp-V) and 2-[2-(4-methylphenyl)-3-[(1-oxidopyrrolidin-1-ium-1-yl)methyl]
oxiran-2-yl]-6-[(1E)-3-oxobut-1-en-1-yl]pyridin-1-ium-1-olate (Imp-VI), and confirmed by their
synthesis followed by spectroscopic analysis, IR, NMR (1H, 13C) and mass. An efficient and selective
high-performance liquid chromatography method has been developed and resolved well the drug
related substances on a Phenomenex Gemini C-18 (250 × 4.6 mm, particle size 5 μm) column. The
mobile phase was composed of sodium dihydrogen phosphate (10 mM) and methanol, temperature at
25 °C, and a PDA detector set at 254 nm used for detection. The method was validated with respect to
specificity, linearity, precision, accuracy, and sensitivity and satisfactory results were achieved.
Identification, synthesis, characterization of impurities and method validation were first reported in
this paper.
Preparation of a monoclonal antibody against amantadine and rimantadine and development of
an indirect competitive enzyme-linked immunosorbent assay for detecting the same in chicken
muscle and liver
Dapeng Peng, Wei Wei, Yuanhu Pan, Yulian Wang, Zonghui Yuan
ABSTRACT
A monoclonal antibody (mAb) was produced in order to monitor the illegal use of amantadine and
rimantadine in animals. The produced mAb 2G3 exhibited an IC50 value of 15.8 μg L−1 for
amantadine and exhibited cross-reactivity to both amantadine (100%) and rimantadine (70.6%).
Standard curves ranged from 5 to 80 μg L−1 for 2G3. The limits of detection of the developed indirect
competitive enzyme-linked immunosorbent assay (ic-ELISA) ranged from 5.0 μg kg−1 to 5.4 μg kg−1
in chicken muscle and liver. The recoveries were 81.3% to 98.1% with a coefficient of variation less
than 15.7%. Good correlations were observed between the results of the ic-ELISA and liquid
chromatography-mass spectrometry in the incurred tissues. These results suggest that ic-ELISA is a
sensitive, accurate, and low-cost method that could be a useful tool for screening the residues of
amantadine and rimantadine in chicken muscle and liver.
18
Determination of pharmaceutical residues in wastewater using high performance liquid
chromatography coupled to quadrupole-Orbitrap mass spectrometry
Iveta Pugajeva, Janis Rusko, Ingus Perkons, Elsa Lundanes, Vadims Bartkevics
ABSTRACT
A multi-class method for the determination of 24 emerging pharmaceutical residues has been
developed and validated. The method is based on solid-phase extraction of wastewater samples using
Strata-X cartridges followed by high performance liquid chromatography coupled to hybrid
quadrupole − Orbitrap high resolution mass spectrometry (HPLC-Q-Orbitrap-HRMS). A single-
laboratory validation procedure showed satisfactory analytical performance. The analysis of 21
samples collected at the wastewater treatment plant in Riga revealed the occurrence of 20 compounds
of different therapeutic classes. The highest concentration was found for the central nervous system
stimulator caffeine − up to 12 μg L−1, the analgesic acetaminophen up to 4.2 μg L−1, the antibiotic
ciprofloxacin in the concentration range of 250–400 ng L−1, and the non-steroidal anti-inflammatory
drug (NSAID) ibuprofen at 100–325 ng L−1.
High-capacity hollow porous dummy molecular imprinted polymers using ionic liquid as
functional monomer for selective recognition of salicylic acid
Haiyan Xiang, Mijun Peng, Hui Li, Sheng Peng, Shuyun Shi
ABSTRACT
The existence of strong intramolecular hydrogen bond in salicylic acid (SA) weakens its
intermolecular hydrogen bonding with functional monomer, then it is a challenge work to fabricate
molecularly imprinted polymers (MIPs) for SA recognition with high capacity and good selectivity.
Here, hollow porous dummy MIPs (HPDMIPs) were prepared using benzoic acid (BA) as dummy
template, ionic liquid (i.e. 1-vinyl-3-methylimidazolium chloride) as functional monomer, and MCM-
48 as sacrificial support. Factors that affected adsorption, such as type of template and porogen, mole
ratio of template–functional monomer–cross-linker and type of binding solvent, were optimized in
detail. Multiple strong interactions between SA and ionic liquid in HPDMIPs deduced higher binding
capacity (29.75 mg/g), imprinting factor (5.61) and selectivity than any previously reported MIPs by
traditional or surface imprinting technology. The large surface area (543.9 m2/g) with hollow porous
structure resulted in faster kinetic binding (25 min). The equilibrium data fitted well to Freundlich
equation and the adsorption process could be described by pseudo-second order model. Finally,
HPDMIPs were successfully applied to selectively extract and enrich SA from Actinidia chinensis
with a relatively high recovery (84.6–94.5%).
19
Demonstration of the IgG antibody repertoire against the bacteria Escherichia coli in Chinese
intravenous immunoglobulins
Shengliang Ye, Min Lei, Peng Jiang, Fengjuan Liu, Changqing Li
ABSTRACT
Intravenous immunoglobulin (IVIg) is produced by pooling plasma from thousands of healthy blood
donors, and the diversity of the antibody is critical for the clinical efficacy of IVIg. This study
investigated the antibody diversity of Chinese IVIg. Firstly, 2-dimensional gel electrophoresis and
immunoblotting with protein extracts of Escherichia coli (E. coli) O157:H7 were used to study IgG
antibody repertoire of 8 IVIg preparations from different Chinese manufacturers. This was followed by
the identification of the antibody-reactive proteins of E. coli by mass spectrometry and the sequence
similarity of the proteins was aligned by bioinformatics analysis. The results showed that all IVIg
preparations expressed a large range of antibody reactivities against E. coli proteins. 94-238 antigens
were recognized by the 8 IVIg preparations. 33 interesting target antigens were selected and identified
as 29 different proteins, mainly including membrane proteins, molecular chaperones, metabolism
enzymes, and proteins involved in cell cycle processes. Additionally, these antigens were highly
conserved proteins which were found extensively in a variety of other pathogenic microorganisms. Our
study indicated that Chinese IVIg preparations recognized a large range of high conserved proteins
which play key roles in pathogenic microorganisms, and showed each IVIg had its own distinct
antibody repertoire.
Metabolites profiling reveals for antimicrobial compositional differences and action mechanism
in the toothbrushing stick ―miswak‖ Salvadora persica
Mohamed A. Farag, Sherifa Fahmy, Mouchira A. Choucry, Mariam O. Wahdan, Mahmoud Fahmi
Elsebai
ABSTRACT
Among many plant species suitable for preparing toothbrushing sticks, miswak (Salvadora persica,
family Salvadoraceae) is found the most effective tool for oral hygiene. S. persica possesses
antibacterial, antiviral and antifungal effects against oral microbes, mostly due to its benzyl
isothiocyanate content. To provide insight into S. persica chemical composition, volatile constituents
from roots and stems of S. persica grown in Egypt and Saudi Arabia were profiled using solid-phase
microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC–MS). A total of 21
volatiles were identified with sulfur compounds amounting for the major volatile class. Orthogonal
projection to latent structures-discriminant analysis (OPLS-DA) revealed for benzyl isothiocyanate
(BITC) enrichment in roots versus stems. Primary metabolites contributing to S. persica taste viz.
sugars and organic acids were profiled using GC–MS with silylation. Polyols (sugars) viz. arabitol,
meso-erythritol, and mannitol were found to predominate sugars composition in S. persica stems being
most enriched in meso-erythritol. The impact of saliva on S. persica aroma profile was further assessed
and revealing for no enhancement in BITC production with salivation, and further not being detected
in toothpaste preparation claimed to contain S. persica extract. This study provides the most complete
profile of volatiles, sugars, and organic acids in S. persica organs and more rationalizing its use as a
toothbrush.
20
The investigation of anti-inflammatory activity of Yi Guanjian decoction by serum
metabonomics approach
Sufang Shui, Xiaorong Cai, Rongqing Huang, Bingkun Xiao, Jianyun Yang
ABSTRACT
Yi Guanjian (YGJ), one of the Chinese herbal medicines most commonly used in western countries,
reported to possess significant anti-inflammatary effects that inhibit the process of inflammation.
However, the mechanisms underlying its anti-inflammation effects remain largely unresolved. This
study was aimed to investigate the anti-inflammatory activity of YGJ and to explore its potential anti-
inflammatory mechanisms by serum metabonomics approach. A xylene-induced mouse right-ear-
edema model was used as an inflammatory response in vivo model. Ear edema, prostaglandin E2
(PGE2) and Tumor-Necrosis-Factor-alpha (TNF-α) were detected. Then, serum metabolic profiling
was analyzed and pathway analysis performed on the biomarkers reversed after YGJ administration
and further integration of metabolic networks. The results showed that YGJ alleviated ear edema and
decreased serum PGE2 and TNF-α level. Fourteen biomarkers were screened, and the levels were all
reversed to different degrees after YGJ administration. These biomarkers were mainly related to
linoleic acid metabolism, taurine and hypotaurine metabolism, glyoxylate and dicarboxylate
metabolism, glycine, serine and threonine metabolism and citrate cycle (TCA cycle). In metabolic
networks, glycine and pyruvate were node molecules. This indicated that YGJ could significantly
inhibit inflammatory response triggered by acute local stimulation and exerted anti-inflammatory
activity mainly by regulating node molecules.
Quantitative profiling of 4'-geranyloxyferulic acid and its conjugate with l-nitroarginine methyl
ester in mononuclear cells by high-performance liquid chromatography with fluorescence
detection
Vito Alessandro Taddeo, Salvatore Genovese, Giuseppe Carlucci, Vincenzo Ferrone, Francesco
Epifano
ABSTRACT
Oxyprenylated natural products were shown to exert in vitro and in vivo remarkable anti-cancer and
anti-inflammatory effects. This paper describes a rapid, selective, and sensitive HPLC method with
fluorescence detection for determination of 4'-geranyloxyferulic acid (GOFA) and its conjugate with l-
nitroarginine methyl ester (GOFA-L-NAME) in mononuclear cells. Analytes were extracted from cells
using methanol and eluted on a GraceSmart RP18 analytical column (250 × 4.6 mm i.d., 5 μm particle
size) kept at 25 °C. A mixture of formic acid 1% in water (A) and methanol (B) were used as mobile
phase, at a flow-rate of 1.2 mL/min in gradient elution. A fluorescence detector (excitation/emission
wavelength of 319/398 nm for GOFA and GOFA-L-NAME), was used for the two analytes.
Calibration curves of GOFA and GOFA-L-NAME were linear over the concentration range of 1.0–50
μg/mL, with correlation coefficients (r2) ≥ 0.9995. Intra- and inter-assay precision do not exceed 6.8%.
The accuracy was from 94% to 105% for quality control samples (2.0, 25.0 and 40 μg/mL). The mean
(RSD%) extraction recoveries (n = 5) for GOFA and GOFA-L-NAME from spiked cells at 2.0, 25.0
and 40.0 μg/mL were 92.4 ± 1.5%, 94.7 ± 0.9% and 93.8 ± 1.1%, for GOFA and 95.3 ± 1.2%, 94.8 ±
1.0% and 93.9 ± 1.3%, for GOFA-L-NAME. The limits of detection and quantification were 0.3
μg/mL and 1.0 μg/mL for GOFA and GOFA-L-NAME. This method was successfully applied to
measure GOFA and GOFA-L-NAME concentrations in a mononuclear cell.
21
A serum nuclear magnetic resonance-based metabolomic signature of antiphospholipid
syndrome
Angelica Palisi, Manuela Grimaldi, Paola Sabatini, Paola Montoro, Anna Maria D‘Ursi
ABSTRACT
Antiphospholipid syndrome (APS) is a rheumatic inflammatory chronic autoimmune disease inducing
hypercoagulable state associated with vascular thrombosis and pregnancy loss in women. Cardiac,
cerebral and vascular strokes in these patients are responsible for reduction in life expectancy. Timely
diagnosis and accurate monitoring of disease are decisive to improve the accuracy of therapy. In the
present work, we present a NMR-based metabolomic study of blood sera of APS patients. Our data
show that individuals suffering APS have a characteristic metabolomic profile with abnormalities
associated to the metabolism of methyl group donors, ketone bodies and amino acids. We have
identified for the first time the metabolomic fingerprint characterizing APS disease having potential
application to improve APS timely diagnosis and appropriate therapeutic approaches.
Development of a multi-matrix LC–MS/MS method for urea quantitation and its application in
human respiratory disease studies
Jianshuang Wang, Yang Gao, Drew W. Dorshorst, Fang Cai, Xiao Ding
ABSTRACT
In human respiratory disease studies, liquid samples such as nasal secretion (NS), lung epithelial lining
fluid (ELF), or upper airway mucosal lining fluid (MLF) are frequently collected, but their volumes
often remain unknown. The lack of volume information makes it hard to estimate the actual
concentration of recovered active pharmaceutical ingredient or biomarkers. Urea has been proposed to
serve as a sample volume marker because it can freely diffuse through most body compartments and is
less affected by disease states. Here, we report an easy and reliable LC–MS/MS method for cross-
matrix measurement of urea in serum, plasma, universal transfer medium (UTM), synthetic absorptive
matrix elution buffer 1 (SAMe1) and synthetic absorptive matrix elution buffer 2 (SAMe2) which are
commonly sampled in human respiratory disease studies. The method uses two stable-isotope-labeled
urea isotopologues, [15N2]-urea and [13C,15N2]-urea, as the surrogate analyte and the internal
standard, respectively. This approach provides the best measurement consistency across different
matrices. The analyte extraction was individually optimized in each matrix. Specifically in UTM,
SAMe1 and SAMe2, the unique salting-out assisted liquid-liquid extraction (SALLE) not only
dramatically reduces the matrix interferences but also improves the assay recovery. The use of an
HILIC column largely increases the analyte retention. The typical run time is 3.6 min which allows for
high throughput analysis.
22
Determination of five potential genotoxic impurities in dalfampridine using liquid
chromatography
Mohit Jain, Vishal Srivastava, Rajesh Kumar, Vishal Dangi, Pramod Kumar
ABSTRACT
A sensitive and selective HPLC method was developed for identification and quantification of five
Potential genotoxic impurities (PGIs) viz. Impurity-I, Impurity-II, Impurity-III, Impurity-IV and
Impurity-V in Dalfampridine (Drug substance). The method utilizes Zorbax silica column (250 mm ×
4.6 mm, 5.0 μm) with UV detector in HILIC (Hydrophilic Interaction Liquid Chromatography) mode
for quantitation of five PGIs. It has been validated as per International Council for Harmonisation
(ICH) guidelines and is able to quantitate all PGIs at 75 ppm with respect to 20 mg/mL of sample
concentration. It is linear in the range of 22.5–112.5 ppm for all PGIs, which matches the range of
LOQ-150% of estimated permitted level (75 ppm). Its accuracy was established in the range from
88.14 to 107.65% for these PGIs. The correlation coefficient of each impurity was >0.999. It is a good
quality control tool for quantitation of PGIs in Dalfampridine at low level.
Isolation and structural characterization of novel photolytic degradation impurities of
Deflazacort using Q-TOF, 2D-NMR and FTIR
Ch Krishnam Raju, Avadhesh Kumar Pandey, Kaushik Ghosh, Arunima Pola, Koduru V.
Surendranath
ABSTRACT
Forced Degradation of Deflazacort drug substance in ultraviolet light condition resulted into a number
of significant degradation products. Two of these degradation products were found to be unknown
during the study and marked as DD-I and DD-II. Thus, the objective of this work is to investigate and
identify these two novel degradation products of DFZ. The isolation method for these new degradation
products were developed using a new reverse-phase high performance liquid chromatography (HPLC).
DD-I and DD-II, eluting at 0.53 and 1.57 relative retention times with respect to Deflazacort (DFZ)
peak respectively, were isolated from reaction mass using preparative HPLC and their structures were
elucidated using high resolution MS, multidimensional NMR and FTIR spectroscopic techniques. To
best of our knowledge, these two degradation products are novel impurities which are not discussed in
any form of publication yet.
Volume 96, Number 1, January 2017
23
Volume 134, February 2017
VLC–ESI–MS/MS evaluation of forced degradation behaviour of silodosin: In vitro anti cancer
activity evaluation of silodosin and major degradation products
Chiguru Vishnuvardhan, Baikadi Saibaba, Lingesh Allakonda, Debasish Swain, N. Satheeshkumar
ABSTRACT
Silodosin (SLD) a novel α1-adrenoceptor antagonist was subjected to forced degradation involving
hydrolysis (acidic, alkaline and neutral), oxidative, photolysis and thermal stress, as per ICH specified
conditions. The drug underwent significant degradation under hydrolytic (acidic, alkaline and neutral)
and oxidative stress conditions whereas, it was found to be stable under other stress conditions. A
rapid, precise, accurate and robust chromatographic method for the separation of the drug and its
degradation products (DPs) was developed on a Fortis C18 analytical column (150 × 4.6 mm, 5 μm)
using 0.1% formic acid and acetonitrile as a mobile phase in gradient elution mode at a flow rate of 1.0
mL/ min. A total of 5 (DP 1 to DP 5) hitherto unknown DPs were identified by LC-ESI-TOF-MS/MS
experiments and accurate mass measurements. The most probable mechanisms for the formation of
DPs have been proposed based on a comparison of the fragmentation of the [M+H]+ ions of silodosin
and its DPs. The major DPs (DP 1 and DP 2) were isolated and evaluated for anticancer activity using
PC3 (human prostate cancer) cell lines by MTT assay. The results revealed that silodosin, DP 1 and
DP 2 have potential anticancer activity with IC50 values (μM) 72.74 (±4.51), 25.21 (±2.36), and,
114.07 (±11.90) respectively.
Quality by Design in the development of hydrophilic interaction liquid chromatography method
with gradient elution for the analysis of olanzapine
Anja Tumpa, Ana Stajić, Biljana Jančić-Stojanović, Mirjana Medenica
ABSTRACT
This paper deals with the development of hydrophilic interaction liquid chromatography (HILIC)
method with gradient elution, in accordance with Analytical Quality by Design (AQbD) methodology,
for the first time. The method is developed for olanzapine and its seven related substances. Following
step by step AQbD methodology, firstly as critical process parameters (CPPs) temperature, starting
content of aqueous phase and duration of linear gradient are recognized, and as critical quality
attributes (CQAs) separation criterion S of critical pairs of substances are investigated. Rechtschaffen
design is used for the creation of models that describe the dependence between CPPs and CQAs. The
design space that is obtained at the end is used for choosing the optimal conditions (set point). The
method is fully validated at the end to verify the adequacy of the chosen optimal conditions and
applied to real samples.
24
Lipophilicity estimation and characterization of selected steroid derivatives of biomedical
importance applying RP HPLC
Lidija R. Jevrić, Milica Ņ. Karadņić, Anamarija I. Mandić, Sanja O. Podunavac Kuzmanović, SrĎan Z.
Stojanović
ABSTRACT
The present paper deals with chromatographic lipophilicity determination of twenty-nine selected
steroid derivatives using reversed-phase high-performance liquid chromatography (RP HPLC)
combined with two mobile phase, acetonitrile-water and methanol-water. Chromatographic behavior
of four groups (triazole and tetrazole, toluenesulfonylhydrazide, nitrile and dinitrile and dione) of
selected steroid derivatives was studied. Investigated compounds were grouped using principal
component analysis (PCA) according to their logk values for both mobile phases. Grouping was in the
very good accordance with the polarity and lipophilicity of the investigated compounds. QSRR
(quantitative structure-retention relationship) approach was used to model chromatographic
lipophilicity behavior using molecular descriptors. Modeling was performed using linear regression
(LR) and multiple linear regression (MLR) methods. The most influential molecular descriptors were
lipophilicity descriptors that are important for molecules ability to pass through biological membranes
and geometrical descriptors. All established LR-QSRR and MLR-QSRR models were statistically
validated by standards, cross- and external validation parameters as well as with two graphical
methods. According to all these assessments, MLR models were better for chromatographic
lipophilicity prediction. It was shown that chromatographic systems with methanol-water were better
for modeling of logk than systems with acetonitrile-water, as well as the systems that contained lower
volume fractions of organic component in mobile phase. Modeling was performed in order to obtain
lipophilicity profiles of investigated compounds as future drug candidates of biomedical importance.
Comparison of the chemical consituents and immunomodulatory activity of ophiopogonis radix
from two different producing areas
Xiaoyan Lu, Wei Tong, Shufang Wang, Jinghui Li, Li Liu
ABSTRACT
Zhemaidong (ZMD) and Chuanmaidong (CMD) are the two genuine cultivation areas of
Ophiopogonis Radix which has been widely used as a traditional Chinese medicine in China for
treating cardiovascular and pulmonary diseases. Differences between ZMD and CMD in chemical
constituents and pharmacological effects have been reported, however, the details remain largely
unknown. The aim of this study was to comprehensively characterize the chemical composition of
Ophiopogonis Radix from the two producing areas and compare their immunomodulatory activities.
An approach of HPLC–MS coupled with multivariate statistical analysis was established to reveal the
characteristic constituents of ZMD and CMD. Furthermore, the effects of ZMD and CMD on the
macrophage phagocytosis and gastrointestinal peristalsis were also determined in zebrafish models for
assessing the immunomodulatory activities of these two strains. The results revealed that the chemical
constituents of ZMD and CMD were much different from each other, and 19 constituents could be
served as chemical markers to distinguish these two strains. Moreover, ZMD showed higher promoting
rates in macrophage phagocytosis and gastrointestinal motility than those of CMD, suggesting ZMD
might possess better immunomodulatory activities. Taken together, the results generated from this
study indicated that the herbs from different producing areas should be evaluated and considered in
preparing TCM prescriptions.
25
Physicochemical analysis in the evaluation of reconstituted dry emulsion tablets
Noémi Anna Niczinger, Barnabás Kállai-Szabó, Miléna Lengyel, Péter Gordon, István Antal
ABSTRACT
The aim of this study was to characterize the formation of emulsions by droplet size analysis and
turbidimetry during reconstitution from a solid dosage form, namely from dry emulsion systems,
which carry an oil phase for poorly soluble active ingredients. For the dry emulsion systems tablets
were prepared either from oil-in-water systems using a freeze-drying process or through direct
compression containing the same oil and excipients. The ratios of oil to emulgents and oil to xanthan
gum were equal in both methods. In the preparation methods applied, mannitol, erythritol and lactose
were used as excipients and mannitol was found to be the most effective excipient based on droplet
size reconstitution, turbidimetry and physical properties. Quality control involved testing the physical
properties of tablets and characterizing the reconstituted emulsions.
Cleaning verification: Exploring the effect of the cleanliness of stainless steel surface on sample
recovery
Imad A. Haidar Ahmad, James Tam, Xue Li, William Duffield, Andrei Blasko
ABSTRACT
The parameters affecting the recovery of pharmaceutical residues from the surface of stainless steel
coupons for quantitative cleaning verification method development have been studied, including active
pharmaceutical ingredient (API) level, spiking procedure, API/excipient ratio, analyst-to-analyst
variability, inter-day variability, and cleaning procedure of the coupons. The lack of a well-defined
procedure that consistently cleaned coupon surface was identified as the major contributor to low and
variable recoveries. Assessment of acid, base, and oxidant washes, as well as the order of treatment,
showed that a base-water-acid-water-oxidizer-water wash procedure resulted in consistent, accurate
spiked recovery (>90%) and reproducible results (Srel ≤ 4%). By applying this cleaning procedure to
the previously used coupons that failed the cleaning acceptance criteria, multiple analysts were able to
obtain consistent recoveries from day-to-day for different APIs, and API/excipient ratios at various
spike levels. We successfully applied our approach for cleaning verification of small molecules (MW
< 1000 Da) as well as large biomolecules (MW up to 50,000 Da). Method robustness was greatly
influenced by the sample preparation procedure, especially for analyses using total organic carbon
(TOC) determination.
26
Raman spectroscopy and capillary zone electrophoresis for the analysis of degradation processes
in commercial effervescent tablets containing acetylsalicylic acid and ascorbic acid
Sabine Neuberger, Kevin Jooß, Dirk Flottmann, Gerhard Scriba, Christian Neusüß
ABSTRACT
In order to ensure the stability of pharmaceutical products appropriate manufacturing and storage
conditions are required. In general, the degradation of active pharmaceutical ingredients (APIs) and
subsequent formation of degradation products affect the pharmaceutical quality. Thus, a fast and
effective detection and characterization of these substances is mandatory. Here, the applicability of
Raman spectroscopy and CZE for the characterization of the degradation of effervescent tablets
containing acetylsalicylic acid (ASA) and ascorbic acid (AA) was evaluated. Therefore, a degradation
study was performed analyzing tablets from two different manufacturers at varying conditions (relative
humidity (RH) 33%, 52% and 75% at 30 °C). Raman spectroscopy combined with principal
component analysis could be successfully applied for the fast and easy discrimination of non-degraded
and degraded effervescent tablets after a storage period of approximately 24 h (RH 52%).
Nevertheless, a clear identification or quantification of APIs and degradation products within the
analyzed tablets was not possible, i.a. due to missing reference materials. CZE-UV enabled the
quantification of the APIs (ASA, AA) and related degradation products (salicylic acid (SA); semi-
quantitative also mono- and diacetylated AA) within the complex tablet mixtures. The higher the RH,
the faster the degradation of ASA and AA as well as the formation of the degradation products. Mono-
and diacetylated AA are major primary degradation products of AA for the applied effervescent
tablets. A significant degradation of the APIs was detected earlier by CZE (6–12 h, RH 52%) than by
Raman spectroscopy. Summarized, Raman spectroscopy is well-suited as quick test to detect
degradation of these tablets and CZE can be utilized for further detailed characterization and
quantification of specific APIs and related degradation products.
RP-HPLC determination of dissociation constant using solely aqueous mobile phase
Tereza Volná, Kamil Motyka, Jan Hlaváč
ABSTRACT
The proposed HPLC method using solely or nearly 100% aqueous mobile buffer as mobile phase
offers fast determination of dissociation constant for compounds in relatively wide range of
lipophilicity (log P from −2.26 to 2.26). The dissociation constant value for simpler chemical
compounds can be determined via only 8 chromatographic runs. The number of needed
chromatographic separations depends on the structural complexity of the tested compound. Moreover,
the proposed method does not require a measurement of Yasuda-Shedlovsky extrapolation that
includes several pKa determinations in solutions with different methanol content which speeds up
considerably the procedure. The methodology is suitable for evaluation of large series of drug
candidates, which can be present as complex mixtures and in small amounts.
27
Quantitative determination of salbutamol sulfate impurities using achiral supercritical fluid
chromatography
Amandine Dispas, Vincent Desfontaine, Bertyl Andri, Pierre Lebrun, Philippe Hubert
ABSTRACT
In the last years, supercritical fluid chromatography has largely been acknowledged as a singular and
performing technique in the field of separation sciences. Recent studies highlighted the interest of SFC
for the quality control of pharmaceuticals, especially in the case of the determination of the active
pharmaceutical ingredient (API). Nevertheless, quality control requires also the determination of
impurities. The objectives of the present work were to (i) demonstrate the interest of SFC as a
reference technique for the determination of impurities in salbutamol sulfate API and (ii) to propose an
alternative to a reference HPLC method from the European Pharmacopeia (EP) involving ion-pairing
reagent. Firstly, a screening was carried out to select the most adequate and selective stationary phase.
Secondly, in the context of robust optimization strategy, the method was developed using design space
methodology. The separation of salbutamol sulfate and related impurities was achieved in 7 min,
which is seven times faster than the LC-UV method proposed by European Pharmacopeia (total run
time of 50 min). Finally, full validation using accuracy profile approach was successfully achieved for
the determination of impurities B, D, F and G in salbutamol sulfate raw material. The validated dosing
range covered 50 to 150% of the targeted concentration (corresponding to 0.3% concentration level),
LODs close to 0.5 μg/mL were estimated. The SFC method proposed in this study could be presented
as a suitable fast alternative to EP LC method for the quantitative determination of salbutamol
impurities.
Hydrogen/deuterium exchange, a unique and effective method for MS fragmentation behavior
elucidation of ginkgolides and its application to systematic research in Ginkgo biloba
Xingliang Niu, Jun Luo, Deran Xu, Hongyan Zou, Lingyi Kong
ABSTRACT
Ginkgolides, the main active constituents of Ginkgo biloba, possess significant selectively inhibition
on platelet-activating factor and pancreatic lipase and attract wide attention in pharmacological
research area. In our study, an effective hydrogen/deuterium (H/D) exchange method was developed
by exchanging the α-Hs of lactone groups in ginkgolides with Ds, which was very useful for the
elucidation of the fragmentation patterns of ginkgolides in Quadrupole Time-of-flight Mass
Spectrometry (Q-TOF-MS), especially in accurately distinguishing the type and position of substituent
in framework of ginkgolides. Then, a systematic research strategy for qualitative and quantitative
analysis of ginkgolides, based on H/D exchange, tandem solid-phase extraction and LC-Q-TOF-MS,
was developed, which was successfully applied in each medicinal part of G. biloba, which indicated
that ginkgolide B was the most abundant ginkgolide in the seeds of G. biloba (60.6 μg/g). This
research was the successful application of H/D exchange in natural products, and proved that H/D
exchange is a potential method for analysis research of complex TCMs active constituents.
28
Identification and characterization of a new dapoxetine impurity by NMR: Transformation of
N-oxide by Cope elimination
András Darcsi, Ákos Rácz, Szabolcs Béni
ABSTRACT
Unknown impurity associated with the degradation process of dapoxetine base was isolated. The
structure elucidation of this new compound using accurate mass data, IR and NMR spectroscopy is
presented herein. The unambiguous resonance assignment concluded to the formation of geometrical
isomers of cinnamyloxynaphtalenes via Cope elimination of dapoxetin-N-oxide, the major oxidative
and metabolic degradation product of dapoxetine. An efficient and simple synthetic approach has also
been developed for the synthesis of dapoxetine-N-oxide for the first time and cinnamyloxynaphtalene
in order to confirm the proposed degradation pathway and structures of the degradation products. It
was observed that the main degradation product of dapoxetine base when exposed to air is 1-(2E)-
cinnamyloxynaphthalene, while its Z isomer was also confirmed as a minor impurity.
A new approach to the rapid separation of isomeric compounds in a Silybum marianum extract
using UHPLC core-shell column with F5 stationary phase
Jakub Fibigr, Dalibor Ńatínský, Petr Solich
ABSTRACT
In this paper, a new ultra-high performance liquid chromatography (UHPLC) method using a core–
shell column with a pentafluorophenyl stationary phase for separation of seven active compounds of a
Silybum marianum extract was developed and validated. Silymarin, an extract of Silybum marianum,
is known for its abilities to protect the liver from toxic substances, hepatitis therapy, and anti-tumour
activity. Silymarin is currently being widely used in commercial preparations and herbal teas.
Separation of seven compounds contained in the Silybum marianum extract (taxifolin, silychristin,
silydianin, silybin A, silybin B, isosilybin A, isosilybin B) and other substances occurring in real
samples was performed on the Kinetex 1.7 μ F5 100A (150 × 2.1 mm), 1.7 μm particle size core–shell
column, with a mobile phase methanol/100 mM phosphate buffer pH 2.0 according to the gradient
program. A mobile phase 0.35 mL min−1 flow rate and 50 °C temperature was used for the separation.
The detection wavelength was set at 288 nm. Under optimal chromatographic conditions, good
linearity with a correlation coefficient of R2 >0.999 for all compounds was achieved. The available
commercial samples of herbal teas and food supplements were extracted with methanol using an
ultrasonic bath. After dilution with water and centrifugation, a 2 μL sample of the filtered supernatant
was directly injected into the UHPLC system. The use of a pentafluorophenyl stationary phase with
methanol as the organic component of the mobile phase showed new ways to effectively separate
isomeric compounds in herbal extracts, which could not be done with the conventional C18 stationary
phase.
29
A comparison report of three advanced methods for drug-cyclodextrin interaction
measurements
Vikramjeet Singh, Yaping He, Caifen Wang, Jianghui Xu, Jiwen Zhang
ABSTRACT
Three advanced methods, high performance affinity chromatography (HPAC), surface plasmon
resonance (SPR) and surface plasmon resonance imaging (SPRi) were compared and evaluated for
determining the drug-cyclodextrin (CD) interactions herein. In total, 18 sparingly soluble drugs were
selected for this comparative study. The three methods share a unique connection in the working
principles and strategies. The same strategies of CD fixation onto solid phase were used in HPAC and
SPR for the measurements, whereas, the SPR and SPRi share identical working principles. However,
whilst these relationships are evident, no strong correlation was found between kinetic constants
obtained from the three methods: Four drugs, namely, prednisolone, pseudolaric acid B, diazepam and
gramisetron failed to show any response on SPR, whereas, the kinetics parameters from SPRi and
HPAC were successfully measured. From a comparative review of all the kinetic data, random results
without any trends were observed (ka, kd and KA) regardless of the relationships between the three
methods: It is apparent that the measurement conditions (volume, flow rate, buffers), non-specific
adsorption and experimental procedures had a strong impact on the generated data. The relative
advantages and limitations of each method are critically presented on the basis of generated data. This
comparative study provides a basis to further upgrade these techniques for confident measurement of
drug-CDs interactions.
Portable near-infrared instruments: Application for quality control of polymorphs in
pharmaceutical raw materials and calibration transfer
Vitor Hugo da Silva, Jailson José da Silva, Claudete Fernandes Pereira
ABSTRACT
This work presents an evaluation of the analytical performance of three different portable near-infrared
(NIR) instruments (denominated Port.1, Port.2 and Port.3) for quantifying mebendazole polymorphs
(A, B and C) in pharmaceutical raw materials using multivariate calibration models. The performance
of the portable instruments was compared with a benchtop one (FT-NIR Frontier spectrometer). In
addition, calibration transfer between the benchtop and one of the portable instruments was also
performed. For polymorph A, the Port.1 presented the lowest RMSEP value (1.01% w/w) even when
compared to the FT-NIR instrument. For polymorphs B and C, the same Port.1 instrument presented
RMSEP values of 2.09% w/w and 2.41% w/w, respectively, which were statistically similar to those
obtained with the benchtop instrument. The LOD ranges (3.9–5.5 for polymorph A, 3.6–5.1 for
polymorph B and 5.7–7.7 for polymorph C) obtained with the Port.1 was higher than those achieved
with the benchtop NIR instrument, with high spectral resolution, signal-to-noise ratio and better
wavelength reproducibility. Calibration transfer was performed between the benchtop NIR and Port.1
instruments. According to the results, the transferability of models is possible. The results obtained for
complete recalibration of the portable instrument and those for the benchtop are comparable. The
methods developed demonstrated a flexible, easy, cheap and fast way for quality control of MBZ
polymorphs in incoming material, mainly in pharmaceutical laboratory chains.
30
Characterization and quantitation of the polyphenolic compounds detected in methanol extracts
of Pistacia atlantica Desf fruits from the Guelmim region of Morocco
Farid Khallouki, Andrea Breuer, Elzemzoumi Merieme, Cornelia M. Ulrich, Robert W. Owen
ABSTRACT
High performance liquid chromatography coupled with electrospray ionization mass spectrometry
(HPLC-ESI–MS) was used for the identification of the major phenolic compounds in mature P.
atlantica fruits from the Guelmim region (southeast of Morocco). In this study twenty seven
polyphenolic compounds are identified and quantitated. To date, this is the most comprehensive report
on the polyphenolic content of Pistacia fruits. The profiles comprise, three major polyphenolic classes,
namely gallates (18.76 g/kg; 63.92%), flavonoids (10.12 g/kg; 34.48%) and ellagic acid derivatives
(0.47 g/kg; 1.60%) with a total of 29.35 g/kg detected. The major gallate was pentagalloyl glucoside
(5.0 g/kg; 17.04% of total polyphenolics), the major flavonoid luteolin (3.18 g/kg; 10.83% of total
polyphenolics) and the major ellagic acid derivative ellagic acid (0.25 g/kg; 0.85% of total
polyphenolics). Identification of galloyl quinate, digalloyl quinates (x 2), galloyl glucoside, digalloyl
glucosides (x 2), trigalloyl glucoside, tetragalloyl glucosides (x 2), pentagalloyl glucoside, 2″-O-
galloyl-quercetin-3-O-galactoside, quercetin-3-O-rhamnogalactoside, quercetin-3-O-galactoside,
ellagic acid diglucoside, luteolin-4′-O-glucoside, 2″-O-galloyl-luteolin-4′-O-glucoside, quercetin-3-O-
glucuronide, kaempferol-3-O-glucoside, eriodictyol, apigenin, ellagic acid diglucoside, ellagic acid
glucoside, methyl ellagic acid glucoside, and ellagic acid are described as phytochemical components
of Pistacia fruits for the first time.
Quantification of biologically active O-prenylated and unprenylated phenylpropanoids in dill
(Anethum graveolens), anise (Pimpinella anisum), and wild celery (Angelica archangelica)
Vito Alessandro Taddeo, Salvatore Genovese, Philippe de Medina, Roberta Palmisano, Serena Fiorito
ABSTRACT
An analytical strategy based on different extraction methodologies and HPLC with spectrophotometric
(UV–vis) detection has been developed to investigate the presence of and to quantitate biologically
active selected unprenylated and O-prenylated phenylpropanoids, namely umbelliferone, 4′-
geranyloxyferulic acid, 7-isopentenyloxycoumarin, auraptene, and umbelliprenin in dill (Anethum
graveolens L.), anise (Pimpinella anisum L.), and wild celery (Angelica archangelica L.). Absolute
ethanol or 7:3 water/ethanol mixtures were seen to be the most powerful extraction solvents to perform
―classic―maceration or ultrasound-assisted one in terms of yields in secondary metabolites. For
anethum and anise, umbelliprenine was found to be the most abundant prenyloxy secondary
metabolite, while in wild celery 4′-geranyloxyferulic acid recorded the highest concentration. Our
experimental approach demonstrated to be efficient for the simultaneous identification and quantitation
of the above mentioned prenyloxyphenylpropanoids in the title plant species that is reported herein for
the first time in the literature.
31
Microfluidic-based G-quadruplex ligand displacement assay for alkaloid anticancer drug
screening
Haihui Shen, Bo Zhang, Huiyan Xu, Yue Sun, Yingchun Liu
ABSTRACT
Some natural heterocyclic alkaloids containing planar group show potential to complex with specific
promoter region of protooncogene for stabilizing the G-quadruplex (G4) structure which nowadays
promises to be a target in anticancer drug design. However, in view of the polymorphic characteristics
and structural complexity of heterocyclic alkaloids, it is desirable to develop high-throughput and low-
consumption approach for anticancer drug screening. In this paper, an intensive study on alkaloid
ligand/G4 DNA interaction has been conducted, demonstrating that the end-stacking interaction is the
favorable binding mode between the oncogene-related Pu22 G4 DNA and the heterocyclic alkaloid
ligand. Based on structural feasibility and energy minimization, a ligand displacement assay for
screening alkaloid ligand in stabilizing the oncogene target G4 has been developed, which also helps to
facilitate the assessment of drug specificity. Coupled with microfluidic-based DNAzyme-catalytic
chemiluminescence detection, the approach showed the advantages of high sensitivity, high throughput
with low sample and reagent consumptions.
Chrysin cocrystals: Characterization and evaluation
Renu Chadha, Yashika Bhalla, Avdesh Nandan, Kunal Chadha, Maninder Karan
ABSTRACT
Solvent free mechanochemical approach is utilized to synthesise new cocrystals of chrysin using
supramolecular chemistry based upon reliable synthons. Chrysin, a flavone nutraceutical with wide
range of beneficial effects has critically low bioavailability on account of its poor aqueous solubility
and consequently poor absorption from the gastrointestinal tract. The present study focuses on this
critical aspect and has exploited non covalent interactions to prepare its cocrystals with cytosine and
thiamine hydrochloride. Various techniques were used for characterization including Differential
Scanning Calorimetry (DSC), Fourier Transform Infrared Spectroscopy (FT-IR), Solid State NMR
Spectroscopy (SSNMR) and Powder X-Ray Diffraction (PXRD). The molecules in the cocrystals
crystallized in neutral forms and assembled in a molecular layer by means of hydrogen bonding which
was confirmed by structural characterization. The cocrystals share a common supramolecular motif
being the OH⋯Narom interaction, involving phenolic moiety of C7 functionality of the parent
molecule. Approximately 3–4 fold increase in solubility and dissolution profile of cocrystals was
observed which was further corroborated by improved in vitro and in vivo activities including
antioxidant, antihaemolytic and anti-inflammatory thus, opening a new viable technique for the
exploitation of useful phytonutrients.
32
Stability behaviour of antiretroviral drugs and their combinations 5: Characterization of novel
degradation products of abacavir sulfate by mass and nuclear magnetic resonance spectrometry
Moolchand Kurmi, Archana Sahu, Saranjit Singh
ABSTRACT
In the present study, degradation behaviour of abacavir sulfate was evaluated in solution and solid
stress conditions. Solution state studies resulted in formation of eleven degradation products; of which
two were also formed on solid stress. The same were separated by high performance liquid
chromatography. They were characterized using liquid chromatography-high resolution mass
spectrometry, liquid chromatography-multistage mass spectrometry and hydrogen/deuterium exchange
mass spectrometry data. Additionally, seven degradation products were isolated and subjected to 1D
and 2D nuclear magnetic resonance studies for their structural confirmation.
Comparison of miRNA signature versus conventional biomarkers before and after off-pump
coronary artery bypas graft
Fatemeh Pourrajab, Fereshteh Torkian Velashani, Masoud Khanaghaei, Seyedhossein
Hekmatimoghaddam, Mohamad Reza Zare-Khormizi
ABSTRACT
Circulating levels of microRNAs (miRNAs) and their expression patterns are supposed to serve as
signatures for diagnosis or prognosis of cardiovascular events. The present study aimed at determining
if there is any correlation between the release pattern of 2 miRNAs and the plasma levels of
conventional biomarkers cardiac troponin I (cTnI), creatine kinase (CK) and uric acid (UA) in patients
undergoing their first off-pump coronary artery bypass graft (OCABG). Seventy OCABG patients
(69% men, aged 59.2 ± 8.2 years) were enrolled. Emergencies, re-operations, abnormal preoperative
serum cTnI and combined procedures were excluded from this study. Pre-operative mean ejection
fraction was 45.8 ± 8.6%, the average number of grafts was 3 ± 0.87/patient, and the internal
mammary artery was used for all. Beside conventional clinical assays, we performed real-time
quantitative PCR to analyze the circulating levels of miR-155, miR-126 and miR-499 at 1 day before
surgery as well as 4 days after surgery. Importantly, there was no report of myocardial infarction in our
patients, pre- or post-operatively. In contrast to conventional biomarkers cTnI and CK, circulating
levels of miRNAs decreased significantly (P < 0.01) after revascularization surgery. A significant
positive correlation was seen between the cTnI and miR-499 (r ∼ 0.53, P < 0.01) and between miR-
126 and UA (r ∼ 0.5, P < 0.01). Time course study of circulating miR-499, miR-126 and miR-155 in
cardiac surgery clarified their advantage and correlations to the traditional biomarkers cTnI, total CK,
CK-MB and UA. Our results suggest that this signature is a novel, early biomarker which indicates
myocardial ischemia in cardiac surgery. It could be postulated that the application of these miRNAs
may be considered for monitoring of response to pharmacological interventions aimed at reducing
cardiac ischemia, especially in OCABG candidates.
33
Microfluidic device for label-free quantitation and distinction of bladder cancer cells from the
blood cells using micro machined silicon based electrical approach; suitable in urinalysis assays
Seied Ali Hosseini, Somayeh Zanganeh, Elaheh Akbarnejad, Fatemeh Salehi, Mohammad Abdolahad
ABSTRACT
This paper introduces an integrated microfluidic chip as a promising tool to measure the concentration
of bladder cancer cells (BCC) in urine samples. Silicon microchannels were used as trapping gates for
both floated BCC and leukocytes which are found in the urine of patients. By the assistance of the gold
electrodes patterned at the bottom of the micro gates, the capacitance of captured cancerous and blood
cells were measured. Different membrane capacitance between BCC and leukocyte was the indicative
signal for diagnosing the nature of captured cells in urine like solution. The concentration range of the
target that could be detected was about 10 BCCs per one chip. Such response has been achieved
without applying any biochemical or florescent markers. Thus, it could be a simple and cheap
approach to support cytological and immune-fluorescent assays. The limit of detection was
approximately 1 cancerous cell/11 leukocytes in 1 ml of the urine like solution. The entire
measurement time was less than an hour. Consequently, this electrical microfluidic device promises
significant potential in urinalysis.
Simultaneous determination of anemoside B4, phellodendrine, berberine, palmatine, obakunone,
esculin, esculetin in rat plasma by UPLC–ESI–MS/MS and its application to a comparative
pharmacokinetic study in normal and ulcerative colitis rats
Lianrong Yang, Xin Meng, Xiaojin Yu, Haixue Kuang
ABSTRACT
A sensitive and rapid ultra-performance liquid chromatography-electrospray ionization-mass
spectrometry (UPLC–ESI–MS/MS) method was developed for the simultaneous analysis of anemoside
B4, phellodendrine, berberine, palmatine, obakunone, esculin, esculetin, toosendanin (IS1 of
anemoside B4), tetrahydropalmatine (IS2 of phellodendrine, berberine, palmatine and obakunone) and
scopoletin (IS3 of esculin and esculetin) and to compare the pharmacokinetics of these active
ingredients in normal and ulcerative colitis rats. After methanol deproteinization, solvents were
evaporated at 40 °C under a gentle stream of nitrogen. Chromatography was performed using a C18
column with a gradient elution of 0.1% aqueous formic acid and acetonitrile at 0.4 ml/min. Detection
and measurement were performed on a 4000 QTRAP UPLC–MS/MS system from AB Sciex in the
multiple reaction monitoring (MRM) mode. Phellodendrine, berberine, palmatine, obakunone, esculin,
esculetin, tetrahydropalmatine (IS2) and scopoletin (IS3) were monitored under positive ionization
conditions. Anemoside B4, and toosendanin (IS1) were monitored under negative ionization
conditions. The optimized mass transition ion-pairs (m/z) were 1221.1/750.7 for anemoside B4,
343.2/193.2 for phellodendrine, 337.1/321.0 for berberine, 353.0/336.9 for palmatine, 455.1/161.1 for
obakunone, 341.2/179.2 for esculin, 179.1/123.0 for esculetin, 573.4/531.4 for toosendanin (IS1),
356.2/192.2 for tetrahydropalmatine (IS2) and 193.0/133.1 for scopoletin (IS3).
34
Simultaneous determination and pharmacokinetic study of four phenolic acids in rat plasma
using UFLC–MS/MS after intravenous administration of salvianolic acid for injection
Xiuman Xie, Jingzhuo Miao, Wanyang Sun, Jingyi Huang, Guoxiang Sun
ABSTRACT
A simple, sensitive and selective ultra-fast liquid chromatography-tandem mass spectrometry (UFLC–
MS/MS) method was established for simultaneous determination and pharmacokinetic study of
rosmarinic acid (RA), salvianolic acid D (Sal D), lithospermic acid (LA) and salvianolic acid B (Sal B)
in rat plasma after intravenous administration of salvianolic acid for injection (SAFI). Three doses of
administration, containing 14, 28 and 56 mg/kg, were investigated in this study. Plasma samples were
pretreated using protein precipitation (PP) with pre-cooled acetonitrile. Chromatographic separation
was achieved on a CORTECS™ UPLC C18 column (1.6 μm, 2.1 × 100 mm) with a mobile phase
composed of 0.1% formic acid aqueous (V/V) and 0.1% formic acid acetonitrile (V/V). Analytes were
detected using electrospray ionization (ESI) source in negative ionization mode and quantified in
multiple reaction monitoring (MRM) mode. The validated method is stable and reliable. No significant
difference of half lives (t1/2) of four analytes at three doses was observed. Area under the curve
(AUC0-∞) and peak concentration (Cmax) of the four analytes demonstrated a linear increase in across
the doses with the linear correlation r of each analyte at three doses were greater than 0.95. It indicated
that the pharmacokinetic behavior of SAFI is positively related to dose at the range of 14–56 mg/kg.
A rapid and sensitive UHPLC–MS/MS method for quantification of 83b1 in plasma and its
application to bioavailability study in rats
Dingsheng Wen, Jing Guo, Fulin Jiang, Caishun Huang, Guoping Zhong
ABSTRACT
Great attentions have been drawn by quinoline for its broad bioactivity as anti-fungal, anti-bacterial
and anti-tumor activities. Compared with cisplatin, 83b1, a quinoline derivative, showed equal activity
in anti-tumor and lower cyctotoxicity in normal cell. In this study, a simple, rapid and sensitive method
for determination of 83b1 in rat plasma using UHPLC–MS/MS was developed for the first time.
Loratadine was used as an internal standard (IS). Separation was performed on an Xterra MS C18
column by isocratic elution using acetonitrile: water solution with 1‰ formic acid (90:10, v/v) as
mobile phase at a flow rate of 0.3 mL/min. A triple quadrupole mass spectrometer operating in the
positive ion-switching electron spray ionization mode with selection reaction monitoring (SRM) was
employed to determine 83b1 and IS transitions of m/z 321.82 → 147.84, 382.71 → 258.76 for 83b1
and Loratadine, respectively. The values of specificity, linearity and lower limit of quantification,
intra- and inter- day precision and accuracy, extraction recovery, matrix effect and stability for this
method satisfied the acceptable limits. The lower limit of quantification was 0.5 ng/mL with a linear
range of 0.5–1500 ng/mL. The validated method was employed to study the bioavailability of 83b1 in
rat by dosing with intravenous injection (1 mg/kg) and gavage (10 mg/kg), and the oral bioavailability
of 83b1 in rat was calculated as 20.9 ± 8.8%.
35
Accurate quantitation of choline and ethanolamine plasmalogen molecular species in human
plasma by liquid chromatography–tandem mass spectrometry
Yurika Otoki, Shunji Kato, Fumiko Kimura, Katsutoshi Furukawa, Kiyotaka Nakagawa
ABSTRACT
Concentration of both choline plasmalogen (PC-Pls) and ethanolamine Pls (PE-Pls) in human
plasma/serum has been getting attention to, since certain patients including those with
neurodegenerative disorders, have been reported to exhibit reduced levels of specific Pls species.
However, despite using liquid chromatography–tandem mass spectrometry (LC–MS/MS), accurate
quantitation of Pls is still difficult because of less product ion from PC-Pls and quantitative issues
(e.g., extraction recoveries and matrix effects). The present study aimed to develop a method for
accurate identification and quantitation of Pls molecular species using LC–MS/MS operated in the
multiple reaction monitoring modes. The LC–MS/MS conditions in the presence of sodium, and the
extraction method using methanol protein precipitation were optimized. Under the optimal condition,
Pls was detected at femtomole levels. The recoveries of Pls from human plasma were nearly 100%,
and matrix effects were not observed. The novel method enabled determination of each Pls species in
human plasma at the concentrations of 0.5–13.6 μM. Then the PC-Pls and PE-Pls species in the plasma
of both healthy subjects and patients with Alzheimer‘s disease were quantitated. The method
developed herein represents a powerful tool for analyzing Pls, which may provide a better
understanding of their physiological roles in vivo.
The profiling of the metabolites of hirsutine in rat by ultra-high performance liquid
chromatography coupled with linear ion trap Orbitrap mass spectrometry: An improved
strategy for the systematic screening and identification of metabolites in multi-samples in vivo
Jianwei Wang, Peng Qi, Jinjun Hou, Yao Shen, Dean Guo
ABSTRACT
Drug metabolites identification and construction of metabolic profile are meaningful work for the drug
discovery and development. The great challenge during this process is the work of the structural
clarification of possible metabolites in the complicated biological matrix, which often resulting in a
huge amount data sets, especially in multi-samples in vivo. Analyzing these complex data manually is
time-consuming and laborious. The object of this study was to develop a practical strategy for
screening and identifying of metabolites from multiple biological samples efficiently. Using hirsutine
(HTI), an active components of Uncaria rhynchophylla (Gouteng in Chinese) as a model and its
plasma, urine, bile, feces and various tissues were analyzed with data processing software (Metwork),
data mining tool (Progenesis QI), and HR-MSn data by ultra-high performance liquid
chromatography/linear ion trap-Orbitrap mass spectrometry (U-HPLC/LTQ-Orbitrap-MS). A total of
67 metabolites of HTI in rat biological samples were tentatively identified with established library, and
to our knowledge most of which were reported for the first time. The possible metabolic pathways
were subsequently proposed, hydroxylation, dehydrogenation, oxidation, N-oxidation, hydrolysis,
reduction and glucuronide conjugation were mainly involved according to metabolic profile. The result
proved application of this improved strategy was efficient, rapid, and reliable for metabolic profiling of
components in multiple biological samples and could significantly expand our understanding of
metabolic situation of TCM in vivo.
36
Metabolic fate and detectability of the new psychoactive substances 2-(4-bromo-2,5-
dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25B-NBOMe) and 2-(4-chloro-2,5-
dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25C-NBOMe) in human and rat
urine by GC–MS, LC–MSn, and LC–HR–MS/MS approaches
Achim T. Caspar, Simon D. Brandt, Andreas E. Stoever, Markus R. Meyer, Hans H. Maurer
ABSTRACT
25B-NBOMe and 25C-NBOMe are potent 5-HT2A receptor agonists that have been associated with
inducing hallucinogenic effects in drug users and severe intoxications. This paper describes the
identification of their metabolites in rat and human urine by liquid chromatography (LC)-high
resolution (HR)-MS/MS, the comparison of metabolite formation in vitro and in vivo and in different
species, the general involvement of human cytochrome-P450 (CYP) isoenzymes on their metabolism
steps, and their detectability by standard urine screening approaches (SUSAs) using GC–MS, LC–
MSn, or LC-HR-MS/MS. Both NBOMe derivatives were mainly metabolized by O-demethylation,
O,O-bis-demethylation, hydroxylation, and combinations as well as by glucuronidation and sulfation
of the main phase I metabolites. For 25B-NBOMe, 66 metabolites could be identified and 69 for 25C-
NBOMe. After application of low doses of both substances to rats, they were detectable mainly via
their metabolites by both LC-based SUSAs. In case of acute intoxication, it was possible to detect
25B-NBOMe and its metabolites in an authentic human urine sample when using the GC–MS SUSA
in addition to the LC-based SUSAs. Initial CYP activity screening revealed the involvement of
CYP1A2 and CYP3A4 in hydroxylation and CYP2C9 and CYP2C19 in O-demethylation. The
presented study demonstrated that 25B-NBOMe and 25C-NBOMe were extensively metabolized and
detectable by both LC-based SUSAs.
Liquid chromatography-tandem mass spectrometric determination of propofol in rat serum and
hair at attogram level after derivatization with 3-bromomethyl-propyphenazone
Alaa Khedr, Soad S.Abd El-Hay, Ahmed K. Kammoun
ABSTRACT
A sensitive, selective and precise liquid chromatography-tandem mass spectrometry (LC-MS/MS)
method for determination of propofol (PRO) in rat serum and hair has been developed. 3-
Bromomethyl-propyphenazone was used as derivatization reagent forming propofol-methyl-
propyphenazone compound. The derivatization reaction was optimized and validated for maximum
MS sensitivity. The MS instrumental sensitivity reached to 10 attogram. The serum samples were
extracted by using Chromabond C8 columns, while hair samples extracted with methanol. The
tendency of volatility of PRO was minimized by adding triethylamine to the extract before the use of
nitrogen gas for evaporation of solvent. The limit of quantitation (LLOQ) was 0.01 pg/mL and the
assay was linear to 10000 pg/mL. The intra-and inter-day precision (RSD%) ranged from 0.33 to
3.44% while the accuracy (Er%, relative error) were −6.4 to 1.1%. The ionization suppression, due to
reagent, was minimized by reacting the excess reagent with methanol, and eluting to waste before MS
ionization source (2–4.5 min). The method was successfully applied for detection and determination of
PRO in rat serum and hair after 7–28 days from administration of only one dose of propofol (10
mg/kg).
37
Investigation of the metabolites of the HIF stabilizer FG-4592 (roxadustat) in five different in
vitro models and in a human doping control sample using high resolution mass spectrometry
Annelie Hansson, Mario Thevis, Holly Cox, Geoff Miller, Mikael Hedeland
ABSTRACT
FG-4592 is a hypoxia-inducible factor (HIF) stabilizer, which can increase the number of red blood
cells in the body. It has not been approved by regulatory authorities, but is available for purchase on
the Internet. Due to its ability to improve the oxygen transportation mechanism in the body, FG-4592
is of interest for doping control laboratories, but prior to this study, little information about its
metabolism was available. In this study, the metabolism of FG-4592 was investigated in a human
doping control sample and in five in vitro models: human hepatocytes and liver microsomes, equine
liver microsomes and S9 fraction and the fungus Cunninghamella elegans. By using liquid
chromatography coupled to a Q-TOF mass spectrometer operated in MSE and MSMS modes, twelve
different metabolites were observed for FG-4592. One monohydroxylated metabolite was detected in
both the human and equine liver microsome incubations. For the fungus Cunninghamella elegans
eleven different metabolites were observed of which the identical monohydroxylated metabolite had
the highest response. This rich metabolic profile and the higher levels of metabolites produced by
Cunninghamella elegans demonstrates its usefulness as a metabolite producing medium. In the doping
control urine sample, one metabolite, which was the result of a direct glucuronidation, was observed.
No metabolites were detected in neither the human hepatocyte nor in the equine liver S9 fraction
incubates.
A quantitative LC–MS/MS method for simultaneous determination of cocaine and its
metabolites in whole blood
Xiabin Chen, Xirong Zheng, Kai Ding, Ziyuan Zhou, Fang Zheng
ABSTRACT
As new metabolic pathways of cocaine were recently identified, a high performance liquid
chromatography tandem mass spectrometry (LC–MS/MS) method was developed to simultaneously
determine cocaine and nine cocaine-related metabolites in whole blood samples. One-step solid phase
extraction was used to extract all of the ten compounds and corresponding internal standards from
blood samples. All compounds and internal standards extracted were separated on an Atlantis T3 (100
Å, 3 μm, 2.1 mm × 150 mm I.D) column and detected in positive ion and high sensitivity mode with
multiple reaction monitoring. This method was validated for its sensitivity, linearity, specificity,
accuracy, precision, recovery, and stability. All of the ten compounds were quantifiable ranging from
the lower limit of quantification (LLOQs) of ∼10 nM (1.9–3.2 ng/ml) to ∼1000 nM (190–320 ng/ml)
without any interfering substance. Accuracy and precision were determined, and both of them were
within the acceptance criteria of the United States (US) Food and Drug Administration (FDA) and
European Medicines Agency (EMA) guidelines. The recovery was above 66.7% for all compounds.
Stability tests demonstrated the stability of compounds under different storage conditions in whole
blood samples. The method was successfully applied to a pharmacokinetic study with co-
administration of cocaine and alcohol in rats.
38
Use of FTIR spectroscopy and PCA-LDC analysis to identify cancerous lesions within the human
colon
E. Kaznowska, J. Depciuch, K. Szmuc, J. Cebulski
ABSTRACT
Colorectal cancer constitutes 33% of all cancer morbidity, so the research of the new methods for
colorectal cancer diagnosis and chemotherapy monitoring is gaining its momentum. Diagnostic
instruments are being sought, which enable the detection of single malignant cells based on the
analysis of tissue material potentially reusable at further stages of diagnostic management. The most
common approach to tissue specimen processing is paraffin-embedding. Yet, paraffin may cause
background noise in spectroscopic measurements with the wavenumber ranging between 900 cm−1
and 3500 cm−1. However, the study by Depciuch et al. (2016) proved that appropriate specimen
processing and paraffin-embedding technique as well as a strict measurement methodology may
eliminate paraffin vibrations. As a result, spectroscopic measurements may become a reliable and
precise method for the diagnosis and treatment monitoring in patients with colorectal cancer as long as
the high standards of specimen processing are maintained. Chemotherapy is the main medical
treatment in colorectal cancer. Unfortunately, the absence of tools which enable monitoring its efficacy
leads to the partial response or non-response frequently seen in affected patients. Hence, diagnostic
instruments are also being sought capable of monitoring treatment efficacy so as to enable early
changes of chemotherapy regimen thus increasing the chance of cure. The paper aims at comparing the
results of FTIR (Fourier Transform Infrared) spectroscopy in several types of colon tissue: healthy
colon, cancerous colon, post-chemotherapy colon and healthy surgical margin of colon cancer sample.
The obtained FTIR spectra along with the Principal Component Analysis-Linear Discriminant
Analysis (PCA-LDC) as well as bandwidth analysis of the primary amide region revealed some
differences between the spectra of healthy tissues as compared to cancerous tissues (pre- or post-
chemotherapy). Apart from confirming that FTIR spectroscopy is a good source of information on the
composition of analysed samples, this fact supports its application as a tool to facilitate understanding
the pathophysiology of various conditions and to monitor efficacy of chemotherapy in cancer patients.
Antibody-free detection of infectious bacteria using quantum dots-based barcode assay
Kristyna Cihalova, Dagmar Hegerova, Ana Maria Jimenez, Vedran Milosavljevic, Vojtech Adam
ABSTRACT
Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Klebsiella pneumoniae are the
most representative bacteria causing infectious diseases. Due to the increased application of
antibiotics, the bacterial resistance is growing causing severe complications. Therefore, a sensitive
determination of these pathogens is crucial for effective treatment. The aim of this study was to design
an effective method for multiplex detection of Staphylococcus aureus, methicillin-resistant
Staphylococcus aureus and Klebsiella pneumoniae taking advantage from properties of magnetic
particles as well as fluorescent nanoparticles (quantum dots). The method was able to detect as low
concentrations of bacteria as 102 CFU/mL using the bacteria-specific genes (fnbA, mecA and wcaG).
39
A simple and sensitive liquid chromatography–tandem mass spectrometry method for trans-ε-
viniferin quantification in mouse plasma and its application to a pharmacokinetic study in mice
Jiseon Kim, Jee Sun Min, Doyun Kim, Yu Fen Zheng, Soo Kyung Bae
ABSTRACT
In this study, a simple and sensitive liquid chromatography-tandem mass spectrometry (LC–MS/MS)
method for the quantification of trans-ε-viniferin in small volumes (10 μl) of mouse plasma using
chlorpropamide as an internal standard was developed and validated. Plasma samples were
precipitated with acetonitrile and separated using an Eclipse Plus C18 column (100 × 4.6 mm, 1.8-μm)
with a mobile phase consisting of 0.1% formic acid in acetonitrile and 0.1% formic acid in water
(60:40 v/v) at a flow rate of 0.5 ml/min. A triple quadrupole mass spectrometer operating in positive
ion mode with selected reaction-monitoring mode was used to determine trans-ε-viniferin and
chlorpropamide transitions of 455.10 → 215.05 and 277.00 → 111.00, respectively. The lower limit of
quantification was 5 ng/ml with a linear range of 5–2500 ng/ml (r ≥ 0.9949). All validation data,
including the selectivity, precision, accuracy, recovery, dilution integrity, and stability, conformed to
the acceptance requirements. No matrix effects were observed. The developed method was
successfully applied to pharmacokinetic studies of trans-ε-viniferin following intravenous (2.5 mg/kg),
intraperitoneal (2.5, 5 and 10 mg/kg), and oral (40 mg/kg) administration in mice. This is the first
report on the pharmacokinetic properties of trans-ε-viniferin. The results provide a meaningful basis
for evaluating the pre-clinical or clinical applications of trans-ε-viniferin.
LC–MS/MS assay for the quantitation of the tyrosine kinase inhibitor neratinib in human
plasma
Brian F. Kiesel, Robert A. Parise, Alvin Wong, Kiana Keyvanjah, Jan H. Beumer
ABSTRACT
Neratinib is an orally available tyrosine kinase inhibitor targeting HER2 (ERBB2) and EGFR (ERBB).
It is being clinically evaluated for the treatment of breast and other solid tumors types as a single agent
or in combination with other chemotherapies. In support of several phase I/II clinical trials
investigating neratinib combinations, we developed and validated a novel LC–MS/MS assay for the
quantification of neratinib in 100 μL of human plasma with a stable isotopic internal standard.
Analytes were extracted from plasma using protein precipitation and evaporation of the resulting
supernatant followed by resuspension. Chromatographic separation was achieved using an Acquity
UPLC BEH Shield RP18 column and a gradient methanol-water mobile phase containing 10%
ammonium acetate. An ABI 4000 mass spectrometer and electrospray positive mode ionization were
used for detection. The assay was linear from 2 to 1,000 ng/mL and proved to be accurate (98.9–
106.5%) and precise (<6.2%CV), and met the FDA guidance for bioanalytical method validation. This
LC–MS/MS assay will be an essential tool to further define the pharmacokinetics of neratinib.
40
Comparative tissue distribution and excretion study of alkaloids from Herba Ephedrae-Radix
Aconiti Lateralis extracts in rats
Mengyue Ren, Shuai Song, Dedong Liang, Weiting Hou, Jiabo Luo
ABSTRACT
Herba Ephedrae-Radix Aconiti Lateralis, composed of Ephedrae (Mahuang in Chinese) and Radix
Aconiti Lateralis (Fuzi in Chinese), is a classical herbal combination proven to be effective in treating
common cold, asthma, and rheumatoid arthritis. Alkaloids, bioactive components of the herbal extract,
have been associated with many side effects. Nine alkaloids, including norephedrine,
norpseudoephedrine, ephedrine, pseudoephedrine, methylephedrine, hypaconitine, benzoylaconine,
benzoylmesaconine and benzoylhypaconine, were simultaneously quantified within 14.5 min, by a
validated ultra-performance liquid chromatography-tandem mass spectrometry method in various rat
tissues, urine, and feces after oral administration of Mahuang-Fuzi and single-herb extracts. The results
indicated that the alkaloids were widely distributed in the heart, liver, spleen, lung, kidney, and brain.
Lower bioavailability and higher clearance of some alkaloids were observed for the herbal
combination, but hypaconitine showed a longer residence time and lower clearance. Elimination
kinetics demonstrated that ephedra and aconitum alkaloids were mainly excreted in urine and feces,
respectively. The tissue distribution and excretion of ephedra and aconitum alkaloids are
comprehensively reported for the first time for the Mahuang-Fuzi combination. Compared with single-
herb extracts, lower extraction efficiencies of alkaloids in vitro were observed which may result in
their lower intake. However, the combination showed a prolonged residence time and delayed
elimination of aconitum alkaloids, which increases the risk of drug accumulation. The study
demonstrated potential risks of intoxication with aconitum alkaloids, associated with the use of Fuzi in
combination with Mahuang. Mahuang-Fuzi is a classical combination used in clinics, further
investigation is needed.
Deep eutectic solvents as green media for extraction of flavonoid glycosides and aglycones from
Platycladi Cacumen
Bo Zhuang, Li-Li Dou, Ping Li, E-Hu Liu
ABSTRACT
Deep eutectic solvents (DESs) are emerging as alternatives to conventional ionic liquids and organic
solvents due to their unique advantages. In the present study, the tuneability of DESs as tailor-made
solvents to efficiently extract polar and non-polar bioactive compounds from Platycladi Cacumen was
detailedly investigated. Totally 12 types of choline chloride-, betaine-, and l-proline-based DESs were
synthesized for initial screening, and extraction conditions was optimized by single-factor experiment.
Experiments with different DESs and principal components analysis demonstrated that the
extractability of both flavonoid glycosides and aglycones was greater with certain designed DESs than
conventional solvents. In addition, the water content in DESs led to significantly different extraction
yields of flavonoid compounds. The target compounds were recovered from DESs by macroporous
resin LX-38 with a satisfactory yield between 77.44% and 98.92%. The knowledge acquired in this
study could contribute to further DES application in extraction of bioactive compounds from natural
sources.
41
Testosterone and its dimers alter tRNA morphology
P. Chanphai, D. Agudelo, A.R. Vesper, G. Bérubé, H.A. Tajmir-Riahi
ABSTRACT
The morphology of tRNA was studied upon conjugation with testosterone and its aliphatic and
aromatic dimers, using multiple spectroscopic methods, transmission electron microscopy (TEM) and
molecular modeling. Structural analysis showed that testosterone binds tRNA through A62, A64, C60,
C61, C63, G51, U50 and U59 bases. The binding affinity was testosterone dimer-aromatic >
testosterone dimer-aliphatic > testosterone. The steroid loading efficacy was 35–45%. Transmission
electron microscopy showed major changes in tRNA morphology upon testosterone interaction with an
increase in the diameter of the tRNA aggregate, indicating encapsulation of testosterone by tRNA.
Development and validation of a simple and robust HPLC method with UV detection for
quantification of the hepatitis C virus inhibitor daclatasvir in human plasma
Giulio Nannetti, Lorenzo Messa, Marta Celegato, Silvana Pagni, Arianna Loregian
ABSTRACT
Daclatasvir is an inhibitor of hepatitis C virus NS5A protein that is used for the therapy of chronic
hepatitis. So far, published methods for analysis of daclatasvir in plasma are exclusively based on mass
spectrometry, which is not always available in standard clinical laboratories. Thus, we wished to
develop and validate a simple, but still reliable and sensitive high-performance liquid chromatography
(HPLC) assay with UV detection for the quantification of daclatasvir, feasible for a wide-spread
clinical routine use. The method consisted of solid-phase extraction of daclatasvir using Waters Oasis
HLB 1cc cartridges, reversed-phase liquid chromatography with a Waters XTerra RP18 (150 mm × 4.6
mm, 3.5 μm) column and a mobile phase of ammonium acetate buffer (pH 5.0, 10 mM) and
acetonitrile (56:44, v/v), and UV detection at 318 nm. This assay proved to be sensitive (lower limit of
quantification of 0.05 μg/mL), linear (correlation coefficients ≥0.997), specific (no interference with
various potentially co-administrated drugs), reproducible (both intra-day and inter-day coefficients of
variation ≤8.9%), and accurate (deviations ranged from −2.2 to 8.0% and from −6.5 to 9.2% for intra-
day and inter-day assays, respectively). The method was applied to therapeutic monitoring of patients
undergoing daclatasvir therapy for hepatitis C and showed to be reliable and robust. Thus, this method
provides a simple, sensitive, precise, and reproducible assay for dosing daclatasvir that can be readily
adaptable to routine use by clinical laboratories with standard equipment. In addition, the stability of
daclatasvir in plasma was evaluated under various conditions, including after the heating procedure
required for inactivation of infectious viruses and in different light exposure conditions. These studies
evidenced photo-instability of the compound under sunlight exposure over time. Thus, blood sampling
and the whole handling procedure have to be performed quickly and with minimal light exposure.
42
Evaluation of ion mobility spectrometry for the detection of mitragynine in kratom products
Nathan Fuenffinger, Melissa Ritchie, Ashley Ruth, Connie Gryniewicz-Ruzicka
ABSTRACT
An ion mobility spectrometry (IMS) method was developed for the rapid detection of mitragynine, the
most abundant alkaloid in Mitragyna speciosa also known as kratom. The peak corresponding to the
mitragynine protonated ion exhibited a reduced ion mobility of 0.95 ± 0.00014 cm2/(V s), and the
mitragynine limit of detection using IMS was 0.5 ng. The IMS method was applied to the analysis of
15 commercial samples suspected of containing kratom. IMS results were compared to those obtained
from liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis of the same samples.
Mitragynine was conclusively detected in 14 of 15 samples using LC–MS/MS and 13 of 15 samples
using IMS. The discrepancy between methods reflected the fact that one sample contained mitragynine
at a concentration below the IMS detection limit. This study demonstrates the utility of IMS for the
rapid screening of products containing kratom as well as the scientific reliability of the IMS screening
method, which was demonstrated by comparing the IMS results to the confirmatory results obtained
using LC–MS/MS.
A single MCR-ALS model for drug analysis in different formulations: Application on diazepam
commercial preparations
Michele De Luca, Giuseppina Ioele, Claudia Spatari, Gaetano Ragno
ABSTRACT
A multivariate curve resolution – alternating least squares (MCR-ALS) analysis was used to quantify
diazepam (DZP) in thirty commercial liquid formulations. MCR calibration was run on the UV
spectrophotometric data of the commercial DZP samples over the range 200–400 nm, allowing the
resolution of the drug signal and then the excipients contained in all the formulations. A single model
MCR for the determination of the drug in all samples was then built through the adoption of the
correlation constraint. This model was optimized by an appropriate selection of the most useful
wavelength ranges and then validated on external samples. DZP concentrations in the pharmaceutical
formulations were measured by HPLC-DAD analysis. The performance of the MCR model was
compared with that from application of classical partial least squares regression (PLSR). The results, in
terms of error of prediction, were very satisfactory, reaching a relative error below of 1.66% against
2.56%, respectively.
43
Selective recognition of cis-trans-isomers of platinum drugs and the detection of triplex DNA
based on fluorescence reversible model of quantum dots
Xiaoling Xu, Fang Gao, Xincai Xiao, Yan Hu, Dan Zhao
ABSTRACT
The identification of spatial structures of drugs and the researches on their interaction mechanism with
DNA are always attractive to the researchers. However, their realization is lack of simple and fast
method. This paper reports the establishment of multiple-functional detection platform based on the
―turn off-on‖ model of ZnCdSe quantum dots. In this system, ZnCdSe quantum dots work as the
fluorescent probe, platinum anti-cancer drugs as the quencher and triplex DNA as the trapping agent.
The seemingly similar cisplatin and transplatin exhibited different fluorescent recovery behaviors due
to their difference in structure, and thus realized the selective detection of cisplatin and transplatin with
the reaction time set at 10 min as well as the quantitation of cisplatin over the range of 2.5 × 10−8–100
× 10−8M. Based on this, the interactions between platinum anti-cancer drugs and ctDNA as well as
polymorphic DNA were further studied, and realized the recognition of triplex DNA. The multiple-
functional detection platform integrates the functions of the filtration of high-efficient platinum anti-
cancer drugs, the researches on interaction mechanism of drugs, and the recognition of polymorphic
DNA, meaningful to the future treatment of viral and cancers based on antisense gene strategy.
Drug-protein binding of Danhong injection and the potential influence of drug combination with
aspirin: Insight by ultrafiltration LC–MS and molecular modeling
Junfeng Zhu, Xiaojiao Yi, Peng Huang, Shuqing Chen, Yongjiang Wu
ABSTRACT
Danhong injection (DHI) is a widely used Chinese medicine injection (CMI) for the clinical treatment
of cardiovascular and cerebrovascular diseases. In this study, a simple and efficient in vitro method
based on ultrafiltration LC–MS and molecular modeling has been developed to study the human serum
albumin (HSA) binding of the compounds in DHI. Seven major components including protocatechuic
aldehyde, p-coumaric acid, salvianolic acid D, rosmarinic acid, salvianolic acid E, lithospermic acid
and salvianolic acid B were identified as HSA ligands and their binding degrees in the proposed non-
saturated model were 26.17, 37.69, 99.77, 91.78, 96.91, 99.42 and 98.10%, respectively. Considering
the drug-HSA binding property of the compounds in DHI may change during drug combination
therapy, competitive binding assay was carried out to evaluate the influence of aspirin on the DHI-
HSA binding. Experimental results revealed that the salvianolic acids in DHI had stronger binding
ability to HSA than sodium salicylate. To further verify the results above, molecular modeling and
probe displacement assay were conducted to investigate the optimum binding site and binding affinity
of the ligands on HSA. Our findings suggested that the established method could be a powerful tool to
study the drug-HSA binding property of CMIs.
44
The importance of vial composition in HPLC analysis: An unusual case of phosphorous
pseudorotation
Rebecca Arvary, Ian Mangion
ABSTRACT
Glass HPLC vials are ubiquitous in analytical laboratories and vendors have developed many varieties
to meet the various needs of scientists. As such there may be multiple types of vials being used
simultaneously in a single laboratory without much consideration as to which is best suited for
analytical method development and validation. This study highlights the possibility of vial
composition as a potential factor that impacts solution stability. Here we describe a case where the
type of HPLC vial used results in an interesting phosphorous pseudorotation driven by the mild
alkalinity of glass.
Supercritical fluid chromatography for separation and preparation of tautomeric 7-epimeric
spiro oxindole alkaloids from Uncaria macrophylla
Wenzhi Yang, Yibei Zhang, Huiqin Pan, Changliang Yao, Dean Guo
ABSTRACT
Increasing challenge arising from configurational interconversion in aqueous solvent renders it rather
difficult to isolate high-purity tautomeric reference standards and thus largely hinders the holistic
quality control of traditional Chinese medicine (TCM). Spiro oxindole alkaloids (SOAs), as the
markers for the medicinal Uncaria herbs, can easily isomerize in polar or aqueous solvent via a retro-
Mannich reaction. In the present study, supercritical fluid chromatography (SFC) is utilized to separate
and isolate two pairs of 7-epimeric SOAs, including rhynchophylline (R) and isorhynchophylline (IR),
corynoxine (C) and corynoxine B (CB), from Uncaria macrophylla. Initially, the solvent that can
stabilize SOA epimers was systematically screened, and acetonitrile was used to dissolve and as the
modifier in SFC. Then, key parameters of ultra-high performance SFC (ultra-performance
convergence chromatography, UPC2), comprising stationary phase, additive in modifier, column
temperature, ABPR pressure, and flow rate, were optimized in sequence. Two isocratic UPC2 methods
were developed on the achiral Torus 1-AA and Torus Diol columns, suitable for UV and MS detection,
respectively. MCI gel column chromatography fractionated the U. macrophylla extract into two
mixtures (R/IR and C/CB). Preparative SFC, using a Viridis Prep Silica 2-EP OBD column and
acetonitrile-0.2% diethylamine in CO2 as the mobile phase, was finally employed for compound
purification. As a result, the purity of four SOA compounds was all higher than 95%. Different from
reversed-phase HPLC, SFC, by use of water-free mobile phase (inert CO2 and aprotic modifier),
provides a solution to rapid analysis and isolation of tautomeric reference standards for quality control
of TCM.
45
Volume 135, February 2017
Renewal of an old European Pharmacopoeia method for Terazosin using modeling with mass
spectrometric peak tracking
Róbert Kormány, Imre Molnár, Jenő Fekete
ABSTRACT
An older method for terazosin was reworked in order to reduce the analysis time from 90 min (2 × 45
min) to below 5 min. The method in European Pharmacopoeia (Ph.Eur.) investigates the specified
impurities separately. The reason of the different methods is that the retention of two impurities is not
adequate in reversed phase, not even with 100% water. Therefore ion-pair-chromatography has to be
applied and since that two impurities absorb at low UV-wavelength they had to be analyzed by
different method than the other specified impurities. In our new method we could improve the
retention with pH elevation using a new type of stationary phases available for high pH applications.
Also a detection wavelength could be selected that is appropriate for the detection and quantification
of all impurities. The method development is the bottleneck of liquid chromatography even today,
when more and more fast chromatographic systems are used. Expert knowledge with intelligent
programs is available to reduce the time of method development and offer extra information about the
robustness of the separation. Design of Experiments (DoE) for simultaneous optimization of gradient
time (tG), temperature (T) and ternary eluent composition (tC) requires 12 experiments. A good
alternative way to identify a certain peak in different chromatograms is the molecular mass of the
compound, due to its high specificity. Liquid Chromatography–Mass Spectrometry (LC–MS) is now a
routine technique and increasingly available in laboratories. In our experiment for the resolution- and
retention modeling the DryLab4 method development software (Version 4.2) was used. In recent
versions of the software the use of (m/z)-MS-data is possible along the UV-peak-area-tracking
technology. The modelled and measured chromatograms showed excellent correlations. The average
retention time deviations were ca. 0.5 s and there was no difference between the predicted and
measured Rs,crit −values.
A revisited structure for nitrosoprodenafil from NMR, mass spectrometry, X-ray and hydrolysis
data
Robert Martino, Christophe Menendez, Stéphane Balayssac, Nathalie Martins-Froment, Myriam
Malet-Martino
ABSTRACT
The sildenafil analogue adulterant previously identified as a nitroso derivative (nitrosoprodenafil) in a
dietary supplement (DS) marketed to increase sexual performance and sold in Europe in the early 2010
s is the same as that found in the same type of DS available in Japan whose structure was established
as a nitro derivative (mutaprodenafil or nitroprodenafil). Indeed, the compound isolated from the Man
Power DS has identical UV, IR, NMR and MS spectroscopic characteristics and hydrolysis behavior
than nitrosoprode-nafil. By revisiting its NMR assignments and MS and MS/MS data interpretation, it
is demonstrated that the compound is actually a nitrothioimidazole-methisosildenafil hybrid, i.e.
nitroprodenafil, whose structure is unequivocally confirmed by X-ray crystallography and synthesis
experiments. Because the product is converted to methisosildenafil by hydrolysis, it is named
nitropromethisosildenafil.
46
The importance of system band broadening in modern size exclusion chromatography
Alexandre Goyon, Davy Guillarme, Szabolcs Fekete
ABSTRACT
In the last few years, highly efficient UHP-SEC columns packed with sub–3 μm particles were
commercialized by several providers. Besides the particle size reduction, the dimensions of modern
SEC stationary phases (150 × 4.6 mm) was also modified compared to regular SEC columns (300 × 6
or 300 × 8 mm). Because the analytes are excluded from the pores in SEC, the retention factors are
very low, ranging from −1 < k < 0, resulting in very small column band variance. Therefore, the
contribution of the system itself to peak variance can become significant under UHP-SEC conditions.
The goal of this study was to evaluate the loss of efficiency observed with three different instruments
(regular HPLC, non-optimized UHPLC and fully optimized UHPLC) offering different system
variances. It appears that the new 150 × 4.6 mm, sub–3 μm SEC columns cannot be employed on a
regular HPLC instrument, since the efficiency loss was equal to 60–85%, when analyzing mAb
sample. Optimized UHPLC systems having very low extra-column volumes (typically Vec < 10 μL)
have therefore to be used to properly operate these columns. Due to the instrument contribution to
band broadening, the apparent efficiency of SEC columns packed with sub–2 μm particles can indeed
be hampered when using inappropriate system. Considering the extra-column band broadening
contribution of current UHPLC instruments, a further decrease of SEC column dimension is therefore
not desired.
Development of a multi-residue method for the determination of human and veterinary
pharmaceuticals and some of their metabolites in aqueous environmental matrices by SPE-
UHPLC–MS/MS
P. Paíga, L.H.M.L.M. Santos, C. Delerue-Matos
ABSTRACT
The aim of the present work was to develop and validate a multi-residue method for the analysis of 33
human and veterinary pharmaceuticals (non-steroidal anti-inflammatory drugs (NSAIDs)/analgesics,
antibiotics and psychiatric drugs), including some of their metabolites, in several aqueous
environmental matrices: drinking water, surface water and wastewaters. The method is based on solid
phase extraction (SPE) followed by ultra-high performance liquid chromatography-tandem mass
spectrometry (UHPLC–MS/MS) and it was validated for different aqueous matrices, namely bottled
water, tap water, seawater, river water and wastewaters, showing recoveries between 50% and 112%
for the majority of the target analytes. The developed analytical methodology allowed method
detection limits in the low nanograms per liter level. Method intra- and inter-day precision was under
8% and 11%, respectively, expressed as relative standard deviation. The developed method was
applied to the analysis of drinking water (bottled and tap water), surface waters (seawater and river
water) and wastewaters (wastewater treatment plant (WWTP) influent and effluent). Due to the
selectivity and sensitivity of the optimized method, it was possible to detect pharmaceuticals in all the
aqueous environmental matrices considered, including in bottled water at concentrations up to 31 ng
L−1 (salicylic acid). In general, non-steroidal anti-inflammatory drugs/analgesics was the therapeutic
group most frequently detected, with the highest concentrations found in wastewaters (acetaminophen
and the metabolite carboxyibuprofen at levels up to 615 and 120 μg L−1, respectively).
47
Development of new efficient method for isolation of phenolics from sea algae prior to their
rapid resolution liquid chromatographic–tandem mass spectrometric determination
Bořivoj Klejdus, Merichel Plaza, Marie Ńnóblová, Lea Lojková
ABSTRACT
The extraction of phenolic compounds from 4 different sea algae samples, three brown algae
(Cystoseira abies-marina, C. abies-marina grinded under cryogenic conditions with liquid nitrogen,
Undaria pinnatifida and Sargassum muticum) and one red algae (Chondrus crispus) via solid phase
extraction using micro-elution solid-phase extraction (μ-SPE) plate method was studied. Prior to μ-
SPE, 50 mg of algae with 80% methanol mixture was extracted in hyphenated series by various
extraction techniques, such as pressurized liquid extraction and Ika Ultra-Turrax® Tube Drive, in
combination with ultrasound assisted extraction. The μ-SPE plate technique reduced the time of
sample pre-treatment thanks to higher sensitivity and pre-concentration effect. Selected groups of
benzoic acid derivatives (p-hydroxybenzoic, protocatechuic, gallic, vanillic, and syringic acids),
hydroxybenzaldehydes (4-hydroxybenzaldehyde, and 3,4-dihydroxybenzaldehyde), and cinnamic acid
derivatives (p-coumaric, caffeic, ferulic, sinapic, and chlorogenic acids) were determined using rapid
resolution liquid chromatography coupled to mass spectrometry detection with negative ion
electrospray ionization (RRLC-ESI–MS) using multiple reactions monitoring. LOQs of measured
samples varied in the range 0.23–1.68 ng/mL and LODs in the range 0.07–0.52 ng/mL. The applied
method allowed a simultaneous determination of phenolics (i.e. free, esters soluble in methanol,
glycosides, and esters insoluble in methanol) in less than 5 min (including alkaline or acidic hydrolysis
of raw extracts) from sea algae extracts.
Physico-chemical profiling of semisynthetic opioids
Károly Mazák, Sándor Hosztafi, Márta Kraszni, Béla Noszál
ABSTRACT
Species-specific acid–base and partition equilibrium constants were experimentally determined for the
therapeutically important semisynthetic opioid receptor agonist hydromorphone, dihydromorphine, and
mixed agonist-antagonist nalorphine and nalbuphine. The acid–base microequilibria were
characterized by combining pH-potentiometry and deductive methods using synthesized auxiliary
compounds. Independent of the pH, there are approximately 4.8 times as many zwitterionic
microspecies than non-charged ones in nalbuphine solutions, while for nalorphine it is the non-charged
form that predominates by the same ratio. The non-charged microspecies is the dominant one also in
the case of hydromorphone, although its concentration exceeds only 1.3 times that of its zwitterionic
protonation isomer. The pH-independent partition coefficients of the individual microspecies were
determined by a combination of experimentally measured, pH-dependent, conditional distribution
constants and a custom-tailored evaluation method, using highly similar auxiliary compounds. The pH-
independent contribution of the zwitterionic microspecies to the distribution constant is 1380, 1070,
3160 and 72,440 times smaller than that of the inherently more lipophilic non-charged one for
hydromorphone, dihydromorphine, nalbuphine and nalorphine, respectively.
48
Interaction of anticancer drug clofarabine with human serum albumin and human α-1 acid
glycoprotein Spectroscopic and molecular docking approach
Mohammad Rehan Ajmal, Saima Nusrat, Parvez Alam, Nida Zaidi, Rizwan Hasan Khan
ABSTRACT
The binding interaction between clofarabine, an important anticancer drug and two important carrier
proteins found abundantly in human plasma, Human Serum Albumin (HSA) and α-1 acid glycoprotein
(AAG) was investigated by spectroscopic and molecular modeling methods. The results obtained from
fluorescence quenching experiments demonstrated that the fluorescence intensity of HSA and AAG is
quenched by clofarabine and the static mode of fluorescence quenching is operative. UV–vis
spectroscopy deciphered the formation of ground state complex between anticancer drug and the two
studied proteins. Clofarabine was found to bind at 298 K with both AAG and HSA with the binding
constant of 8.128 × 103 and 4.120 × 103 for AAG and HSA, respectively. There is stronger interaction
of clofarabine with AAG as compared to HSA. The Gibbs free energy change was found to be
negative for the interaction of clofarabine with AAG and HSA indicating that the binding process is
spontaneous. Binding of clofarabine with HSA and AAG induced ordered structures in both proteins
and lead to molecular compaction. Clofarabine binds to HSA near to drug site II. Hydrogen bonding
and hydrophobic interactions were the main bonding forces between HSA-clofarabine and AAG-
clofarabine as revealed by docking results. This study suggests the importance of binding of anticancer
drug to AAG spatially in the diseases like cancers where the plasma concentration of AAG increases
many folds. Design of drug dosage can be adjusted accordingly to achieve optimal treatment outcome.
A novel method for the determination of chemical purity and assay of menaquinone-7
Comparison with the methods from the official USP monograph
Łukasz Jedynak, Maria Jedynak, Magdalena Kossykowska, Joanna Zagrodzka
ABSTRACT
An HPLC method with UV detection and separation with the use of a C30 reversed phase analytical
column for the determination of chemical purity and assay of menaquinone-7 (MK7) in one
chromatographic run was developed. The method is superior to the methods published in the USP
Monograph in terms of selectivity, sensitivity and accuracy, as well as time, solvent and sample
consumption. The developed methodology was applied to MK7 samples of active pharmaceutical
ingredient (API) purity, MK7 samples of lower quality and crude MK7 samples before purification.
The comparison of the results revealed that the use of USP methodology could lead to serious
overestimation (up to a few percent) of both purity and MK7 assay in menaquinone-7 samples.
49
Structure and pharmaceutical formulation development of a new long-acting recombinant
human insulin analog studied by NMR and MS
Elżbieta Bednarek, Jerzy Sitkowski, Wojciech Bocian, Piotr Borowicz, Lech Kozerski
ABSTRACT
A monomer structure of novel human insulin analog A22S-B3K-B31R (SK3R) has been characterized
by NMR in water/acetonitrile solution and compared with the structure of human insulin (HIS)
established in the same medium. The composition of the oligomer ensemble for neat insulins in water
was qualitatively assessed by monitoring, derived from NMR experiment, translational diffusion
coefficient Di x 10−10 m2 s−1, whose value is a population averaged of individual coefficients for
species in oligomeric ensemble. Nanospray ESI/MS experiment was used to establish the masses of
oligomers in pharmaceutical formulation of the SK3R insulin. The pharmacodynamic data were
established and compared to insulin glargine characterized by the same profile of action in diabetics.
The oligomerization process of insulin during development of pharmaceutical formulation with
routinely used excipients has been studied using translation diffusion coefficient Di x 10−10 m2 s−1
established in water solution. These properties were compared with those of human insulin (HIS)
which is a standard reference for novel recombinant insulins.
Simultaneous HPLC assay for pretomanid (PA-824), moxifloxacin and pyrazinamide in an
inhaler formulation for drug-resistant tuberculosis
Mohammad A.M. Momin, Sim J. Thien, Woravimol Krittaphol, Shyamal C. Das
ABSTRACT
A simple and sensitive reversed phase HPLC method has been developed for the simultaneous
quantitation of pretomanid (PA-824), moxifloxacin and pyrazinamide in a combination spray-dried
powder formulation for inhalation, without any use of an internal standard. Good resolution of the
analytes was achieved on a Luna C18 (2), 150 × 4.6 mm, 5 μm, 100 Å column using gradient elution
with a mobile phase containing methanol and triethylamine phosphate buffer (pH 2.5) at a flow rate of
1.0 mL/min in a total run time of 25 min. Pyrazinamide, moxifloxacin and pretomanid (PA-824) were
detected at wavelengths (retention times) of 269 nm (3.80 min), 296 nm (7.94 min) and 330 nm (17.46
min), respectively. The assay was linear for all analytes in the concentration range 2.5–100 μg/mL
(correlation coefficients >0.999) with LODs and LLOQs (μg/mL) of pretomanid (PA-824) 0.51 and
1.56, moxifloxacin 0.06 and 0.19 and pyrazinamide 0.35 and 1.06, respectively. Recoveries of the
three drugs were 99.6–106.8% with intra- and inter-day precisions (as relative standard deviation) of
<7%. The method was successfully applied to an evaluation of content uniformity and freedom from
interference by l-leucine of a spray-dried combination powder for inhalation.
50
Levothyroxine sodium revisited: A wholistic structural elucidation approach of new impurities
via HPLC-HRMS/MS, on-line H/D exchange, NMR spectroscopy and chemical synthesis
M. Ruggenthaler, J. Grass, W. Schuh, C.G Huber, R.J. Reischl
ABSTRACT
The structural elucidation of unknown pharmaceutical impurities plays an important role in the quality
control of newly developed and well-established active pharmaceutical ingredients (APIs). The United
States Pharmacopeia (USP) monograph for the API Levothyroxine Sodium, a synthetic thyroid
hormone, features two high pressure liquid chromatography (HPLC) methods using UV-VIS
absorption detection to determine organic impurities in the drug substance. The impurity profile of the
first USP method (―Procedure 1‖) has already been extensively studied, however for the second
method (―Procedure 2‖), which exhibits a significantly different impurity profile, no wholistic
structural elucidation of impurities has been performed yet. Applying minor modifications to the
chromatographic parameters of USP ―Procedure 2‖ and using various comprehensive structural
elucidation methods such as high resolution tandem mass spectrometry with on-line hydrogen-
deuterium (H/D) exchange or two-dimensional nuclear magnetic resonance spectroscopy (NMR) we
gained new insights about the complex impurity profile of the synthetic thyroid hormone. This resulted
in the characterization of 24 compounds previously unknown to literature and the introduction of two
new classes of Levothyroxine Sodium impurities. Five novel compounds were unambiguously
identified via isolation or synthesis of reference substances and subsequent NMR spectroscopic
investigation. Additionally, Collision-Induced Dissociation (CID)-type fragmentation of identified
major impurities as well as neutral loss fragmentation patterns of many characterized impurities were
discussed.
Verification of the authenticity of drugs by means of NMR relaxometry—Viagra® as an example
S. Wilczyńki, M. Petelenz, M. Florek-Wojciechowska, S. Kulesza, D. Kruk
ABSTRACT
1H spin-lattice Nuclear Magnetic Resonance (NMR) relaxometry, vibrational spectroscopy and
Atomic Force Microscopy (AFM) have been applied to differentiate between original and counterfeit
Viagra®. The relaxation studies have been performed in a frequency range covering four orders of
magnitude, from 4 kHz to 40 MHz. It has been shown that for the counterfeit product the relaxation is
bi-exponential in the whole frequency range, while for the original Viagra® the relaxation process is
always single exponential. Thus, even a qualitative analysis of the relaxation data makes it possible to
identify the falsified medicine. Moreover, it has been demonstrated that vibrational spectroscopy does
not allow for differentiating between the products, while AFM studies are likely to lead one to
deceptive conclusions regarding the originality of the medicine. Furthermore, a quantitative analysis of
the relaxation data has been performed to describe in detail the relaxation properties of the original and
falsified products.
51
Metabolomics study of Populus type propolis
Boban AnĎelković, Ljubodrag Vujisić, Ivan Vučković, Vele Teńević, Dejan GoĎevac
ABSTRACT
Herein, we propose rapid and simple spectroscopic methods to determine the chemical composition of
propolis derived from various Populus species using a metabolomics approach. In order to correlate
variability in Populus type propolis composition with the altitude of its collection, NMR, IR, and UV
spectroscopy followed by OPLS was conducted. The botanical origin of propolis was established by
comparing propolis spectral data to those of buds of various Populus species. An O2PLS method was
utilized to integrate two blocks of data. According to OPLS and O2PLS, the major compounds in
propolis samples, collected from temperate continental climate above 500 m, were phenolic glycerides
originating from P. tremula buds. Flavonoids were predominant in propolis samples collected below
400 m, originating from P. nigra and P. x euramericana buds. Samples collected at 400–500 m were of
mixed origin, with variable amounts of all detected metabolites.
Hydrophilic interaction liquid chromatography method development and validation for the
assay of HEPES zwitterionic buffer
Xiaolong Xu, Bert Gevaert, Nathalie Bracke, Han Yao, Bart De Spiegeleer
ABSTRACT
HEPES is a zwitterionic buffer component used as a raw material in the GMP-manufacturing of
advanced therapy medicinal products (ATMPs), hence requiring an adequate assay method with
sufficient selectivity toward related impurities. Therefore, a hydrophilic interaction chromatography
(HILIC) method was developed. Different factors were investigated towards the retention behavior of
HEPES, its analogue EPPS and its starting material isethionate: pH, ion concentration and organic
solvent ratio of the mobile phase, as well as column temperature. Moreover, stress testing resulted in
the N-oxide degradant, identified by high resolution MS. The final method consisted of an isocratic
system with an aqueous (pH 2.0 with H3PO4) acetonitrile (35:65, v/v) mobile phase on a zwitterionic
HILIC (Obelisc N) column with a flow rate of 0.5 mL/min and UV detection at 195 nm. The assay
method of HEPES was validated, obtaining adequate linearity (R2 = 0.999), precision (RSD of 0.5%)
and accuracy (recovery of 100.08%). Finally, the applicability of the validated method was
demonstrated by analysis of samples from different suppliers.
52
Simultaneous analysis of glucocorticosteroid fluticasone propionate and its metabolite
fluticasone propionate 17β-carboxylic acid in human plasma by UPLC–MS/MS at sub pg/mL
level
Sneha G. Nair, Daxesh P. Patel, Mallika Sanyal, Puran Singhal, Pranav S. Shrivastav
ABSTRACT
A highly sensitive and rapid ultra performance liquid chromatography-tandem mass spectrometry
method has been developed for the simultaneous determination of fluticasone propionate (FP) and its
major metabolite, fluticasone propionate-17beta-carboxylic acid (FP 17β-CA) in human plasma. The
analytes and their deuterated internal standards, FP-d3 and FP 17β-CA-d3 were extracted from 500 μL
plasma samples by solid phase extraction on Oasis MAX cartridges. The chromatographic analysis
was performed on ACQUITY UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm) column using methanol-
acetonitrile (50:50, v/v) and 2.0 mM ammonium trifluroacetate (ATFA) (85:15, v/v) as the mobile
phase. Following separation of the analytes, protonated precursor → product ion transitions (FP: m/z
501.1 → 293.2, FP17β-CA: m/z 453.3 → 293.2, FP-d3: m/z 504.2 → 293.2, FP 17β-CA-d3: m/z 456.3
→ 293.2) were monitored on FP 17β-CA a triple quadrupole mass spectrometer, operating in multiple
reaction monitoring (MRM) and positive ionization mode. The calibration curves were established in
the range of 0.5–100 pg/mL with a correlation coefficient (r2) ≥ 0.9992 for both the analytes. The
intra-batch and inter-batch accuracy and precision varied from 95.5-103.4% and 0.74-5.06% across
quality controls for both the analytes. The mean assay recoveries for FP and FP 17β-CA were 84.2%
and 93.5% respectively. The validated method was successfully applied to support a bioequivalence
study of 200 μg FP, administered using nasal spray formulation in 18 healthy Indian subjects.
Reproducibility of the method was assessed by reanalysis of 98 incurred study samples.
Fast Screening of Tissue Samples for Glycogen
Raluca-Ioana Stefan-van Staden, Amalia Gabriela Diaconeasa, Camelia Stanciu-Gavan
ABSTRACT
Screening of tissue samples for glycogen is very important in assessing the ageing, but also the state of
health of the tissue. Therefore, two needle stochastic sensors based on maltodextrins presenting
different dextrose equivalence (DE) MD-m (DE 13.0-17.0), and MD-M (DE 16.5-19.5) immobilized
in diamond paste (obtained from synthetic diamond and paraffin oil) were designed and characterized.
These stochastic sensors were used reliable for both qualitative and quantitative analysis for the assay
of glycogen in tissue samples with limits of determination as low as 1 fmol L−1. select article Urinary
metabolic profiling of cisplatin nephrotoxicity and nephroprotective effects of <em>Orthosiphon
stamineus</em> leaves elucidated by <sup>1</sup>H NMR spectroscopy
53
Urinary metabolic profiling of cisplatin nephrotoxicity and nephroprotective effects of
Orthosiphon stamineus leaves elucidated by 1H NMR spectroscopy
Raghunath Pariyani, Intan Safinar Ismail, Amalina Azam, Alfi Khatib, Hazilawati Hamza
ABSTRACT
Orthosiphon stamineus (OS) is a popular medicinal herb used in traditional Chinese medicine as a
diuretic agent and for renal system disorders. This study employed 1H NMR based metabolomics
approach to investigate the possible protective activity of OS in cisplatin induced nephrotoxicity owing
to its diuretic and antioxidant activities. Aqueous (OSAE) and 50% aqueous ethanolic (OSFE) extracts
of OS leaves were orally administered at 400 mg/kg BW doses to rats which were then
intraperitoneally injected with cisplatin at 5 mg/kg BW dose. The 1H NMR profile of the urine
samples collected on day 5 after cisplatin administration were analyzed by multivariate pattern
recognition techniques, whereby 19 marker metabolites suggestive in the involvement of TCA cycle,
disturbed energy metabolism, altered gut microflora and BCAA metabolism pathways were identified.
It was observed that OSFE caused significant changes (p < 0.05) in the levels of 8 markers namely
leucine, acetate, hippurate, lysine, valine, 2-oxoglutarate, 3-HBT and acetoacetate resulting in a
moderate ameliorative effect, however, it did not completely protect from nephrotoxicity. OSAE did
not demonstrate significant down regulatory effects on any markers, albeit, it potentiated the cisplatin
nephrotoxicity by inducing significant increase in glucose, glycine, creatinine, citrate, TMAO, acetate
and creatine levels. A Principal Component Analysis (PCA) of the 1H NMR spectra of OS extracts
identified that OSFE had higher concentrations of the secondary metabolites such as caffeic acid,
chlorogenic acid, protocatechuic acid and orthosiphol, among others. Whereas, OSAE was
characterized by higher concentrations of acetate, lactate, succinic acid, valine and
phosphatidylcholine. This research denotes the first comprehensive analysis to identify the effects of
OS extracts on cisplatin nephrotoxicity.
54
Preparation, characterization and in vivo evaluation of a formulation of dantrolene sodium with
hydroxypropyl-β-cyclodextrin
Mengmeng Chen, Qijuan Wu, Juan Jiang, Xin Jin, Chunjie Zhao
ABSTRACT
Dantrolene sodium (Da) is an effective skeletal muscle relaxant. However, its pharmacological effects
are severely limited owing to its poor solubility and low oral bioavailability. In order to solve these
problems, an inclusion complex using hydroxypropyl-β-cyclodextrin (HP-β-CD) to improve the oral
bioavailability of Da was prepared successfully by freeze-drying. The prepared complex was
characterized by Powder X-ray diffractometry (PXRD), Fourier transform infrared spectroscopy
(FTIR) and evaluated by a dissolution test and a pharmacokinetic study. The results of PXRD and
FTIR proved the formation of a complex between Da and HP-β-CD. The dissolution rate of Da was
markedly improved from inclusion complex with more than 90% being released within 5 min. The in
vivo pharmacokinetics of Da and dantrolene sodium-hydroxypropyl-β-cyclodextrin (Da-HP-β-CD)
inclusion complex were investigated in rats using a UPLC/MS/MS method. The Cmax and AUC0-t of
the Da-HP-β-CD inclusion complex were 5- and 3-fold higher than that of the Da. These results
suggested that the Da-HP-β-CD inclusion complex markedly improved the dissolution rate and
bioavailability of Da. select article Promotion of classic neutral bile acids synthesis pathway is
responsible for cholesterol-lowing effect of Si-miao-yong-an decoction: Application of LC–MS/MS
method to determine 6 major bile acids in rat liver and plasma
Promotion of classic neutral bile acids synthesis pathway is responsible for cholesterol-lowing
effect of Si-miao-yong-an decoction: Application of LC–MS/MS method to determine 6 major
bile acids in rat liver and plasma
Ziying Liu, Yu Zhang, Ruowen Zhang, Liqiang Gu, Xiaohui Chen
ABSTRACT
Si-miao-yong-an decoction (SMYAD), a traditional Chinese medicine formula, significantly reduced
plasma TC, LDL-c levels and increased HDL-c level in hyperlipidemia rats. Liver function test and
tissue section examination indicated that SMYAD improved liver function and reduced fat
accumulation in hyperlipidemia rat liver. A LC–MS/MS method was established and well validated to
evaluate major bile acids derived from cholesterol metabolism through the classic neutral pathway and
the alternative acidic pathway (cholic acid, chenodeoxycholic acid and their taurine and glycine
conjugates) in liver and plasma. Increased total 6 bile acids concentrations in both liver and plasma
were observed after oral administration of 12 g/kg/d, 24 g/kg/d and 36 g/kg/d of SMYAD in a dose
dependent manner which contributed to eliminate of cholesterol. Cholic acid, taurocholic acid and
glycocholic acid act as the main products of bile acid classic neutral synthesis pathway and show sharp
increase (p < 0.01) after treatment of SMYAD at dosage of 24–36 g/kg/d. For liver samples,
taurocholic acid level act as the largest growth section, while in plasma samples, cholic acid act as the
largest growth section after SMYAD treatment, compared with Model group. By contrast, the main
products of alternative acidic pathway (chenodeoxycholic acid and its glycine and taurine conjugates)
show no significant increase after treatment of SMYAD. In conclusion, the cholesterol lowing effect of
SMYAD may be related with the accelerated transformation of cholesterol into bile acids through the
classic neutral pathway.
55
Analysis of amino acid and monoamine neurotransmitters and their metabolites in rat urine of
Alzheimer’s disease using in situ ultrasound-assisted derivatization dispersive liquid-liquid
microextraction with UHPLC–MS/MS
Xian-En Zhao, Yongrui He, Meng Li, Guang Chen, Jinmao You
ABSTRACT
Neurotransmitters (NTs) may play an important role in neurodegenerative disorders such as
Alzheimer‘s disease (AD). In order to investigate the potential links, a new simple, fast, accurate and
sensitive analytical method, based on in situ ultrasound-assisted derivatization dispersive liquid-liquid
microextraction (in situ UA-DDLLME) coupled with ultra high-performance liquid chromatography
tandem mass spectrometry (UHPLC–MS/MS), has been developed and validated. The quantitation of
amino acid neurotransmitters (AANTs) and monoamine neurotransmitters (MANTs) in urine of AD
rats were performed in this work. The in situ UA-DDLLME procedure involved the rapid injection of
the mixture of low toxic 4-bromoanisole (extractant) and acetonitrile (dispersant), which containing
the new designed and synthesized 4′-carbonyl chloride rosamine (CCR) as derivatization reagent, into
the aqueous phase of real sample and buffer. Under the selected conditions, the derivatization and
microextraction of analytes were simultaneously completed within 1 min. Good linearity for each
analyte (R > 0.992) was observed with low limit of detections (LODs, S/N > 3). Moreover, the
proposed method was compared with direct detection or other reported methods, and the results
showed that low matrix effects and good recoveries results were obtained in this work. Taken together,
in situ UA-DDLLME coupled with UHPLC–MS/MS analysis was demonstrated to be a good method
for sensitive, accurate and simultaneous monitoring of AANTs and MANTs. This method would be
expected to be highly useful in AD diseases‘ clinical diagnostics and may have potential value in
monitoring the efficacy of treatment.
Rapid determination of alkaloids in Macleaya cordata using ionic liquid extraction followed by
multiple reaction monitoring UPLC–MS/MS analysis
Linqiu Li, Mingyuan Huang, Junli Shao, Bokun Lin, Qing Shen
ABSTRACT
The ultrasonic-assisted extraction (UAE) and ionic liquid based dispersive liquid–liquid
microextraction (IL-DLLME) have been successfully applied in extracting of six alkaloids from M.
cordata. 1-hexyl-3-methylimidazolium tetrafluoroborate ([C6MIM][BF4]) aqueous solution was used
as extraction solvent. The target analytes in raw material were deposited into a single drop of 1-hexyl-
3-methylimidazolium hexafluorophosphate ([C6MIM][PF6]), which was in situ formed by mixing
[C6MIM][BF4] and potassium hexafluorophosphate ([K][PF6]. Afterwards, the extract was analyzed
by ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) in multiple-
reaction monitoring (MRM) mode. The proposed method was fully validated in terms of linearity
(0.9983–0.9992), LOD (0.080 ng mL−1), LOQ (0.25 ng mL−1), intra-day precision (<5.46%), inter-
day precision (<6.36%), and recovery (86.42–112.48%). The results indicate that the approach of
combining IL-DLLME with UPLC–MS/MS is powerful and practical for analyzing alkaloids in M.
cordata., and it also has great potential for comprehensive quality control of other herbal medicines.
56
Volumetric absorptive microsampling (VAMS) as an alternative to conventional dried blood
spots in the quantification of miltefosine in dried blood samples
A.E. Kip, K.C. Kiers, H. Rosing, J.H.M. Schellens, T.P.C. Dorlo
ABSTRACT
Miltefosine is an oral agent against the neglected tropical disease leishmaniasis, which is mostly
endemic in resource-poor areas. Dried blood spot (DBS) sampling is an attractive alternative to plasma
sampling for pharmacokinetic studies in these remote areas, but introduces additional variability in
analyte quantification due to possible blood spot inhomogeneity and variability in blood spot volume
and haematocrit values. Volumetric absorptive microsampling (VAMS) potentially overcomes a few
of these issues as the VAMS device absorbs a fixed volume that is processed as a whole. We
developed and validated an LC–MS/MS method for the quantification of miltefosine with this novel
sampling technique with good performance in terms of linearity, selectivity, accuracy (bias within
±10.8%), precision (CV% ≤ 11.9%), recovery, carry-over and matrix effect. VAMS samples were
stable for at least one month at room temperature and 37 °C. The impact of haematocrit on assay
accuracy was reduced compared to conventional DBS sampling, but indicated a declining recovery
with increased haematocrit due to haematocrit dependency in recovery from the sampling device. A
clinical validation will be required to investigate whether VAMS is an appropriate and cost-effective
alternative sampling method to conventional DBS sampling.
The integration of GC–MS and LC–MS to assay the metabolomics profiling in Panax ginseng
and Panax quinquefolius reveals a tissue- and species-specific connectivity of primary
metabolites and ginsenosides accumulation
Jia Liu, Yang Liu, Yu Wang, Ann Abozeid, Zhong-Hua Tang
ABSTRACT
The traditional medicine Ginseng mainly including Panax ginseng and Panax quinquefolius is the most
widely consumed herbal product in the world. Despite the extensive investigation of biosynthetic
pathway of the active compounds ginsenosides, our current understanding of the metabolic interlink
between ginsenosides synthesis and primary metabolism at the whole-plant level. In this study, the
tissue-specific profiling of primary and the secondary metabolites in two different species of ginseng
were investigated by gas chromatography- and liquid chromatography coupled to mass spectrometry.
A complex continuous coordination of primary- and secondary-metabolic network was modulated by
tissues and species factors during growth. The results showed that altogether 149 primary compounds
and 10 ginsenosides were identified from main roots, lateral roots, stems, petioles and leaves in P.
ginseng and P. quinquefolius. The partial least squares-discriminate analysis (PLS-DA) revealed
obvious compounds distinction among tissue-specific districts relative to species. To survey the
dedication of carbon and nitrogen metabolism in different tissues to the accumulation of ginsenosides,
we inspected the tissue-specific metabolic changes. Our study testified that the ginsenosides content
was dependent on main roots and lateral roots energy metabolism, whereas independent of leaves and
petiole photosynthesis during ginsenosides accumulation. When tow species were compared, the
results indicated that high rates of C assimilation to C accumulation are closely associated with
ginsenosides accumulation in P. ginseng main roots and P. quinquefolius lateral roots, respectively.
Taken together, our results suggest that tissue-specific metabolites profiling dynamically changed in
process of ginsenosides biosynthesis, which may offer a new train of thoughts to the mechanisms of
the ginsenosides biosynthesis at the metabolite level.
57
Hierarchical identification of bioactive components in a medicinal herb by preparative high-
performance liquid chromatography and selective knock-out strategy
Cheng Qu, Lin-Yan Wang, Hang Lin, Er-Xin Shang, Jin-Ao Duan
ABSTRACT
A novel and generally applicable approach was established to hierarchically identify the bioactive
components of a medicinal herb by preparative high-performance liquid chromatography (prep-HPLC)
and a selective knock-out strategy. In this study, the targeted components of an herbal medicine were
separated and knocked out using prep-HPLC. Subsequently, the contributions of the different target
components to the overall effect of the medicinal herb were comparatively evaluated and differentiated
by a heat map and a 3D score plot. This approach was successfully applied to investigate the bioactive
constituents of safflower. The contributions of 11 components to the overall effect of safflower were as
follows: anhydrosafflor yellow B (10) > 6-hydroxykaempferol 3,6-di-O-β-d-glucoside (8) >
hydroxysafflor yellow A (3) > kaempferol 3-O-β-rutinoside (11) > 6-hydroxykaempferol 3-O-β-
rutinoside (9) > 6-hydroxykaempferol 3,6-di-O-β-d-glucoside-7-O-β-d-glucuronide (4) > 6-
hydroxyapigenin 6-O-β-d-glucoside-7-O-β-d-glucuronide (6) > cytidine (1) > 6-hydroxykaempferol 3-
O-β-rutinoside-6-O-β-d-glucoside (7) > 6-hydroxykaempferol 3,6,7-tri-O-β-d-glucoside (5) >
adenosine (2). These results demonstrate that quinochalcone C-glycosides (3 and 10) and some
flavonoid glycosides containing C7-OH (such as 8, 9 and 11) made a greater contribution to the overall
effect of safflower than the other components that were knocked out. The results provided an
important reference for improving quality control and further development of safflower products. And
this approach should also be useful for investigating the bioactive constituents of other medicinal
herbs.
Volume 136, March 2017
Metabolomics: A potential way to know the role of vitamin D on multiple sclerosis
Diego Luque-Córdoba, María D. Luque de Castro
ABSTRACT
The literature about the influence of vitamin D on multiple sclerosis (MS) is very controversial,
possibly as a result of the way through which the research on the subject has been conducted. The
studies developed so far have been focused exclusively on gene expression: the effect of a given
vitamin D metabolite on target receptors. The influence of the vitamin D status (either natural or after
supplementation) on MS has been studied by measurement of the 25 monohydroxylated metabolite
(also known as circulating form), despite the 1,25 dihydroxylated metabolite is considered the active
form. In the light of the multiple metabolic pathways in which both forms of vitamin D (D2 and D3)
are involved, monitoring of the metabolites is crucial to know the activity of the target enzymes as a
function of both the state of the MS patient and the clinical treatment applied. The study of
metabolomics aspects is here proposed to clarify the present controversy. In ―omics‖ terms, our
proposal is to take profit from up-stream information—thus is, from metabolomics to genomics—with
a potential subsequent step to systems biology, if required.
58
Impact of space environment on stability of medicines: Challenges and prospects
Priti Mehta, Dhara Bhayani
ABSTRACT
To upkeep health of astronauts in a unique, isolated, and extreme environment of space is the primary
goal for a successful space mission, hence, safe and efficacious medications are essential for the
wellness of astronauts. Space medication has been challenged with problems related to efficacy. Along
with altered physiology, one of the possible reasons could be instability of space medications in the
presence of harsh spaceflight environmental conditions. Altered physical and chemical stability can
result in reduced potency which can result in reduced efficacy. Right now, medicines from the
International Space Station are replaced before their expiration. But, for longer duration missions to
Mars or any other asteroid, there will not be any chance of replacement of medicines. Hence, it is
desired that medicines maintain the shelf-life throughout the space mission. Stability of medicines used
for short term or long term space missions cannot be judged by drug stability guidelines based on
terrestrial environmental factors. Unique environmental conditions related to spaceflight include
microgravity, excessive vibration, hard vacuum, humidity variation, temperature differences and
excessive radiation, which may cause instability of medicines. This write-up provides a review of the
problem and countermeasure approaches for pharmaceuticals exposed to the space environment. The
first part of the article discusses thought processes behind outlining of International Conference on
Harmonization drug stability guidelines, Q1A (R2) and Q1B, and its acceptance limits for accelerated
stability study. The second part of the article describes the difference in the radiation environment of
deep space compared to radiation environment inside the space shuttle based on penetration power of
different types of radiation. In the third part of the article, various promising approaches are listed
which can be used for assurance of space medicine stability. One of the approaches is the use of
ground-based space simulation analogues and statistical treatment to data to calculate failure rate of
drugs and probabilistic risk assessment. Another approach is to innovate storage and packaging
technology using radiation hardens polymer or using cryogenic temperatures.
Evaluation of size-exclusion chromatography for the analysis of phosphorothioate
oligonucleotides
Atsuko Shimoyama, Aki Fujisaka, Satoshi Obika
ABSTRACT
We evaluated size exclusion chromatography (SEC) for the detection of high-order structure of
phosphorothioate oligonucleotides (PS-oligo). Because of strong interaction between PS-oligo and
column packing material, peaks were broader and elution time was longer than those of the
corresponding natural DNA oligonucleotides. However, single- and double-stranded structures of PS-
oligo were clearly separated and discriminated, while single-stranded with high-order structures such
as G-quadruplex and hairpin structure were not distinguished from each other. select article Molecular
insight into atypical instability behavior of fixed-dose combination containing amlodipine besylate and
losartan potassium.
59
Molecular insight into atypical instability behavior of fixed-dose combination containing
amlodipine besylate and losartan potassium
Tarun Handa, Shalu Jhajra, Shweta Bhagat, P.V. Bharatam, Saranjit Singh
ABSTRACT
Combination therapy with the use of fixed-dose combinations (FDCs) is evincing increasing interest of
prescribers, manufacturers and even regulators, evidently due to the primary benefit of improved
patient compliance. However, owing to potential of drug-drug interaction, FDCs require closer
scrutiny with respect to their physical and chemical stability. Accordingly, the purpose of the present
study was to explore stability behavior of a popular antihypertensive combination of amlodipine
besylate (AML) and losartan potassium (LST). Physical mixtures of the two drugs and multiple
marketed formulations were stored under accelerated conditions of temperature and humidity (40
°C/75% RH) in a stability chamber and samples were withdrawn after 1 and 3 months. The physical
changes were observed visibly, while chemical changes were monitored by HPLC employing a
method that could separate the two drugs and all other components present. The combination revealed
strong physical instability and also chemical degradation of AML in the presence of LST.
Interestingly, three isomeric interaction products of AML were formed in the combination, which
otherwise were reported in the literature to be generated on exposure of AML free base above its
melting point. The same unusual products were even formed when multiple marketed FDCs were
stored under accelerated conditions outside their storage packs. However, these were absent when
AML alone was stored in the same studied conditions. Therefore, reasons for physical and chemical
incompatibility and the mechanism of degradation of AML in the presence of LST were duly explored
at the molecular level. The outcomes of the study are expected to help in development of stable FDCs
of the two drugs.
Qualification of HSQC methods for quantitative composition of heparin and low molecular
weight heparins
Lucio Mauri, Giovanni Boccardi, Giangiacomo Torri, Michael Karfunkle, Marco Guerrini
ABSTRACT
An NMR HSQC method has recently been proposed for the quantitative determination of the mono-
and disaccharide subunits of heparin and low molecular weight heparins (LMWH). The focus of the
current study was the validation of this procedure to make the 2D-NMR method suitable for
pharmaceutical quality control applications. Pre-validation work investigated the effects of several
experimental parameters to assess robustness and to optimize critical factors. Important experimental
parameters were pulse sequence selection, equilibration interval between pulse trains and temperature.
These observations were needed so that the NMR method was sufficiently understood to enable
continuous improvement. A standard validation study on heparin then examined linearity,
repeatability, intermediate precision and limits of detection and quantitation; selected validation
parameters were also determined for LMWH.
60
Selecting optimal columns for clarithromycin impurity analysis according to the quantitative
relationship of hydrophobic subtraction model
Xia Zhang, Changqin Hu
ABSTRACT
Hydrophobic subtraction model (HSM) is widely applied to select columns of equivalent or different
selectivity compared with a reference column, but its application in identifying optimal columns for
specific separations of real samples is rare. In this work, a column selection method was proposed by
firstly directly correlating separation selectivity of different pairs of solutes to column parameters
based on the quantitative relationship of HSM and then selecting the optimal columns according to the
predicted selectivity in consideration of the total separation of all critical pairs of solutes. Three critical
pairs of solutes in clarithromycin impurity analysis were evaluated as examples. Starting with the
analysis of clarithromycin impurities on 15 columns with different selectivities, ten optimal columns
were finally identified for clarithromycin impurity analysis from the HSM column characterization
database containing nearly 600 columns and two of them were validated with satisfactory separations
for all critical peak pairs. The proposed methodology was also compared to the traditional column
selection procedure based on calculations of scalar measures of the Euclidean distance between
chromatographic columns. Results showed that our method provides an effective way to find the
desired columns that may be overlooked by the traditional column selection due to selection of an
inappropriate reference column or overestimation of column similarity, such as Fs introduced in HSM.
Efficacy of metformin in human single hair fibre by ATR-FTIR spectroscopy coupled with
statistical analysis
Kamatchi Sundaramoorthi, Gunasekaran Sethu, Sailatha Ethirajulu, Pavithra Raja Marthandam
ABSTRACT
Diabetes mellitus is chronic metabolic disorder, resulting from insulin deficiency, characterized by
hyperglycemia altered metabolism of carbohydrates, proteins and lipids and an increased risk of
vascular complications. There are different classes of anti-diabetic drugs in allopathic system of
medicine. Metformin (dimethyl biguanide) is a blood glucose lowering agent used in the treatment of
non-insulin dependent diabetes mellitus. Almost in all diseases the blood serves as the primary
metabolic transport system in the body. Its composition is the preferred indicator with respect to the
pathophysiological condition of the patient. Instead of analyzing blood to diagnose diabetes, hair could
be used to detect diabetes using FTIR-ATR technique. The most important components of hair are
fibrous proteins (keratins), melanins, glycogen, and lipids. Hair follicles are located 3–4 mm below the
surface of the skin and are surrounded by rich blood capillary system. In the present study, ten diabetic
subjects were considered to evaluate the efficacy of metformin hydrochloride for the treatment of
diabetes mellitus using FTIR-ATR spectroscopy. The spectra of diabetic hair fibre samples have been
recorded in the mid infrared region of 4000–450 cm−1. The hair samples of the diabetic subjects
before medication were taken as pre-treatment samples. The hair samples of diabetic subjects referred
to medication with metformin for a period of three month were taken as post-treatment sample. Some
remarkable spectral differences were elucidated between pre- and post-treatment hair fibre samples. A
comparative study on the FTIR-ATR hair spectra of patients (pre- and post-treatment) along with the
healthy subjects has been made.
61
Primer design for SNP genotyping based on allele-specific amplification—Application to organ
transplantation pharmacogenomics
Luis A. Tortajada-Genaro, Rosa Puchades, Ángel Maquieira
ABSTRACT
Diagnostic methods based on single nucleotide polymorphism (SNP) biomarkers are essential for the
real adoption of personalized medicine. Allele specific amplification in a homogeneous format or
combined to microarray hybridization are powerful approaches for SNP genotyping. However, primers
must be properly selected to minimize cross-reactivity, dimer formation and nonspecific hybridization.
This study presents a design workflow diagram for the selection of required oligonucleotides for
multiplex assays. Based on thermodynamic restrictions, the oligonucleotide sets are chosen for a
specific amplification of wild- and mutant-type templates. Design constraints include the structural
stability of primer-template duplexes, template-probe duplexes and self-annealing complexes or
hairpins for each targeted gene. The performance of the design algorithm was evaluated for the
simultaneous genotyping of three SNPs related to immunosuppressive drugs administered after solid
organ transplantation. The assayed polymorphisms were rs1045642 (ABCB1 gene), rs1801133
(MTHFR gene) and rs776746 (CYP3A5 gene). Candidates were confirmed by discriminating
homozygote and heterozygote populations using a fluorescence solution method and two colorimetric
microarray methods on polycarbonate chips. The analysis of patient samples provided excellent
genotyping results compared to those obtained by a reference method. The study demonstrates that the
development of the allele-specific methods as pharmacogenetic tools can be simplified.
Development and validation of a liquid chromatography–mass spectrometric assay for
simultaneous determination of tacrolimus and 13-O-desmethyl tacrolimus in rat kidney tissue
Tatian Kirresh, Sony Tuteja, D. Russo, P.D. Brophy, D.J. Murry
ABSTRACT
A sensitive and robust LC–MS/MS method has been developed and validated to determine the
concentrations of tacrolimus and its major metabolite 13-O-desmethyl tacrolimus (13-ODMT) in
kidney tissue from rats who received tacrolimus intra-peritoneally at doses of 0.5 mg/kg and 2 mg/kg.
The samples were prepared by a liquid-liquid extraction procedure using ethyl ether as the extraction
solvent and ascomycin as the internal standard. Chromatographic separation was achieved using
Phenomenex Kinetex column (2.6 μm C18 100 Å, 100 × 2.1 mm, Phenomenex, Torrance CA) and a
gradient mobile phase of water and methanol-acetonitrile (50:50, v/v) both containing 0.1% formic
acid. The limit of quantification was 0.25 ng/ml and the calibration curves covered a concentration
range from 0.25 to 50 ng/ml. Intra-and inter-assay precision and accuracy for both tacrolimus and 13-
ODMT were all within FDA guidelines for bioanalysis. Extraction efficiency for tacrolimus ranged
from 67.00 to 74.90% and from 66.70 to 78.40% for 13-ODMT. Several challenges interfering with
the performance of the method such as phospholipid build-up have also been addressed. Kidney tissue
samples from six rats receiving either 0.5 or 2 mg/kg dose were analyzed and resulted in a median
concentration of 11.54 and 0.72 ng/ml for tacrolimus and 13-ODMT, respectively, for the lower dose
level, and a median concentration of 8.89 ng/ml and 1.50 ng/ml for tacrolimus and 13-ODMT,
respectively, at the higher dose level.
62
Towards interference free HPLC-SERS for the trace analysis of drug metabolites in biological
fluids
Waleed A. Hassanain, Emad L. Izake, Arumugam Sivanesan, Godwin A. Ayoko
ABSTRACT
Sofosbuvir metabolite, 2′-deoxy-2′-fluoro-2′-C-methyluridine (PSI-6206) was studied for the first time
by surface enhanced Raman spectroscopy (SERS) using the paper-based SERS substrate. The
quantification limit of PSI-6206 by SERS was found to be 13 ng L−1 (R2 value = 0.959, RSD =
5.23%). For the structural and quantitative analysis of PSI-6206 in blood plasma, an interference-free
HPLC-SERS method was developed and compared to HPLC-DAD and HPLC–MS methods. The
SERS quantification of the drug by the paper substrate was 4 orders of magnitude more sensitive than
that by the diode array detector. In addition, the SERS detection provided unique structural
identification of the drug in blood plasma, similar to Mass spectroscopy detector. Due to the
disposable nature of the SERS substrate, the new method does not suffer from the known ―memory
effect‖ which is known to lead to false positive identification in traditional HPLC-SERS methods.
Therefore, the presented HPLC-paper SERS platform holds great potential for the sensitive and cost
effective determination of drugs and their metabolites in biological fluids.
1H NMR-based metabolomics study of liver damage induced by ginkgolic acid (15:1) in mice
Lei Jiang, Zhi-Hong Si, Ming-Hui Li, He Zhao, Jun-Song Wang
ABSTRACT
Ginkgolic acid (15:1) is a major toxic component in extracts obtained from Ginkgo biloba (EGb) that
has allergic and genotoxic effects. This study is the first to explore the hepatotoxicity of ginkgolic acid
(15:1) using a NMR (nuclear magnetic resonance)-based metabolomics approach in combination with
biochemistry assays. Mice were orally administered two doses of ginkgolic acid (15:1), and mouse
livers and serum were then collected for NMR recordings and biochemical assays. The levels of
activity of alanine aminotransferase (ALT) and glutamic aspartate transaminase (AST) observed in the
ginkgolic acid (15:1)-treated mice suggested that it had induced severe liver damage. An orthogonal
signal correction partial least-squares discriminant analysis (OSC-PLSDA) performed to determine the
metabolomic profile of mouse liver tissues indicated that many metabolic disturbances, especially
oxidative stress and purine metabolism, were induced by ginkgolic acid (15:1). A correlation network
analysis combined with information related to structural similarities further confirmed that purine
metabolism was disturbed by ginkgolic acid (15:1). This mechanism might represent the link between
the antitumour activity and the liver injury-inducing effect of ginkgolic acid (15:1). A SUS (Shared
and Unique Structure) plot suggested that a two-dose treatment of ginkgolic acid (15:1) had generally
the same effect on metabolic variations but that its effects were dose-dependent, revealing some of the
common features of ginkgolic acid (15:1) dosing. This integrated metabolomics approach helped us to
characterise ginkgolic acid (15:1)-induced liver damage in mice.
63
Dose-response characteristics of Clematis triterpenoid saponins and clematichinenoside AR in
rheumatoid arthritis rats by liquid chromatography/mass spectrometry-based serum and urine
metabolomics
Rui Li, Lin-Xiu Guo, Yi Li, Wen-Qi Chang, Gui-Zhong Xin
ABSTRACT
Clematidis Radix et Rhizoma is a traditional Chinese medicine widely used for treating arthritic
disease. Clematis triterpenoid saponins (TS) and clematichinenoside AR (C-AR) have been considered
to be responsible for its antiarthritic effects. However, the underling mechanism is still unclear because
of their low bioavailability. To address of this issue, metabolomics tools were performed to determine
metabolic variations associated with rheumatoid arthritis (RA) and responses to Clematis TS, C-AR
and positive drug (Triptolide, TP) treatments. This metabolomics investigation of RA was conducted
in collagen-induced arthritis (CIA) rats. Liquid chromatography/mass spectrometry and multivariate
statistical tools were used to identify the alteration of serum and urine metabolites associated with RA
and responses to drug treatment. As a result, 45 potential metabolites associated with RA were
identified. After treatment, a total of 24 biomarkers were regulated to normal like levels. Among these,
PC(18:0/20:4), 9,11-octadecadienoic acid, arachidonic acid, 1-methyladenosine, valine, hippuric acid
and pantothenic acid etc, were reversed in Clematis TS and C-AR groups. Tetrahydrocortisol was
regulated to normal levels in Clematis TS and TP groups, while 3,7,12-trihydroxycholan-24-oic acid
was regulated in C-AR and TP groups. Biomarkers like citric acid, p-cresol glucuronide, creatinine,
cortolone were reversed in TP group.
Enhancing analysis throughput, sensitivity and specificity in LC/ESI–MS/MS assay of plasma
25-hydroxyvitamin D3 by derivatization with triplex 4-(4-dimethylaminophenyl)-1,2,4-triazoline-
3,5-dione (DAPTAD) isotopologues
Shoujiro Ogawa, Hiroki Kittaka, Akiho Nakata, Kenji Komatsu, Tatsuya Higashi
ABSTRACT
The plasma/serum concentration of 25-hydroxyvitamin D3 [25(OH)D3] is a diagnostic index for
vitamin D deficiency/insufficiency, which is associated with a wide range of diseases, such as rickets,
cancer and diabetes. We have reported that the derivatization with 4-(4-dimethylaminophenyl)-1,2,4-
triazoline-3,5-dione (DAPTAD) works well in the liquid chromatography/electrospray ionization-
tandem mass spectrometry (LC/ESI–MS/MS) assay of the serum/plasma 25(OH)D3 for enhancing the
sensitivity and the separation from a potent interfering metabolite, 3-epi-25-hydroxyvitamin D3 [3-epi-
25(OH)D3]. However, enhancing the analysis throughput remains an issue in the LC/ESI–MS/MS
assay of 25(OH)D3. The most obvious restriction of the LC/MS/MS throughput is the
chromatographic run time. In this study, we developed an enhanced throughput method for the
determination of the plasma 25(OH)D3 by LC/ESI–MS/MS combined with the derivatization using the
triplex (2H0-, 2H3- and 2H6-) DAPTAD isotopologues. After separate derivatization with 1 of 3
different isotopologues, the 3 samples were combined and injected together into LC/ESI–MS/MS.
Based on the mass differences between the isotopologues, the derivatized 25(OH)D3 in the 3 different
samples were quantified within a single run. The developed method tripled the hourly analysis
throughput without sacrificing assay performance, i.e., ease of pretreatment of plasma sample (only
deproteinization), limit of quantification (1.0 ng/mL when a 5 μL-plasma was used), precision (intra-
assay RSD ≤ 5.9% and inter-assay RSD ≤ 5.5%), accuracy (98.7–102.2%), matrix effects, and
capability of separating from an interfering metabolite, 3-epi-25(OH)D3.
64
A highly sensitive quantum dots-DNA nanobiosensor based on fluorescence resonance energy
transfer for rapid detection of nanomolar amounts of human papillomavirus 18
Mojtaba Shamsipur, Vahid Nasirian, Kamran Mansouri, Ali Barati, Soheila Kashanian
ABSTRACT
A very sensitive and convenient nanobiosensor based on fluorescence resonance energy transfer
(FRET) was developed for the detection of a 22-mer oligonucleotides sequence in Human
Papillomavirus 18 virus (HPV18) gene. For this purpose, water-soluble CdTe quantum dots (QDs)
were synthesized and, subsequently, amino-modified 11-mer oligonucleotide as one of the two
necessary probes was attached to QDs surface to form functional QDs-DNA conjugates. Right after
addition of the QDs-DNA and a second Cyanine5 (Cy5)-labeled 11-mer oligonucleotide probe to the
DNA target solution, the sandwiched hybrids were formed. The resulting hybridization brings the Cy5
fluorophore as the acceptor to close proximity of the QDs as donor, so that an effective transfer of
energy from the excited QDs to the Cy5 probe would occur via FRET processing. The fluorescence
intensity of Cy5 found to linearly enhance by increasing the DNA target concentration from 1.0 to 50.0
nM, with a detection limit of 0.2 nM. This homogeneous DNA detection method does not require
excessive washing and separation steps of un-hybridized DNA, due to the fact that no FRET can be
observed when the probes are not ligated. Finally, feasibility and selectivity of the proposed one-spot
DNA detection nanobiosensor were investigated by analysis of derived nucleotides from HPV18 and
mismatched sequences.
Development and validation of stability indicating HPLC methods for related substances and
assay analyses of amoxicillin and potassium clavulanate mixtures
Esen Bellur Atici, Yücel Yazar, Çağan Ağtaş, Nurten Ridvanoğlu, Bekir Karlığa
ABSTRACT
Antibacterial combinations consisting of the semisynthetic antibiotic amoxicillin (amox) and the β-
lactamase inhibitor potassium clavulanate (clav) are commonly used and several chromatographic
methods were reported for their quantification in mixtures. In the present work, single HPLC method
for related substances analyses of amoxicillin and potassium clavulanate mixtures was developed and
validated according to international conference on harmonization (ICH) guidelines. Eighteen
amoxicillin and six potassium clavulanate impurities were successfully separated from each other by
using triple gradient elution using a Thermo Hypersil Zorbax BDS C18 (250 mm × 4.6 mm, 3 μm)
column with 50 μL injection volumes at a wavelength of 215 nm. Commercially unavailable impurities
were formed by degradation of amoxicillin and potassium clavulanate, identified by LC–MS studies
and used during analytical method development and validation studies. Also, process related
amoxicillin impurity-P was synthesized and characterized by using nuclear magnetic resonance
(NMR) and mass spectroscopy (MS) for the first time. As complementary of this work, an assay
method for amoxicillin and potassium clavulanate mixtures was developed and validated; stress-testing
and stability studies of amox/clav mixtures was carried out under specified conditions according to
ICH and analyzed by using validated stability-indicating assay and related substances methods.
65
Rapid analysis of drug dissolution by paper spray ionization mass spectrometry
Yang Liu, Ning Liu, Ya-nan Zhou, Lan Lin, Lan He
ABSTRACT
With a great quantity of solid dosage tested by dissolution technology, developing a rapid and sensitive
method to access the content of drug within dissolution media is highly desired by analysts and
scientists. Traditionally, dissolution media is not compatible with mass spectrometry since the
inorganic salts in the media might damage the mass spectrometer. Here, paper spray ionization mass
spectrometry (PSI-MS), one of the ambient mass spectrometry technologies, is developed to
characterize the content of drugs in dissolution media. The porous structure of paper can effectively
retain salts from entering mass spectrometer. This makes the measurement of drug content within
dissolution media by mass spectrometer possible. After the experimental parameters were optimized,
calibration curves of model drugs − enalapril, quinapril and benazepril were established by using
corresponding deuterated internal standards. PSI-MS was then deployed to characterize the content of
enalapril from the dissolution testing of enalapril tablets. The results from PSI-MS are comparable to
those from HPLC characterization. More importantly, the analysis time of 6 samples is shortened from
90 min to 6 min. Detection limit of enalapril maleate tablets by PSI-MS is 1/300 of LC. PSI-MS is
rapid, sensitive and accurate in analyzing drug content from dissolution tests.
Development and validation of a liquid chromatography-MS/MS method for simultaneous
quantification of tenofovir and efavirenz in biological tissues and fluids
Luisa Barreiros, Cassilda Cunha-Reis, Eduarda M.P. Silva, Joana R.B. Carvalho, Marcela A.
Segundo
ABSTRACT
Millions of people worldwide live with human immunodeficiency virus (HIV) infection thus justifying
the continuous search for new prevention and treatment strategies, including topical microbicide
products combining antiretroviral drugs (ARVs) such as tenofovir (TFV) and efavirenz (EFV).
Therefore, the aim of this work was to develop and validate a high performance liquid chromatography
method coupled to triple quadrupole-tandem mass spectrometry (HPLC–MS/MS) for the
quantification of TFV and EFV in biological matrices (mouse vaginal tissue, vaginal lavage and blood
plasma). Chromatographic separation was achieved using a reversed phase C18 column (3 μm, 100 ×
2.1 mm) at 45 °C and elution in gradient mode using a combination of 0.1% (v/v) formic acid in water
and 0.1% (v/v) formic acid in acetonitrile at 0.35 mL min−1. Total run time was 9 min, with retention
time of 2.8 and 4.1 min for TFV and EFV, respectively. The MS was operated in positive ionization
mode (ESI+) for TFV and in negative ionization mode (ESI-) for EFV detection. Data were acquired
in selected reaction monitoring (SRM) mode and deuterated ARVs were employed as internal
standards. Calibration curves were linear for ARV concentrations ranging from 4 to 500 ng mL−1 with
LOD and LOQ for both analytes ≤0.4 and ≤0.7 ng mL−1 in sample extracts, respectively. The method
was found to be specific, accurate (96.0–106.0% of nominal values) and precise (RSD < 2.4%) in all
matrices. Both TFV and EFV were found to be stable in all matrices after standing 24 h at room
temperature (20 °C) or in the autosampler, and after three freeze-thawing cycles. Mean recovery values
of ARVs spiked in mice tissues or fluids were ≥88.4%. Matrix effects were observed for EFV
determination in tissue and plasma extracts but compensated by the use of deuterated internal
standards. The proposed methodology was successfully applied to a pharmacokinetic study following
intravaginal administration of both ARVs.
66
Isolation, characterization using LC-ESI-QTOF, NMR and in vitro cytotoxicity assay of
niclosamide forced degradation products
Johnsirani P., Vishnuvardhan Ch., Lingesh A., Naidu V.G.M., Satheeshkumar N.
ABSTRACT
The present study describes the isolation, characterization and in vitro cytotoxic effect of all forced
degradation products of niclosamide (NCM) an anthelmintic class of drug used specifically to treat
tapeworms. NCM was subjected to forced degradation involving hydrolysis (acidic, alkaline and
neutral), oxidative, photolysis and thermal stress, as per ICH (Q1A (R2)) suggested conditions. The
drug under hydrolytic (acidic and basic) conditions showed extensive degradation, while it was stable
under neutral hydrolytic, oxidative, photolytic and thermal stress conditions. A total of four
degradation products (DPs) were observed and chromatographic separation of drug and its degradation
products were achieved on a reverse phase Fortis diphenyl column (150 × 4.6 mm, 5 μm) using 0.1%
formic acid and acetonitrile as mobile phase in gradient mode. All the four degradation products were
isolated by semi preparative LC and its structures were characterized and confirmed by high resolution
MS and 1H NMR spectroscopic techniques. In view of safety aspects, cytotoxicity assay were carried
out for NCM and its four degradation products on human mononuclear cells and cell lines depicting
the major organelle: neuronal (Neuro 2a), hepatic (HepG 2) and alveolar (A549). NCM was found to
be non toxic on human mononuclear cells and cell lines at tested concentrations. However DP-1, DP-2,
DP-3 and DP-4 showed significant increase in LDH release as compared to control at a concentration
of 100 μM. DP-1 and DP-3 exhibited toxicity on A549 cells with an IC50 of 92.18 ± 4.93 μM and
65.42 ± 6.29 μM respectively. DP-2, DP-3 and DP-4 were cytotoxic to Neuro 2a cells with an IC50 of
63.62 ± 3.85 μM, 86.09 ± 6.19 μM and 42.81 ± 8.10 μM respectively. The degradation products were
found to be nontoxic on HepG 2 cells.
Quantitative determinations using portable Raman spectroscopy
Chelliah V. Navin, Chaitanya Tondepu, Roxana Toth, Latevi S. Lawson, Jason D. Rodriguez
ABSTRACT
A portable Raman spectrometer was used to develop chemometric models to determine percent (%)
drug release and potency for 500 mg ciprofloxacin HCl tablets. Parallel dissolution and
chromatographic experiments were conducted alongside Raman experiments to assess and compare
the performance and capabilities of portable Raman instruments in determining critical drug attributes.
All batches tested passed the 30 min dissolution specification and the Raman model for drug release
was able to essentially reproduce the dissolution profiles obtained by ultraviolet spectroscopy at 276
nm for all five batches of the 500 mg ciprofloxacin tablets. The five batches of 500 mg ciprofloxacin
tablets also passed the potency (assay) specification and the % label claim for the entire set of tablets
run were nearly identical, 99.4 ± 5.1 for the portable Raman method and 99.2 ± 1.2 for the
chromatographic method. The results indicate that portable Raman spectrometers can be used to
perform quantitative analysis of critical product attributes of finished drug products. The findings of
this study indicate that portable Raman may have applications in the areas of process analytical
technology and rapid pharmaceutical surveillance.
67
Screening active compounds from Corydalis yanhusuo by combining high expression VEGF
receptor HEK293 cell membrane chromatography with HPLC - ESI - IT - TOF - MSn method
Fen Wei, Qi Hu, Jing Huang, Shengli Han, Sicen Wang
ABSTRACT
Corydalis Thizoma,or Yuanhu in China, is a common herbal drug used for thousands of years as
analgesic in Chinese medicine that has been reported to have potential anti-angiogenic effects. In this
study, a VEGFR/cell membrane chromatography (VEGFR/CMC) coupled with HPLC- ESI-IT-TOF-
MSn system was developed and successfully applied for identifying active components from YuanHu
extract acting on VEGFR. We identified tetrahydropalmatine and corydaline as bioactive components
with VEGFR activity, thus confirming their inhibitory activity on VEGFR engineered HEK293 cell
growth by MTT assay. The activity of tetrahydropalmatine and corydaline was compared with the
positive control sorafenib in a range of concentration from 6.25 to 50.0 μM, showing a dose-dependent
inhibitory trend. These results indicate that the VEGFR/CMC coupled with HPLC- ESI-IT-TOF-MSn
system can purify and identify specific bioactive components from complex systems, thus representing
a promising tool for screening molecules active towards VEGFR from natural herbs.
Volume 137, April 2017
Powerful combination of analytical and chemometric methods for the photodegradation of 5-
Fluorouracil
Cristian Gómez-Canela, Gabino Bolivar-Subirats, Romà Tauler, Silvia Lacorte
ABSTRACT
The photodegradation of the antineoplastic drug 5-fluorouracil (5-FU) under the action of UV light
was studied using UV–vis spectroscopy and High Performance Liquid Chromatography coupled to
Diode Array Detector and High Resolution Mass Spectrometry (HPLC-DAD-HRMS). To analyze,
integrate and interpret the degradation kinetics, the Multivariate Curve Resolution-Alternating Least
Squares (MCR-ALS) chemometric method has been applied. The high complexity of the
photodegradation process of this drug involving up to seven different photoproducts showed the
advantages of the proposed approach which provides simultaneously mechanistic and structural
information explaining the 5-FU photodegradability. Moreover, this study provides a new tool to be
used for elucidating the photodegradation kinetics at real time.
68
Separation of antibody drug conjugate species by RPLC: A generic method development
approach
Szabolcs Fekete, Imre Molnár, Davy Guillarme
ABSTRACT
This study reports the use of modelling software for the successful method development of IgG1
cysteine conjugated antibody drug conjugate (ADC) in RPLC. The goal of such a method is to be able
to calculate the average drug to antibody ratio (DAR) of and ADC product. A generic method
development strategy was proposed including the optimization of mobile phase temperature, gradient
profile and mobile phase ternary composition. For the first time, a 3D retention modelling was
presented for large therapeutic protein. Based on a limited number of preliminary experiments, a fast
and efficient separation of the DAR species of a commercial ADC sample, namely brentuximab
vedotin, was achieved. The prediction offered by the retention model was found to be highly reliable,
with an average error of retention time prediction always lower than 0.5% using a 2D or 3D retention
models. For routine purpose, four to six initial experiments were required to build the 2D retention
models, while 12 experiments were recommended to create the 3D model. At the end, RPLC can
therefore be considered as a good method for estimating the average DAR of an ADC, based on the
observed peak area ratios of RPLC chromatogram of the reduced ADC sample.
Time domain NMR as a new process monitoring method for characterization of pharmaceutical
hydrates
Stefanie Ulrike Schumacher, Benno Rothenhäusler, Alf Willmann, Jürgen Thun, Martin Kuentz
ABSTRACT
Hydrates are of great pharmaceutical relevance and even though they have been characterized
thoroughly by various analytical techniques, there is barely literature available on molecular mobility
of the hydrate water studied by NMR relaxation in the time domain. The aim of this work was to
examine the possibility of differentiating hydration states of drugs by 1H time domain NMR (TD-
NMR) regarding spin-spin and spin-lattice relaxation times (T2 and T1) using benchtop equipment.
Caffeine and theophylline were selected as model compounds and binary mixtures of hydrate to
anhydrate were analyzed for each drug using a spin echo and inversion recovery pulse sequence. It was
possible to extract a signal that was specific for the water in the hydrates so that differentiation from
anhydrous solid forms was enabled. Excellent calibrations were obtained for quantitative analysis of
hydrate/anhydrate mixtures and predicted water contents were in good agreement with water amounts
determined in desiccator sorption experiments. TD-NMR was therefore found to be a suitable new
technique to characterize pharmaceutical hydrates in a non-invasive and hence sample-sparing manner.
Quantification of the hydrate content in pharmaceutical mixtures appears highly attractive for product
development and process monitoring. TD-NMR provides here a valuable and complementary
technique to established process analytics, such as for example Raman spectroscopy.
69
LC-method development for the quantification of neuromedin-like peptides Emphasis on column
choice and mobile phase composition
Yannick Van Wanseele, Johan Viaene, Leslie Van den Borre, Kathleen Dewachter, Ann Van
Eeckhaut
ABSTRACT
In this study, the separation of four neuromedin-like peptides is investigated on four different core-
shell stationary phases. Moreover, the effect of the mobile phase composition, i.e. organic modifier
(acetonitrile and methanol) and additive (trifluoroacetic acid, formic acid, acetic acid, ammonium
formate and ammonium acetate) on the chromatographic performance is studied. An improvement in
chromatographic performance is observed when using the ammonium salt instead of its corresponding
acid as additive, except for the column containing a positively charged surface (C18+). In general, the
RP-Amide column provided the highest separation power with different mobile phases. However, for
the neuromedin-like peptides of interest, the C18+ column in combination with a mobile phase
containing methanol as organic modifier and acetic acid as additive provided narrower and higher
peaks. A three-factor, three-level design is applied to further optimize the method in terms of increased
peak height and reduced solvent consumption, without loss in resolution. The optimized method was
subsequently used to assess the in vitro microdialysis recovery of the peptides of interest. Recovery
values between 4 and 8% were obtained using a perfusion flow rate of 2 μL/min.
2D-LC as an on-line desalting tool allowing peptide identification directly from MS unfriendly
HPLC methods
Hao Luo, Wendy Zhong, Jiong Yang, Ping Zhuang, Christopher J. Welch
ABSTRACT
The increasing interest in peptides and proteins in pharmaceutical research and development has led to
many challenges for the researchers tasked with characterizing and analyzing these larger molecules.
Due to the more complicated impurity profile of peptides and proteins, multiple liquid chromatography
techniques are often needed to achieve comprehensive analysis. However, many of these separation
conditions require buffers, salts or additives that render them incompatible with mass spectrometry
(MS) detection. Previous researchers have demonstrated proof of concept for the use of two
dimensional liquid chromatography (2D-LC) to provide convenient second dimension online desalting
of components purified in the first chromatographic dimension. In this paper, we evaluated the Agilent
heart-cutting 2D-LC system connected with an Agilent Q-TOF mass spectrometer to address this
frequently encountered analytical challenge. On this 2D-LC/MS system, fractions containing the
compounds of interest are separated by the first dimension using an MS incompatible mobile phase,
then sent to a second dimension HPLC method where fast desalting using an MS compatible mobile
phase is performed prior to MS analysis. The system allows for fast and direct collection of MS
information for chromatographic peaks eluted in MS incompatible mobile phases, without requiring
difficult, time consuming and error-prone translation of chromatographic methods from MS
incompatible to MS compatible eluents, or off-line fraction collection and preparation. Several
examples showing the application of the approach to complex mixtures containing peptides with
impurities and positional isomers are presented.
70
Quantification of EC-18, a synthetic monoacetyldiglyceride (1-palmitoyl-2-linoleoyl-3-acetyl-rac-
glycerol), in rat and mouse plasma by liquid-chromatography/tandem mass spectrometry
Heon-Min Ryu, Yoo-Seong Jeong, Chang-Soon Yim, Jong-Hwa Lee, Suk-Jae Chung
ABSTRACT
EC-18 (i.e., 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol), an active ingredient in Rockpid®, has been
reported to be useful in controlling various types of inflammations, particularly those caused by
neutropenia. Although this product was originally approved as a functional food in Korea, it is
currently in phase II clinical trials for use in managing the severe chemotherapy-induced neutropenia
in patients with advanced breast cancer who are receiving intermediate febrile neutropenia risk
chemotherapy. The objective of this study was to develop a rapid, sensitive method for the
determination of EC-18 in rat and mouse plasma and to evaluate the applicability of the assay in
pharmacokinetic studies. EC-18 was extracted with MeOH from rat and mouse plasma samples, and
the extract directly introduced onto an LC–MS/MS system. The analyte and EC-18-d3, an internal
standard, were analyzed by multiple reaction monitoring (MRM) at m/z transitions of 635.4 → 355.4
for EC-18 and 638.4 → 338.4 for the internal standard, respectively. The lower limit of quantification
(LLOQ) was determined at 50 ng/mL, with an acceptable linearity in the range from 50 to 10,000
ng/mL (r > 0.999) for both matrices. Validation parameters such as accuracy, precision, dilution,
recovery, matrix effects and stability were found to be within the acceptance criteria of the assay
validation guidelines, indicating that the assay is applicable for estimating EC-18 in concentrations in
the range examined. EC-18 was readily determined in plasma samples for periods of up to 8 h
following an intravenous bolus injection of 1 mg/kg in rats and at 5 mg/kg in mice, respectively, and
up to 24 h following the oral administration of 2000 mg/kg in mice. The findings indicate that the
current analytical method is applicable for pharmacokinetic studies of EC-18 in small animals.
Compatibility study of a parenteral microdose polyethylene glycol formulation in medical
devices and identification of degradation impurity by 2D-LC/MS
Lulu Dai, Geoffrey K. Yeh, Yingqing Ran, Peter Yehl, Kelly Zhang
ABSTRACT
Polyethylene glycol (PEG) based formulation and polyvinylchloride (PVC) tubing are frequently used
for drug delivery and administration. The compatibility of a parenteral drug microdose formulation in
intravenous infusion (IV) devices was studied to support the clinical determination of absolute
bioavailability by the microdosing method. The investigational microdose formulation containing PEG
was found prone to significant loss of potency within hours of storage in the PVC IV tubing due to
degradation. Degradation occurred only when both PEG and PVC tubing were present. The
degradation product could not be detected by LC/MS due to the significant interference from the high
concentration of PEG (4%) matrix and the extremely low level of drug (0.6 ppm). To obtain structural
information of the degradation impurity and understand the cause of the degradation, a simple heart-
cutting 2D-LC/MS approach was utilized to effectively separate the impurity from the complex PEG
oligomers and overcome the matrix interference, enabling mass spectrometric analysis of the impurity.
An oxidation- dominated mechanism was proposed in which the combination of PEG auto-oxidation
and dehydrochlorination of the PVC tubing yielded an oxidative environment that enhanced radical
propagation and accelerated degradation of the investigational parent drug.
71
Use of mixture design in drug-excipient compatibility determinations: Thymol nanoparticles
case study
Felipe Q. Pires, Tamara Angelo, Joyce K.R. Silva, Lívia C.L. Sá-Barreto, Marcílio S.S. Cunha-Filho
ABSTRACT
The objective of this work was to access thymol-excipient compatibility using an alternative protocol
of mixture design subsidizing the development of nanostructures lipid carriers containing this drug.
Simultaneous DTA-TG analyses associated with infrared spectroscopy were performed according to
simplex centroid mixture designs with three components. Two designs were used: the design A
containing stearic acid (SA), soybean lecithin (LC), and sodium taurodeoxycholate (TAU) and the
design B, where TAU was replaced by polysorbate 80 (P80). Assays allowed for a quantitative
evaluation of thermal events involved with thymol (TML) – melting and evaporation –, as well as
events related to excipients decomposition and overall system stability. Although the anionic
surfactant TAU did not interact with TML in solid state, chemical and physical stability were
compromised after drug melting. Alternatively, nonionic surfactant P80 could be a good excipient
option, as TML formulation stability was not influenced by it. Fatty acid SA did not compromise TML
stability alone, but, when in combination with other formulation components, negative interaction
leading to a possible decomposition of the system was observed. Finally, phospholipid LC solubilizes
TML extending its evaporation to higher temperatures; hence, drug stability may be increased. In
conclusion, the use of mixture design in the evaluation of multicomponent systems is a valuable tool
for identification of synergistic effects of excipients, providing more complete information on
formulation development. In addition, the association of techniques employed allowed inferring with
certainty if thermal interactions could compromise formulation stability.
Rapid analysis of Aurantii Fructus Immaturus (Zhishi) using paper spray ionization mass
spectrometry
Xuemei Liu, Zhixin Gu, Yuan Guo, Jingjing Liu, Liping Wang
ABSTRACT
Paper spray-mass spectrometry (PS-MS) is a rapid, solvent-efficient, and high-throughput analytical
method for analyzing complex samples. In this study, a PS-MS method was developed to obtain MS
profiles of Aurantii Fructus Immaturus (aka Zhishi in Chinese) in positive and negative ion modes. In
combination with multivariate analyses, including principal component analysis and cluster analysis,
the PS-MS profiles of 25 batches of Zhishi were discriminated in 25 batches of Citri Reticulatae
Pericarpium Viride (aka Qingpi in Chinese; an adulterant of Zhishi). Moreover, a rapid quantitative
analysis of synephrine, a prescriptive quality control component of Zhishi listed in the Chinese
Pharmacopoeia, was conducted with PS-MS using synephrine-d2 as an internal standard (IS). The
linearity range was 1.68–16.8 μg/mL (R2 = 0.9985), the limit of quantitation was 0.5 μg/mL. Relative
standard deviations in the intra- and inter-day precision of the MS were 4.87 and 4.90%, respectively.
Compared with HPLC results, there was no significant difference in the quantitation of synephrine.
This study demonstrated that the PS-MS method is useful for the rapid discrimination and quality
control of Zhishi samples.
72
Rapid determination of 30 bioactive constituents in XueBiJing injection using ultra high
performance liquid chromatography-high resolution hybrid quadrupole-orbitrap mass
spectrometry coupled with principal component analysis
Lihua Zuo, Zhi Sun, Yurong Hu, Ya Sun, Xiaojian Zhang
ABSTRACT
Xuebijing injection (XBJ) is a traditional Chinese herbal prescription widely used in the treatment of
sepsis. Extensive chemical studies revealed that XBJ injection contains amino acids, phenolic acids,
flavonoid glycosides, terpeneglycosides and phthalides. In this study, the applicability of ultra high
performance liquid chromatography coupled with high resolution hybrid quadruple-orbitrap mass
spectrometry (UHPLC-Q-Orbitrap MS) for the simultaneous quantitative analysis of 30 bioactive
constituents in XueBiJing injection (XBJ) was investigated. The mass spectrometer was operated in
full MS scan mode. The use of 70,000 FWHM mass resolution and narrow mass windows (5 ppm)
could effectively improve the selectivity of the method. Separation was achieved on a Waters
ACQUITY UPLC® HSS C18 column (2.1 mm × 100 mm, 1.8 μm) with a gradient mobile phase
consisting of acetonitrile-water (containing 10 mM ammonium acetate) at a flow rate of 0.2 mL/min.
Satisfactory linearity was achieved within wide linear range and all correlation coefficients (r) of
analytes were more than 0.9996. The limits of detection (LODs) were in the range of 0.1180–27.82
ng/mL for different analytes. The relative standard deviations (RSDs) of inter- and intra-day precisions
were less than 3.0% and the recoveries of the assay were in the range of 98.5%–101.5%. The validated
method was successfully applied for simultaneous determination of 30 bioactive compounds in
XueBiJing injection from 10 batches samples by UHPLC-Q-Orbitrap MS within 10 min. Moreover,
the results were evaluated principal component analysis and two compounds might be the most
important chemical markers for chemical quality control of XBJ injection.
Generic DART-MS platform for monitoring the on-demand continuous-flow production of
pharmaceuticals: Advancing the quantitative protocol for caffeates in microfluidic biocatalysis
Yan Xu, Dong-Yang Zhang, Xiang-Yun Meng, Xi Liu, Fu-An Wu
ABSTRACT
Today, continuous processing is regarded as an effective on-demand production technique of
pharmaceuticals. Homemade microreactors packed with immobilized lipase under continuous-flow
conditions were first applied to tailor the production of high-value caffeic acid phenethyl ester (CAPE)
from methyl caffeate (MC) and 2-phenylethanol (PE) in cyclohexane via transesterification; however,
this method is challenging due to the lack of a rapid platform for monitoring caffeates in microfluidic
biocatalysis. The reactants were directly analyzed using Direct Analysis in Real Time Mass
Spectrometry (DART-MS), and the corresponding ionization parameters were investigated. Special
ions produced from MC (parent ion m/z 192.87 and product ion m/z 133.44) and CAPE (parent ion
m/z 282.93 and product ion m/z 178.87) were determined using DART-MS2 in the negative ion mode.
The peak areas of the select reaction monitoring (SRM) signals were calculated to develop the
standard curves for quantitative analyses of the concentration. Reasonable linear regression equations
of MC and CAPE were obtained in the range of 3.125–50.000 mg/L, with linear coefficients (R2) of
0.9515 and 0.9973, limits of detection (LOD) of 0.005 and 0.003 mg/L, limits of quantification (LOQ)
of 0.02 and 0.01 mg/L, and recovery ranges of 92.50–97.11% and 90.11–97.60%, respectively.
73
Potential impurities of anxiolytic drug, clobazam: Identification, synthesis and characterization
using HPLC, LC-ESI/MSn and NMR
Neeraj Kumar, Subba Rao Devineni, Shailendra Kumar Dubey, Pramod Kumar
ABSTRACT
During the optimization of process, eight impurities (CLB Imp-A to CLB Imp-H) were detected in few
of the laboratory batches of clobazam, used as anxiolytic agent, in the range of 0.02–0.12% using
gradient HPLC method with UV detection. On the basis of co-spiking analysis, six impurities (CLB
Imp-A to -F) enumerated by European Pharmacopoeia, however, not reported in the earlier literature,
have been harmonized and found to be two impurities are completely unknown (CLB Imp-G and -H).
These two new impurities structures were presumed based on LC-ESI/MSn study as 8-chloro-1-
methyl-5-phenyl-1,5-dihydro-3H-1,5-benzodiazepine-2,4-dione (CLB Imp-G) and 5-chloro-1-methyl-
3-phenyl-1H-benzo[d]imidazol-2(3H)-one (CLB Imp-H). The presumed impurities structures were
confirmed by their synthesis followed by the complete spectral analysis such as ESI–MS, 1D NMR
(1H, 13C and DEPT), 2D NMR (HSQC, HMBC and COSY) and IR, and chromatographic retention
time profile. Identification, synthesis, structural characterization, prospects to the formation and
controlling of these new impurities were described in detail and reported first in this paper.
Simultaneous quantification of twenty-one ginsenosides and their three aglycones in rat plasma
by a developed UFLC–MS/MS assay: Application to a pharmacokinetic study of red ginseng
Qi-Le Zhou, Di-Na Zhu, Yan-Fang Yang, Wei Xu, Xiu-Wei Yang
ABSTRACT
To track the pharmacokinetic features of red ginseng (RG), a rapid and sensitive ultra fast liquid
chromatographic coupled with electrospray ionization triple quadrupole tandem mass spectrometry
(UFLC–MS/MS) method was developed for simultaneous quantification of twenty-one ginsenosides
and their three aglycones, including 18 prototype compounds (ginsenosides Rb1, Rb2, Rc, Rd, Re,
Rg1, Rg5, Rh4, Rk1, Rk3, 20(S)-Rf, 20(S)-Rg2, 20(R)-Rg2, 20(S)-Rg3, 20(R)-Rg3, 20(S)-Rh1,
20(R)-Rh1, 20(S)-NG-R2), and 6 metabolites (ginsenosides 20(S)-Rh2 and Rh3, 20(S)-
protopanaxadiol (PPD), 20(S)-protopanaxatriol (PPT), 20(R)-PPT, ginseng saponin compound K) of
RG in rat plasma after oral administration of RG water extract at a single dose of 4 g/kg body weight
to rats. All analytes with internal standard (digoxin) were detected by multiple reaction monitoring in
negative ionization mode and separated on an ACQUITY UPLC® BEH RP-C18 column (1.7 μm, 100
× 2.1 mm). This established method was well validated in terms of linearity, sensitivity, intra- and
inter-day precisions, accuracy, recovery, matrix effect, stability, and had a lower limit of quantification
at the concentration range of 0.12–8.12 ng/mL for all of analytes. This UFLC–MS/MS approach was
successfully applied to the pharmacokinetic study for RG water extract in rats. We firstly proposed that
Rb1, Rb2, Rc, Rd, Rg1, Rg5, 20(S)-Rg3, 20(S)-Rh2, and 20(S)-PPD measured in rat plasma were
suitable pharmacokinetic markers of RG extract in rats due to their high systemic exposure levels.
Thus, this specific and reliable method will be useful for future applications to pharmacokinetic studies
for various sources of ginsenoside samples and Panax herbs in vivo.
74
Metabolic profiles of exudates from chronic leg ulcerations
Adam Junka, Wojciech Wojtowicz, Adam Ząbek, Grzegorz Krasowski, Marzenna Bartoszewicz
ABSTRACT
Chronic leg ulceration is a disease usually associated with other comorbidities, and significantly
reduces patient quality of life. Infected leg ulcers can lead to limb-threatening sequelae or mortality.
Leg ulcerations are colonized by a number of microbes that are able to cause life-threating infections
in susceptible patients. Wound exudate is a body fluid that collects metabolites from patient eukaryotic
cells and from prokaryotic bacterial communities inhabiting the wound. This study aimed at
identification of metabolites in exudates collected from chronic leg ulcers, and correlation of this
metabolome with patient comorbidities and microbiological status of the wound. By means of NMR
spectroscopy we detected 42 metabolites of microbial or patient origin. The metabolites that were in
abundance in exudates analyzed were lactate, lysine, and leucine. Metabolites were associated with the
presence of neutrophils in wounds and destruction of high quantities of microbes, but also with
hypoxia typical for venous insufficiency. The combination of nuclear magnetic resonance
spectroscopy technique and partial least squares discriminant analysis allowed us to further
discriminate groups of metabolites with regards to potential clinical meaning. For example, to
discriminate between S.aureus versus all other isolated microbial species, or between patients suffering
from type I or II diabetes versus patients without diabetes. Therefore, wound exudate seems to be
highly applicable material for discriminant analysis performed with the use of NMR technique to
provide for rapid metabolomics of chronic wound status.
Development and validation of a sensitive and fast UPLC–MS/MS method for simultaneous
determination of seven bioactive compounds in rat plasma after oral administration of Guizhi-
gancao decoction
Bin Ji, Limeng Zhuo, Bin Yang, Yang Wang, Zhiguo Yu
ABSTRACT
Rapid, sensitive, selective and accurate UPLC–MS/MS method was developed and fully validated for
simultaneous determination of cinnamaldehyde, cinnamic acid, 2-methoxy cinnamic acid, glycyrrhizic
acid, glycyrrhetinic acid, liquiritigenin and isoliquiritin in rat plasma after oral administration of
Guizhi-gancao decoction. Plasma samples were processed with a simple protein precipitation
technique using acetonitrile, followed by chromatographic separation using a Thermo Hypersil GOLD
C18 column. A 11.0 min linear gradient elution was used at a flow rate of 0.2 mL/min with a mobile
phase of 0.1% acetic acid containing 0.2 mM ammonium acetate in water and acetonitrile. The
analytes and internal standard, schisandrin, were detected using both positive and negative ion
electrospray ionization in multiple reaction monitoring mode. The developed method was validated for
intra-day and inter-day accuracy and precision whose values fell in the acceptable limits. Matrix effect
was found to be minimal. Recovery efficiency of all the analytes was found to be >60%. Stability
results showed that the analytes were stable at all the conditions. This validated method was
successfully used to study the pharmacokinetics of multiple compounds in rat plasma after oral
administration of Guizhi-gancao decoction.
75
Universal efavirenz determination in transport study, rat placenta perfusion and placenta lysate
by HPLC-UV
Lucie Zelena, Josef Reznicek, Martina Ceckova, Hana Sklenarova
ABSTRACT
Efavirenz is an antiretroviral drug used in the treatment of HIV-positive patients. A simple, fast and
sensitive high-performance liquid chromatography (HPLC) method was developed in order to
determine efavirenz in three types of samples provided from pharmacokinetic studies. The analysis
took 5 min and was performed using a C18 analytical column (Discovery HS C18, 150 × 4.6 mm,
particle size of 5 μm) in isocratic mode with a mobile phase containing acetonitrile and water (65:35,
v/v), a flow rate of 1.6 mL min−1, a sample volume of 10 μL and UV detection at 245 nm. Three
different sample matrices (Opti-MEM medium, Krebs perfusion liquid and tissue lysate) and their
treatment (dilution, SPE) were considered. The validated method was applied for the analysis of 805
real samples arising from in vitro transcellular transport assays and in vivo organ perfusion
experiments in order to evaluate the interaction of efavirenz with ATP-dependent drug efflux
transporters. The lack of interaction of efavirenz with ABCB1, ABCG2 and ABCC2 transporters as
well as technical aspects of this analysis, including the adhesion of efavirenz to the plastic materials
and the stability of the drug during different tissue lysis approaches are discussed.
Metabolite characterization of a novel sedative drug, remimazolam in human plasma and urine
using ultra high-performance liquid chromatography coupled with synapt high-definition mass
spectrometry
Ying Zhou, Pei Hu, Ji Jiang
ABSTRACT
Remimazolam is a new chemical entity belonging to the benzodiazepine class of sedative drugs, which
shows faster-acting onset and recovery than currently available short-acting sedatives. In the present
study, ultra high performance liquid chromatography with synapt high-definition mass spectrometry
method combined with MassLynx software was established to characterize metabolites of
remimazolam in human plasma and urine. In total, 5 human metabolites were detected, including 3
phase I and 2 phase II metabolites. There was no novel human metabolite detected compared to that in
rat. Hydrolysis, glucuronidation and oxidation were the major metabolic reactions. To our knowledge,
this is the first report of the human metabolic profile of remimazolam.
76
Chiral separation of new sulfonamide derivatives and evaluation of their enantioselective affinity
for human carbonic anhydrase II by microscale thermophoresis and surface plasmon resonance
Tiphaine Rogez-Florent, Catherine Foulon, Anne-Sophie Drucbert, Nadège Schifano, Jean-François
Goossens
ABSTRACT
The aim of this study was to develop a method combining chiral separation and biophysical techniques
to evaluate the enantioselective affinity of original sulfonamide derivatives towards their therapeutic
target, the human carbonic anhydrase II (hACII). The first step consisted in the preparation of the
enantiomers by chromatographic separation. The performances of HPLC and Supercritical Fluid
Chromatography (SFC) were studied at the analytical scale by optimization of various experimental
conditions using adsorbed polysaccharide chiral stationary phases (amylose AD-H and cellulose OD-
H). Since SFC allowed obtaining higher enantioresolutions per time unit, it was selected for the semi-
preparative scale and successfully used to isolate each enantiomer with a satisfactory enantiomeric
purity (>98%). Secondly, microscale thermophoresis (MST) method and surface plasmon resonance
(SPR) used as reference method were developed to measure potential enantioselective affinities of
these enantiomers towards the hACII. The optimizations of both methods were performed using a
reference compound, i.e. acetazolamide, which affinity for hCAII has previously been demonstrated.
For all compounds, KD values obtained using MST and SPR were in good agreement, leading to
similar affinity scales despite both approaches totally differ (labeling for MST versus immobilization
of the protein for SPR). The equilibrium dissociation constants of our original compounds for the
hCAII were in the range 100–1000 nM and an enantioselectivity was observed using the MST and
SPR methods for the diarylpyrazole 2.
Ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS)
for determination of GHB, precursors and metabolites in different specimens: Application to
clinical and forensic cases
Francesco Paolo Busardò, Chrystalla Kyriakou, Emilia Marchei, Roberta Pacifici, Simona Pichini
ABSTRACT
Gamma-hydroxybutyric acid (GHB) acts as a precursor and metabolite of the inhibitory central
nervous system (CNS) neurotransmitter gamma-aminobutyric acid (GABA). Sodium salt of GHB has
been used as a medication for narcolepsy and alcohol withdrawal. Moreover, GHB and its precursor
gamma-butyrolactone (GBL), are illegal recreational drugs of abuse. A procedure based on ultra-high-
performance liquid chromatography tandem mass spectrometry has been developed and validated in
plasma, urine, cerebrospinal fluid and hair for acute and chronic exposure to GHB and in seized
preparations coming from black market. In biological matrices, GHB was investigated together with its
glucuronide (GHB-Gluc) as a potential marker of exposure, GABA as endogenous precursor and
metabolite and GBL as eventual exogenous precursor. GBL was sought together with GHB in illegal
preparations. Chromatographic separation was achieved at ambient temperature using a reverse-phase
column and an isocratic elution with two solvents: 0.1% formic acid in water and pure methanol.
Multiple reaction monitoring (MRM) was used. The method was linear for all analytes under
investigation from limit of quantification (LOQ) to 500 μg mL−1 plasma, urine and cerebrospinal
fluid, from LOQ to 100 ng mg−1 hair and from LOQ to 10 mg mL−1 illicit preparations with good
correlation coefficients (r2 = 0.99) for all substances.
77
Impedimetric nanostructured genosensor for detection of schistosomiasis in cerebrospinal fluid
and serum samples
Giselle S. Santos, Cesar A.S. Andrade, Igor S. Bruscky, Leandro B. Wanderley, Maria D.L. Oliveira
ABSTRACT
Schistosomiasis is a neglected disease closely related to the low levels of social development and a
serious public health problem. In this work, we performed an electrochemical detection of
Schistosoma mansoni DNA with a self-assembled monolayer of mercaptobenzoic acid (MBA)
immobilizing nanostructures composed of gold nanoparticles (AuNPs) and magnetite nanoparticles
(Fe3O4_NPs). Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used
to monitor the hybridization process. MBA-Fe3O4_NPs-AuNPs-DNAprobe system reveals an
effective electrochemical response indicating the surface modification. The proposed biosystem was
capable to recognize specific nucleotide sequence of S. mansoni present in cerebrospinal fluid (CFS)
and serum samples at different genome DNA concentrations. The biorecognition resulted in an
increase in the electron transfer resistance and a decrease of the current peaks at higher DNA
concentrations during electrochemical measurements. The developed platform showed a DNA
detection limit of 0.781 and 0.685 pg μL−1 for serum and CFS, respectively. Therefore, the obtained
biosensor can be considered as a useful tool for specific detection of S. mansoni at low concentrations
in various biological fluids.
LC–MS/MS method for the simultaneous determination of Lys-MCC-DM1, MCC-DM1 and
DM1 as potential intracellular catabolite of the antibody-drug conjugate trastuzumab emtansine
(T-DM1)
Yazhong Liu, Fang Zhou, Hua Sang, Hui Ye, Jingwei Zhang
ABSTRACT
Lysine-MCC-DM1, MCC-DM1 and DM1 are potential catabolites of trastuzumab emtansine (T-
DM1). A convenient liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was
developed and validated to detect these catabolites simultaneously in in vitro investigations for the first
time. Protein precipitation was utilized to prepare the samples. Chromatographic separation was
achieved on a Phenomenex Kinetex C18 column (100 × 2.1 mm, 2.6 μm) with mobile-phase gradient
elution. The calibration curves of each analyte ranging from 1 to 100 nM showed good linearity (r2 >
0.995). The method was validated successfully and applied to the intracellular catabolism and
regulation of T-DM1.
78
Quantification of hydroxyurea in human plasma by HPLC–MS/MS and its application to
pharmacokinetics in patients with chronic myeloid leukaemia
Xin Hai, Meihua Guo, Chunlu Gao, Jin Zhou
ABSTRACT
Hydroxyurea (HU) has been used in the treatment of chronic myeloid leukaemia (CML) and other
myeloproliferative malignancies. Considering patient‘s wide variation in clinical response to HU, a
new and simple liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was
developed and validated to monitor patients‘ compliance to treatment and investigate the
pharmacokinetics of HU in patients with CML. Stable isotope labeled HU-13C1,15N2 was used as
internal standard. Plasma samples were treated with acetonitrile to precipitate protein. The supernatant
was injected directly without derivatization and separated on a hydrophilic interaction liquid
chromatography column. HU was quantitatively analyzed with a mobile phase of acetonitrile-1.5 mM
ammonium formate (90:10, V:V) within 3 min. The proposed method provided a linearity range of 1–
200 μg/mL. The coefficients of variation for intra- and inter-day precision were less than 2.07% and
4.28%, respectively, while the accuracy (bias) was in the range of −3.77 to 2.96%. This method was
satisfactorily applied to the determination of HU in two patients with CML. It is suitable for
supporting pharmacokinetic studies and clinical therapeutic monitoring.
Determination of rodenticides and related metabolites in rabbit liver and biological matrices by
liquid chromatography coupled to Orbitrap high resolution mass spectrometry
Marina López-García, Roberto Romero-González, Antonia Garrido Frenich
ABSTRACT
An analytical method based on ultra-high performance liquid chromatography (UHPLC) coupled to
Orbitrap high resolution mass spectrometry was developed for the determination of rodenticides
(bromadiolone, brodifacoum, difenacoum, chlorophacinone, diphacinone, coumachlor and warfarin) in
liver matrix. Different extraction conditions were tested, obtaining the best results when the ―dilute and
shoot‖ method (acidified acetonitrile as extraction solvent) and a clean-up step with primary secondary
amine (PSA) were used. The optimized method was validated, obtaining recoveries ranging from 60 to
120%. Repeatability and reproducibility were evaluated obtaining values lower than 20%, except for
brodifacoum at 10 μg/kg. Limits of quantification (LOQs) ranged from 0.1 to 0.5 μg/kg, except for
brodifacoum, which was 100 μg/kg. Six liver samples were analyzed and diphacinone and
chlorophacinone were detected in three samples at concentrations ranging from 4 μg/kg to 13 μg/kg.
Moreover a retrospective screening of rodenticide metabolites in those samples and in animal forensic
samples was developed based on Orbitrap capabilities. Brodifacoum was detected in three samples,
and warfarin alcohol, which is a metabolite of warfarin, was also detected in one sample.
79
Comparative pharmacokinetic profiles of selected irreversible tyrosine kinase inhibitors,
neratinib and pelitinib, with apigenin in rat plasma by UPLC–MS/MS
Hadir M. Maher, Nourah Z. Alzoman, Shereen M. Shehata, Ashwag O. Abahussain
ABSTRACT
Neratinib (NER) and pelitinib (PEL) are irreversible tyrosine kinase inhibitors (TKIs) that have been
recently employed in cancer treatment. Apigenin (API), among other flavonoids, is known to have
antioxidant, anti-proliferative, and carcinogenic effect. API can potentiate the antitumor effect of
chemotherapeutic agents and/or alleviate the side effects of many anticancer agents. Since TKIs are
mostly metabolized by CYP3A4 enzymes and that API could alter the enzymatic activity, potential
drug interactions could be expected following their co-aministration. In the present study, a
bioanalytical UPLC–MS/MS method has been developed and validated for the quantification of NER
and PEL in rat plasma, using domperidone (DOM) as an internal standard. Sample preparation was
carried out using solid phase extraction (SPE) with C18 cartridges with good extraction recovery of
not less than 92.42% (NER) and 89.73% (PEL). Chromatographic analysis was performed on a Waters
BEH C18 column with a mobile phase composed of acetonitrile and water, (70:30, v/v), each with
0.1% formic acid. Quantitation was performed using multiple reaction monitoring (MRM) of the
transitions from protonated precursor ions [M+H]+, at m/z 557.30 (NER), m/z 468.21 (PEL), and at
m/z 426.27 (DOM), to selected product ions at m/z 112.05 (NER), m/z 395.22 (PEL), and at m/z
175.18 (DOM). The method was fully validated as per the FDA guidelines over the concentration
range of 0.5–200 ng/mL with very low lower limit of quantification (LLOQ) of 0.5 ng/mL for both
NER and PEL. The intra- and inter-day assay precision and accuracy were evaluated for both drugs
and the calculated values of percentage relative standards deviations (%RSD) and relative errors (%Er)
were within the acceptable limits (<15%) for concentrations other than LLOQ and 20% for LLOQ.
The applicability of the method was extended to study the possibility of drug interactions following the
oral co-administration of NER/PEL with API. Thus, this study could be readily applied in therapeutic
drug monitoring (TDM) of cancerous patients receiving such drug combinations.
LC–MS/MS determination of d-mannose in human serum as a potential cancer biomarker
Lyndsey White, Jing Ma, Su Liang, Beatriz Sanchez-Espiridion, Dong Liang
ABSTRACT
Several metabolites in human serum have been identified as potential cancer biomarkers for early
detection. This study focuses on the LC–MS/MS method development and validation of d-mannose in
human serum. Surrogate blank serum, coupled with stable isotope d-mannose-13C6, as internal
standard, was used for generating standard curves ranging from 1 to 50 μg/mL. Separation was
achieved by an Agilent 1200 series HPLC equipped with a SUPELCOGELTM Pb, 6% Crosslinked
column with HPLC water as a mobile phase at flow rate of 0.5 mL/min at 80 °C. Mass detection was
performed under negative ionization electrospray. Inter- and intra-day accuracy and precision were
<2%. The extraction recovery and matrix effect were 104.1%–105.5% and 97.0%–100.0%,
respectively. This method was successfully applied for the quantification of d-mannose in the serum
samples of 320 esophageal cancer patients and 323 healthy volunteers. We report a simple, specific
and reproducible LC–MS/MS method for the quantification of d-mannose in human serum as a
potential cancer biomarker.
80
Assessment of in vitro cardiotoxicity of extract fractions and diterpene alkaloids from Aconitum
leucostomum Worosch: A short communication
Jihong Nie, Fang Wang, Tengfei Ji, Jun Zhao, Feicui Zhao
ABSTRACT
Aconitum leucostomum Worosch is a traditional Chinese medicine (TCM) and has a broad spectrum
of health effects, but with a narrow therapeutic window. It is important to identify both the therapeutic
ingredients and the toxic components to better utilize this TCM. The present study investigated the
cardiotoxicity of the selected compounds in Aconitum leucostomum Worosch. The effects of extract of
A. leucostomum Worosch and the isolated compounds on cardiocardiomyocytes were evaluated in
vitro. Five known compounds in this TCM, including three C18-diterpene alkaloids, lappaconitine (2),
N-deacetyllappaconitine (3), and ranaconitine (5), and two C19-diterpene alkaloids, delvestidine (1)
and anthranoyllycoctonine (4), were isolated from A. leucostomum Worosch. The cardiotoxicity of
these components and extract fractions, as measured by lactate dehydrogenase release and apoptosis,
was ranked as follows, in descending order: delvestidine > anthranoyllycoctonine > pH 4 fraction > pH
8 fraction > aconitine > N-deacetyllappaconitine > ranaconitine > lappaconitine. The cytotoxicity of
these compounds was shown to be dose-dependent, with delvestidine (1) and anthranoyllycoctonine
(4) being the two most toxic compounds to cardiomyocytes in our assays. These results provide a basis
for future rational use of this TCM, reducing side effects while retaining therapeutic effects.
Development and validation of a liquid chromatography–tandem mass spectrometry method for
the assay of tafamidis in rat plasma: Application to a pharmacokinetic study in rats
Hun-Chan Hyun, Jong-Woo Jeong, Hye-Rim Kim, Ji-Hoon Oh, Tae-Sung Koo
ABSTRACT
Tafamidis is a first-in-class inhibitor of transthyretin amyloid fibril formation. It has been available in
Argentina, Japan, and Mexico for the treatment of transthyretin amyloidosis in adult patients with
early-stage symptomatic polyneuropathy. In this study, a rapid and sensitive liquid chromatography-
tandem mass spectrometry method was developed and validated for the assay of tafamidis in rat
plasma. The method was also assessed for its applicability to pharmacokinetic studies in rats.
Tafamidis was extracted from rat plasma by the liquid-liquid extraction method using hydrochloric
acid and ethyl acetate. A reversed-phase C18 column and a mobile phase consisting of 10 mM
ammonium formate and acetonitrile were used to achieve chromatographic separation. The flow rate
for the mobile phase was set at 0.3 mL/min. Tafamidis and 2-CBC, which was used as the internal
standard (IS), were analyzed by multiple reaction monitoring in negative ESI mode at m/z transitions
of 305.4 → 261.4 for tafamidis and 271.7 → 227.8 for the IS. The lower limit of quantification of
tafamidis was obtained as 3 ng/mL, and the calibration curve was linear over a concentration range of
3–3000 ng/mL (R2 > 0.99). The validation parameters investigated, which were specificity, precision,
accuracy, matrix effect, recovery, and stability, were well within acceptable limits. The method was
successfully used for the evaluation of the pharmacokinetics of tafamidis in rats.
81
Isolation and characterization of a novel dithio-carbodenafil analogue from a health supplement
Chee-Leong Kee, Min-Yong Low, Xiaowei Ge
ABSTRACT
A novel dithio-carbodenafil compound was isolated and identified from a health supplement. The
structure of the unknown compound has been characterized using LC-UV, Fourier Transform Infrared
(FTIR), high-resolution mass spectrometry, 1D and 2D nuclear magnetic resonance (NMR)
spectroscopy. It has been revealed as 3,5-dimethylpiperazinyl dithio-desmethylcarbodenafil as a result
of two additional methyl groups on the piperazine ring.
Preparation and 68Ga-radiolabeling of porous zirconia nanoparticle platform for PET/CT-
imaging guided drug delivery
Andras Polyak, Lívia Naszalyi Nagy, Judith Mihaly, Sebastian Görres, Tobias L. Ross
ABSTRACT
This paper describes the preparation of gallium-68 (68Ga) isotope labeled porous zirconia (ZrO2)
nanoparticle (NP) platform of nearly 100 nm diameter and its first pharmacokinetic and biodistribution
evaluation accomplished with a microPET/CT (μPet/CT) imaging system. Objectives of the
investigations were to provide a nanoparticle platform which can be suitable for specific delivery of
various therapeutic drugs using surface attached specific molecules as triggering agents, and at the
same time, suitable for positron emission tomography (PET) tracing of the prospective drug delivery
process. Radiolabeling was accomplished using DOTA bifunctional chelator. DOTA was successfully
adsorbed onto the surface of nanoparticles, while the 68Ga-radiolabeling method proved to be simple
and effective. In the course of biodistribution studies, the 68Ga-labeled DOTA-ZrNPs showed proper
radiolabeling stability in their original suspension and in blood serum. μPet/CT imaging studies
confirmed a RES-biodistribution profile indicating stable nano-sized labeled particles in vivo. Results
proved that the new method offers the opportunity to examine further specifically targeted and drug
payload carrier variants of zirconia NP systems using PET/CT imaging.
82
Determination of panduratin A in rat plasma by HPLC–MS/MS and its application to a
pharmacokinetic study
Minsoo Kim, Seungmok Choi, Keumhan Noh, Changhee Kim, Wonku Kang
ABSTRACT
Although panduratin A, a chalcone derivative isolated from the rhizome of Kaempferia pandurata
Roxb, represents a variety of pharmacological activities, no validated method for determination of
panduratin a levels in biological samples is yet available. Thus, we developed a liquid
chromatographic method using a tandem mass spectrometry for the determination of panduratin A in
rat plasma. After simple protein precipitation with methanol including flufenamic acid (internal
standard, IS), the analytes were chromatographed on a reversed-phased column with a mobile phase of
distilled water and acetonitrile including 2% of formic acid (2:8, v/v). The ion transitions of the
precursor to the product ion were principally deprotonated ions [M−H] − at m/z 405.2 → 165.9 for
panduratin A and 280.1 → 236.0 for the IS. The accuracy and precision of the assay were in
accordance with FDA regulations for the validation of bioanalytical methods. This analytical method
was successfully applied to monitor the plasma concentrations of panduratin a following oral
administration in rats.
Simultaneous determination of creatinine and acetate by capillary electrophoresis with
contactless conductivity detector as a feasible approach for urinary tract infection diagnosis
Wojciech Grochocki, Michał J. Markuszewski, Joselito P. Quirino
ABSTRACT
Urinary tract infection (UTI) is one of the most common bacterial infections in human but its diagnosis
is difficult. Metabolomic studies with nuclear magnetic resonance of urine have shown that acetic
acid/creatinine ratio may be used for early UTI diagnosis. Here, a method for simultaneous
determination of acetate and creatinine by capillary zone electrophoresis with contactless conductivity
detector was developed for the first time. The separation was with 40 mM MES and 20 mM l-histidine
as a background solution. The total time of a single run, including capillary conditioning, was less than
12 min. The method was successfully demonstrated for analysis of actual and fortified human urine
samples after methanol dilution. Analytical figures of merit such as linearity, LOQ, and repeatability
(intraday and interday) were studied.
83
Analytical method development and validation for the analysis of verapamil hydrochloride and
its related substances by using ultra perfomance liquid chromatography
S. Vijayabaskar, V. Mahalingam, Kalaivani
ABSTRACT
A novel, economic, and time-efficient stability-indicating, reverse-phase ultra-performance liquid
chromatographic (RP-UPLC) method has been developed for the analysis of verapamil hydrochloride
in the presence of both impurities and degradation products generated by forced degradation. When
verapamil hydrochloride was subjected to acid hydrolysis, oxidative, base hydrolysis, photolytic, and
thermal stress, degradation was observed only in oxidative and base hydrolysis. The drug was found to
be stable to other stress conditions. Successful chromatographic separation of the drug from impurities
formed during synthesis and from degradation products formed under stress conditions was achieved
on a Shimpak XR ODS, 75 mm × 3.0 mm, 1.7 μ particle size column, UV detection at 278 nm and a
gradient elution of ammonium formate, orthophosphoric acid and acetonitrile as mobile phase. The
method was validated for specificity, precision, linearity, accuracy, robustness and can be used in
quality control during manufacture and for assessment of the stability samples of verapamil
hydrochloride. To the best of our knowledge, a validated UPLC method which separates all the sixteen
impurities disclosed in this investigation has not been published elsewhere. Total elution time was
about 18 min which allowed quantification of more than 100 samples per day. The analytical method
discussed in British Pharmacopeia was pH sensitive and not compatible to LC–MS analysis but the
method reported in this study is not involved any pH adjustment.
Evaluation of in silico pharmacokinetic properties and in vitro cytotoxic activity of selected
newly synthesized N-succinimide derivatives
Natasa P. Milosevic, Vesna Kojic, Jelena Curcic, Dimitar Jakimov, Roman Kaliszan
ABSTRACT
Design of a new drug entity is usually preceded by analysis of quantitative structure activity
(properties) relationships, QSA(P)R. Six newly synthesized succinimide derivatives have been
determined for (i) in silico physico-chemical descriptors, pharmacokinetic and toxicity predictors, (ii)
in vitro biological activity on four different carcinoma cell lines and on normal fetal lung cells and (iii)
lipophilicity on liquid chromatography. All compounds observed were predicted for good permeability
and solubility, good oral absorption rate and moderate volume of distribution as well as for modest
blood brain permeation, followed by acceptable observed toxicity. In silico determined lipophilicity,
permeability through jejunum and aqueous solubility were correlated with experimentally obtained
lipophilic constants (by use of high pressure liquid chromatography) and linear correlations were
obtained. Absorption rate and volume of distribution were predicted by chromatographic lipophilicity
measurements while permeation through blood bran barrier was predicted dominantly by molecular
size defined with molecular weight. Five compounds have demonstrated antiproliferative activity
toward cervix carcinoma HeLa cell lines; three were cytotoxic against breast carcinoma MCF-7 cells,
while one inhibited proliferation of colon carcinoma HT-29 cell lines. Only one compound was
cytotoxic toward normal cell lines, while other compounds were proven as safe. Antiproliferative
potential against HeLa cells was described as exponential function of lipophilicity. Based on obtained
results, lead compounds were selected.
84
Biopharmaceutical characterization of praziquantel cocrystals and cyclodextrin complexes
prepared by grinding
Martina Cugovčan, Jasna Jablan, Jasmina Lovrić, Dominik Cinčić, Mario Jug
ABSTRACT
Mechanochemical activation using several different co-grinding additives was applied as a green
chemistry approach to improve physiochemical and biopharmaceutical properties of praziquantel
(PZQ). Liquid assisted grinding with an equimolar amount of citric acid (CA), malic acid (MA),
salicylic acid (SA) and tartaric acid (TA) gained in cocrystal formation, which all showed pH-
dependent solubility and dissolution rate. However, the most soluble cocrystal of PZQ with MA was
chemically unstable, as seen during the stability testing. Equimolar cyclodextrin complexes prepared
by neat grinding with amorphous hydroxypropyl-β-cyclodextrin (HPβCD) and randomly methylated β-
cyclodextrin (MEβCD) showed the highest improvement in drug solubility and the dissolution rate, but
only PZQ/HPβCD product presented an acceptable chemical and photostability profile. A combined
approach, by co-grinding the drug with both MA and HPβCD in equimolar ratio, also gave highly
soluble amorphous product which again was chemical instable and therefore not suitable for the
pharmaceutical use. Studies on Caco-2 monolayer confirmed the biocompatibility of PZQ/HPβCD
complex and showed that complexation did not adversely affect the intrinsically high PZQ
permeability (Papp(PZQ) = (3.72 ± 0.33) × 10−5 cm s−1 and Papp(PZQ/HPβCD) = (3.65 ± 0.21) ×
10−5 cm s−1; p > 0.05). All this confirmed that the co-grinding with the proper additive is as a
promising strategy to improve biopharmaceutical properties of the drug.
Development of an enzyme-linked immunosorbent assay for the detection of isomiroestrol, an
identical marker, in White Kwao Krua using a monoclonal antibody
Tharita Kitisripanya, Kamonthip Jutathis, Chadathorn Inyai, Jukrapun Komaikul, Waraporn Putalun
ABSTRACT
Pueraria candollei var. mirifica or White Kwao Krua (WKK) is a phytoestrogen-rich plant widely used
among women to improve climacteric symptoms. Additionally, the tuberous roots of this plant have
been added as an active ingredient for skin rejuvenation and breast enlargement effects in various
functional foods. However, most of the products on the market containing WKK have not been
sufficiently standardized with respect to the active compound or identical marker. To control the
quality of these plant materials, an enzyme-linked immunosorbent assay (ELISA) using anti-
isomiroestrol antibodies was established for the determination of isomiroestrol, an identical marker in
WKK. Polyclonal and monoclonal antibodies against isomiroestrol were generated and their specificity
characterized in this study. Monoclonal antibody 12C1 showed higher specificity to isomiroestrol and
was thus selected to develop the ELISA. Based on the validation analysis and the tested performance
of the developed ELISA in variably sourced WKK samples, the assay can provide an alternative
approach that is reliable and highly sensitive for the quantitative analysis of isomiroestrol in plant.
85
Volume 138 May 2017
Fabrication of a novel hemin-based monolithic column and its application in separation of
protein from complex bio-matrix
Xiaoya Jiang, Doudou Zhang, Xueying Li, Xixi Wang, Hongyuan Yan
ABSTRACT
A novel polymer-based monolithic column was prepared via redox initiation system within the
confines of a stainless steel column with 4.6 mm i.d. In the processes, hemin and lauryl methacrylate
were used as co-monomers; ethylene dimethacrylate as crosslinking agent; n-butyl alcohol, ethanediol,
and N, N-dimethylformamide as tri-porogens; benzoyl peroxide and N, N-dimethyl aniline as redox
initiation system. The resulting polymer-based monolithic columns were characterized by scanning
electron microscopy, nitrogen adsorption-desorption instrument, and mercury intrusion porosimeter,
respectively. The results illustrated that the improved monolith had relative uniform porous structure,
good permeability, and low back pressure. Aromatic compounds were used to test the chromatographic
behavior of the monolith, resulting in highest column efficiency of 19 880 plates per meter with
reversed-phase mechanism. Furthermore, the homemade monolith was used as the stationary phase of
high performance liquid chromatography to separate proteins from complex bio-matrix, including
human plasma, egg white, and snailase. The results showed that the monolithic column occupied good
separation ability with these complex bio-samples.
High-throughput NIR-chemometric methods for chemical and pharmaceutical characterization
of sustained release tablets
Alina Porfire, Cristina Filip, Ioan Tomuta
ABSTRACT
The aim of this study was the development and validation of methods based on near-infrared
spectroscopy (NIRS) and chemometry, useful for characterization of sustained release (SR) tablets
with indapamide, in terms of tablet composition (API and two excipients), in vitro drug release
mechanism (k and n Peppas) and crushing strength. A calibration set consisting of 25 different tablets
formulations containing API, HPMC and lactose at five different content levels in the range 100 ±
20% relative to a targeted tablet composition, were manufactured by direct compression in order to
develop the methods for prediction of tablet composition, and in vitro drug release mechanism. On the
other hand, a 15 batches calibration set prepared at five different compression forces was used for
development of methods for prediction of crushing strength. Moreover, independent batches were
manufactured for validation of all methods Intact tablets were analyzed by transmission mode with
NIRS, the spectra were pre-processed, and partial least square (PLS) regression was used to build
prediction models. Cross-validation was carried out in order to select the optimal number of PLS
factors for all models, and the best model was chosen based on their RMSECV and bias. All developed
methods were validated in terms of trueness, precision and accuracy. Based on the validation results,
the methods proposed in this work can successfully be applied for routine determination of
indapamide, HPMC and lactose content of sustained release tablets, as well as for prediction of their in
vitro drug release mechanism (k and n Peppas) and crushing strength.
86
Optimized multi-step NMR-crystallography approach for structural characterization of a stable
quercetin solvate
Xenia Filip, Maria Miclaus, Flavia Martin, Claudiu Filip, Ioana Georgeta Grosu
ABSTRACT
Herein we report the preparation and solid state structural investigation of the 1,4-dioxane-quercetin
solvate. NMR crystallography methods were employed for crystal structure determination of the
solvate from microcrystalline powder. The stability of the compound relative to other reported
quercetin solvates is discussed and found to be in perfect agreement with the hydrogen bonding
networks/supra-molecular architectures formed in each case. It is also clearly shown that NMR
crystallography represents an ideal analytical tool in such cases when hydrogen-bonding networks are
required to be constrained at a high accuracy level.
Study on the forced degradation behaviour of ledipasvir: Identification of major degradation
products using LC–QTOF–MS/MS and NMR
Debasish Swain, Gananadhamu Samanthula
ABSTRACT
Ledipasvir, a novel NS5A inhibitor is used in the management of hepatitis C virus infections. The drug
was subjected to forced degradation studies as per the conditions prescribed in ICH Q1 (R2) guideline.
Ledipasvir degraded in hydrolytic (acid, alkaline and neutral) and oxidative stress conditions. The drug
was found to be stable in thermal and photolytic conditions. Eight novel degradation products were
obtained and were well separated using an HPLC C18 stationary phase (150 × 4.6 mm, 5 μm) and
mobile phase composed of formic acid/acetonitrile in gradient elution mode. All the degradation
products were characterised using tandem mass spectrometry with a time-of-flight analyser and the
major degradation products of hydrolytic and oxidative stress were isolated and their structural
confirmation was studied using 1H and 13C NMR. A well resolved chromatographic method proposed
in this study suggests that the proposed analytical method finds its application as a stability indicating
assay method for the drug and can be used in routine analysis.
87
Raman scattering-based multiconformational analysis for probing the structural differences
between acetylcholine and acetylthiocholine
Belén Hernández, Pascal Houzé, Fernando Pflüger, Sergei G. Kruglik, Mahmoud Ghomi
ABSTRACT
Acetylcholine is the first discovered neurotransmitter that has received a great attention regarding its
capability of binding to several cellular targets. The chemical composition of acetylcholine, including
a positively charged trimethylammonium and a carbonyl group, as well as its conformational
flexibility was pointed out as the key factors in the stabilization of its interactions. Here, the
possibilities offered by a Raman scattering-based multiconformatioal analysis to access the most stable
conformers of acetylcholine, is discussed. To control the validity of this protocol, acetylcholine and
one of its closely structured analogues, acetylthiocholine, were simultaneously analyzed. Solution
Raman spectra revealed distinct and well resolved strong markers for each molecule. Density
functional theory calculations were consistent with the fact that the energy order of the low energy
conformers is considerably affected by the acyloxy oxygen → sulfur atom substitution. Raman spectra
were calculated on the basis of the thermal average of the spectra arising from the low energy
conformers. It has been evidenced that the carbonyl and trimethylammonium groups are the most
favorable hydration sites in aqueous environment. Taking into account the large gap between the
carbonyl bond-stretch and aliphatic bending bands, Raman spectra also allowed separation of the HOH
bending vibrations arising from the bound and bulk water molecules.
Characterization of metabolites in different kiwifruit varieties by NMR and fluorescence
spectroscopy
Nur Ashikin Abdul Hamid, Ahmed Mediani, M. Maulidiani, Faridah Abas, S. Gorinstein
ABSTRACT
It is known from our previous studies that kiwifruits, which are used in common human diet, have
preventive properties of coronary artery disease. This study describes a combination of 1H NMR
spectroscopy, multivariate data analyses and fluorescence measurements in differentiating of some
kiwifruit varieties, their quenching and antioxidant properties. A total of 41 metabolites were identified
by comparing with literature data Chenomx database and 2D NMR. The binding properties of the
extracted polyphenols against HSA showed higher reactivity of studied two cultivars in comparison
with the common Hayward. The results showed that the fluorescence of HSA was quenched by Bidan
as much as twice than by other fruits. The correlation between the binding properties of polyphenols in
the investigated fruits, their relative quantification and suggested metabolic pathway was established.
These results can provide possible application of fruit extracts in pharmaceutical industry.
88
Comparison of bioactive components and pharmacological activities of ophiopogon japonicas
extracts from different geographical origins
Min Zhao, Wan-feng Xu, Han-yuan Shen, Pei-qiang Shen, Li-juan Cao
ABSTRACT
Ophiopogon japonicus (Linn. f.) Ker-Gawl (O. japonicas), mainly cultivated in Sichuan and Zhejiang
province in China, has different bioactive components and therefore their pharmacological activities.
To explain the different clinical efficacy of O. japonicas derived preparations, herein we report
differences of pharmacological activities between Sichuan and Zhejiang O. japonicas and behind them
the exact differences of bioactive components. Based on a LC/MS-IT-TOF method, the differences of
bioactive components between Sichuan and Zhejiang O. japonicas extracts were analyzed and
respective characteristic components were picked out. We determined 39 ophiopogonones and 71
ophiopogonins compounds in Sichuan and Zhejiang O. japonicas extracts and found the contents of
these compositions have several times difference. Evidenced by experimental data of pharmacological
activities in inhibiting cardiomyocyte damage induced by H2O2, mouse macrophage cell inflammation
induced by lipopolysaccharide and cytotoxicity in vitro, Zhejiang O. japonicas extract had a stronger
antioxidant and anti-inflammatory capacity than Sichuan O. japonicas extract, and the two O.
japonicas extracts exhibited selective cytotoxicity on different cancer cell lines in vitro. These data
shed light on the links between bioactive components and pharmacological activities of O. japonicas
derived preparations. Thus, geographical origin of O. japonicas should be considered to be a key factor
in efficacy studies and further clinical application.
Tiered analytics for purity assessment of macrocyclic peptides in drug discovery: Analytical
consideration and method development
Jingfang (Jenny) Qian Cutrone, Xiaohua (Stella) Huang, Edward S. Kozlowski, Ye Bao, Adrienne A.
Tymiak
ABSTRACT
Synthetic macrocyclic peptides with natural and unnatural amino acids have gained considerable
attention from a number of pharmaceutical/biopharmaceutical companies in recent years as a
promising approach to drug discovery, particularly for targets involving protein–protein or protein–
peptide interactions. Analytical scientists charged with characterizing these leads face multiple
challenges including dealing with a class of complex molecules with the potential for multiple isomers
and variable charge states and no established standards for acceptable analytical characterization of
materials used in drug discovery. In addition, due to the lack of intermediate purification during solid
phase peptide synthesis, the final products usually contain a complex profile of impurities. In this
paper, practical analytical strategies and methodologies were developed to address these challenges,
including a tiered approach to assessing the purity of macrocyclic peptides at different stages of drug
discovery. Our results also showed that successful progression and characterization of a new drug
discovery modality benefited from active analytical engagement, focusing on fit-for-purpose analyses
and leveraging a broad palette of analytical technologies and resources.
89
Enantiomeric separation of the antiuremic drug colchicine by electrokinetic chromatography
Method development and quantitative analysis
Nuria Menéndez-López, Jesús Valimaña-Traverso, María Castro-Puyana, Antonio Salgado, María
Luisa Marina
ABSTRACT
Two analytical methodologies were developed by CE enabling the enantiomeric separation of
colchicine, an antiuremic drug commercialized as pure enantiomer. Succinyl-γ-CD and Sulfated-γ-CD
were selected as chiral selectors after a screening with different anionic CDs. Under the optimized
conditions, chiral resolutions of 5.6 in 12 min and 3.2 in 8 min were obtained for colchicine with
Succinyl-γ-CD and Sulfated-γ-CD, respectively. An opposite enantiomeric migration order was
observed with these two CDs being S-colchicine the first-migrating enantiomer with Succinyl-γ-CD
and the second-migrating enantiomer with Sulfated-γ-CD. H NMR experiments showed a 1:1
stoichiometry for the enantiomer-CD complexes in both cases. However, the apparent and averaged
equilibrium constants for the enantiomer-CD complexes could be calculated only for Succinyl-γ-CD.
The developed methods were applied to the analysis of pharmaceutical formulations but only the use
of Succinyl-γ-CD enabled to detect a 0.1% of enantiomeric impurity in colchicine formulations.
Integrative hepatoprotective efficacy comparison of raw and vinegar-baked Radix Bupleuri
using nuclear magnetic resonance-based metabolomics
Jie Xing, Hui-Min Sun, Jin-Ping Jia, Xue-Mei Qin, Zhen-Yu Li
ABSTRACT
Radix Bupleuri (RB), with a Chinese name Chaihu, is one of the most popular Traditional Chinese
herbal drug. It can be baked with vinegar to afford vinegar-baked Radix Bupleuri (VBRB), which is
used in Traditional Chinese Medicine (TCM) for liver diseases treatment. In the present study, nuclear
magnetic resonance-based metabolomic approach was used to compare the liver protective effect of
RB and two types of VBRBs, which were prepared by two kinds of vinegar. The contents of 14
metabolites in the liver of carbon tetrachloride (CCl4) treated mice were significantly altered in
comparison with control group, and VBRB prepared by Shanxi vinegar showed best effect as revealed
by the amount and regulatory degree of the perturbed metabolites. The metabolism pathways analysis
showed that the liver protective effect was related with the energy metabolism, lipid metabolism,
ketone body metabolism, glutathione metabolism, amino acids metabolism and nucleotide synthesis.
The results presented here showed that metabolomic approach made it possible to disclose the subtle
biological difference between two types of VBRB, which highlight the potential of metabolomic
approach to quantitatively compare the pharmacological effect of the herbal drugs.
90
Systematic identification of flavonols, flavonol glycosides, triterpene and siraitic acid glycosides
from Siraitia grosvenorii using high-performance liquid chromatography/quadrupole-time-of-
flight mass spectrometry combined with a screening strategy
Zhi-Xing Qing, Huan Zhao, Qi Tang, Chang-ming Mo, Jian-Guo Zeng
ABSTRACT
The fruits of Siraitia grosvenorii are considered to be health-promoting because of the diversity of their
bioactive ingredients. In the present study, a screening method, using high-performance liquid
chromatography/quadrupole-time-of-flight mass spectrometry (HPLC-Q-TOF-MS) combined with a
screening strategy, has been established. The technology was used to systematically screening the
targeted metabolites, primarily from the complex matrix of S. grosvenorii. The compounds were then
identified by their exact masses and characteristic fragment ions, in comparison with the fragmentation
behaviors of 19 references. Finally, 122 compounds, including 53 flavonols and flavonol glycosides,
59 triterpene glycosides and 10 siraitic acid glycosides, were screened and identified in 10-, 50- and
80-day fruits, roots, stems and leaves of S. grosvenorii. 98 of them were reported for the first time.
Additionally, the distribution of all identified components in different parts of the plant was
determined and metabolic networks for flavonol and triterpene glycosides were proposed.
Rapid discrimination and determination of antibiotics drugs in plastic syringes using near
infrared spectroscopy with chemometric analysis: Application to amoxicillin and penicillin
Laetitia Minh Mai Lê, Luc Eveleigh, Ikram Hasnaoui, Patrice Prognon, Eric Caudron
ABSTRACT
The aim of this study was to investigate near infrared spectroscopy (NIRS) combined to chemometric
analysis to discriminate and quantify three antibiotics by direct measurement in plastic
syringes.Solutions of benzylpenicillin (PENI), amoxicillin (AMOX) and amoxicillin/clavulanic acid
(AMOX/CLAV) were analyzed at therapeutic concentrations in glass vials and plastic syringes with
NIR spectrometer by direct measurement. Chemometric analysis using partial least squares regression
and discriminative analysis was conducted to develop qualitative and quantitative calibration models.
Discrimination of the three antibiotics was optimal for concentrated solutions with 100% of accuracy.
For quantitative analysis, the three antibiotics furnished a linear response (R²>0.9994) for
concentrations ranging from 0.05 to 0.2 g/mL for AMOX, 0.1 to 1.0 MUI/mL for PENI and 0.005 to
0.05 g/mL for AMOX/CLAV with excellent repeatability (maximum 1.3%) and intermediate precision
(maximum of 3.2%). Based on proposed models, 94.4% of analyzed AMOX syringes, 80.0% of
AMOX/CLAV syringes and 85.7% of PENI syringes were compliant with a relative error including
the limit of ± 15%.NIRS as rapid, non-invasive and non-destructive analytical method represents a
potentially powerful tool to further develop for securing the drug administration circuit of healthcare
institutions to ensure that patients receive the correct product at the right dose.
91
Identification of UV-absorbing extractables from rubber closures used in containers of
injectable powder and safety assessment of leachables in the drug
Yulei Wei, Ying Wu, Tingli Zhu, Zhiyan Li, Yilan Zhang
ABSTRACT
Rubber closures have been of great concern to regulatory authorities on account of their potential
safety risks to patients. The aim of our work is to provide part of data about the compatibility of the
injectable powder and its packaging materials for the drug registration. In this report, methodologies
were established to study the system of the preparation. Firstly, three major extractables were isolated
by semi-preparative HPLC method combined with silica gel-based chromatographic methods. NMR
spectra including 1D NMR (1H, 13C, DEPT135) and 2D NMR (COSY, HSQC, HMBC) were
introduced to identify the extractables, besides HPLC, GC–MS, ESI–MS/MS and HRMS. The
extractables were determined to be N-(2-(2,2,4,4-tetramethylcyclohexyl)allyl) benzo[d]thiazol-2-
amine (1), 2,6-Di-tert-butyl-4-methylphenol (2) and sulfur (3) respectively. Then, to address safety
concerns, approaches including QSAR analysis, the TTC and comprehensive literature evaluation
methods to toxicological safety evaluation of the target compounds were established, where the safety
threshold such as TTC and PDE values were developed. Finally, the migration testing of the
extractables were performed to assess the leaching behavior of the rubber closures. An optimized
analysis method was proposed using SPE and HPLC with an ultraviolet detector, which demonstrated
good linearity, acceptable accuracy and precision.
Solid state characterization of azelnidipine–oxalic acid co-crystal and co-amorphous complexes:
The effect of different azelnidipine polymorphs
Yahui Pan, Wenzhe Pang, Jie Lv, Jing Wang, Wei Guo
ABSTRACT
In present study, based on the two polymorphs (α and β form) of azelnidipine (AZE), 12 complexes of
AZE and oxalic acid (OXA) were prepared by solvent-assisted grinding (SG) and neat powder
grinding (NG) methods at the AZE/OXA molar ratios of 2:1, 1:1, and 1:2. The effect of the different
polymorphs of AZE on the micro-structure of the complexes were investigated by powder X-ray
diffraction (PXRD), tempreture modulated differential scanning calorimetry and thermogravimetric
analysis, cryo-field emission scanning electron microscope system, fourier transform infrared (FTIR),
and solid-state nuclear magnetic resonance spectroscopy. β-AZE-OXA co-crystal was produced at β-
AZE/OXA molar ratio of 2:1 when SG method was used; while α-AZE was used to produce α-AZE-
OXA co-crystal at same condition. However, the other 10 combinations were in co-amorphous forms,
including the NG samples with α (or β)-AZE/OXA molar ratios of 2:1, 1:1 (SG and NG), and 1:2 (SG
and NG). Although the XRD pattern and IR spectra of the two co-crystals showed no difference, the
melting enthalpy and specific heat cp of the β-AZE-OXA co-crystal was higher than that of the α-
AZE-OXA co-crystal, indicating that the numbers of solvent molecules which entered the two co-
crystal lattices were different. Interestingly, obvious difference occurred in the IR spectra between the
α-AZE-OXA and β-AZE-OXA co-amorphous systems. 1745 cm−1 wave-numbers, which were
assigned to the free CO groups, appeared in the α-AZE-OXA co-amorphous systems even when just a
small amount of OXA was introduced, thereby indicating the presence of different intermolecular
forces in the two series of co-amorphous forms.
92
Biorelevant physicochemical profiling of (E) - and (Z)-resveratrol determined from isomeric
mixtures
Gábor Orgován, Imre Gonda, Béla Noszál
ABSTRACT
Biorelevant, isomer-specific physicochemical parameters of resveratrol, a multifunctional component
in red wines, with cardioprotective, anti-Alzheimer and several other pharmacologic activities were
determined. The parameters include site-specific basicities, lipophilicities, solubilities and diffusion
constants for the two geometric isomers. The protonation equilibria of (E)- and (Z)-resveratrol were
monitored by 1H NMR-pH titrations. Five closely related auxiliary compounds ((E)-pinostilbene, (Z)-
pinostilbene, (E)-pterostilbene, (Z)-pterostilbene and resorcinol) were also studied. Combining the
datasets, the group-specific protonation constants of resveratrol isomers were determined. The results
show that (Z)-resveratrol is more basic at every protonation site than the (E)-isomer. Lipophilicities are
quantified in terms of logP values and were determined by octanol/water partition experiments and
quantitative NMR spectroscopy: (E)-resveratrol was found to be more lipophilic. Since the molecular
geometries of the isomers differ, diffusion ordered NMR spectroscopy (DOSY) experiments were also
carried out to quantify the diffusion capabilities of the isomers: (Z)-resveratrol of bent shape has a
slightly higher diffusion coefficient than its extended (E) counterpart. A striking 10-fold difference of
water solubility was found in favor of the (Z) isomer, due obviously to the reduced water-repellent
character in the more compact molecule.
An improved size exclusion-HPLC method for molecular size distribution analysis of
immunoglobulin G using sodium perchlorate in the eluent
Hsiaoling Wang, Mark S. Levi, Alfred V. Del Grosso, William M. McCormick, Lokesh Bhattacharyya
ABSTRACT
Size exclusion (SE) high performance liquid chromatography (HPLC) is widely used for the molecular
size distribution (MSD) analyses of various therapeutic proteins. We report development and
validation of a SE-HPLC method for MSD analyses of immunoglobulin G (IgG) in products using a
TSKgel SuperSW3000 column and eluting it with 0.4 M NaClO4, a chaotropic salt, in 40 mM
phosphate buffer, pH 6.8. The chromatograms show distinct peaks of aggregates, tetramer, and two
dimers, as well as the monomer and fragment peaks. In addition, the method offers about half the run
time (12 min), better peak resolution, improved peak shape and more stable base-line compared to
HPLC methods reported in the literature, including that in the European Pharmacopeia (EP). A
comparison of MSD analysis results between our method and the EP method shows interactions
between the protein and the stationary phase and partial adsorption of aggregates and tetramer on the
stationary phase, when the latter method is used. Thus, the EP method shows lower percent of
aggregates and tetramer than are actually present in the products. In view of the fact that aggregates
have been attributed to playing a critical role in adverse reactions due to IgG products, our observation
raises a major concern regarding the actual aggregate content in these products since the EP method is
widely used for MSD analyses of IgG products. Our method eliminates (or substantially reduces) the
interactions between the proteins and stationary phase as well as the adsorption of proteins onto the
column. Our results also show that NaClO4 in the eluent is more effective in overcoming the
protein/column interactions compared to Arg-HCl, another chaotropic salt. NaClO4 is shown not to
affect the molecular size and relative distribution of different molecular forms of IgG.
93
Assessment of the structure of pegylated-recombinant protein therapeutics by the NMR
fingerprint assay
Derek J. Hodgson, Yves Aubin
ABSTRACT
A number of recombinant protein therapeutic products, such as filgrastim (methionyl granulocyte
colony stimulating factor [Met-GCSF] used to boost the immune system in chemotherapy treated
cancer patients), and interferon alpha-2 (used for the treatment of various viral infections), have been
chemically modified with the addition of a polyethylene glycol (PEG) chain. This modification
prolongs residency of the drug in the body and reduces metabolic degradation, which allows less
frequent administration of the products. Here we show how NMR spectroscopy methods can assess the
higher order structure (HOS) of pegylated-filgrastim (Neulasta®), pegylated interferon-α2a
(Pegasys®) pegylated interferon-α2b (PEG-Intron®) purchased from the marketplace. The addition of
the PEG moiety effectively doubles the molecular weight of the three products. This presents a
significant challenge for the application of NMR techniques. Nevertheless, the results showed that
high-resolution spectra could be recorded for two of the three products. Comparison of the spectra of
the pegylated protein and the non-pegylated protein shows that the chemical modification did not alter
the HOS of these proteins.
Prediction of the hydroxypropyl cellulose—poly (vinyl alcohol) ratio in aqueous solution
containing papaverine hydrochloride in terms of drug loaded electrospun fiber formation
Adrienn Kazsoki, Péter Szabó, Romána Zelkó
ABSTRACT
Papaverine hydrochloride loaded electrospun fibers were prepared for buccal drug delivery with the
aim of improving the oral bioavailability of the crystalline drug, which can be achieved by the
increased solubility and by the circumvention of the intensive first pass metabolism. The water soluble
hydroxypropyl cellulose (HPC) was chosen as a mucoadhesive polymer. In order to improve the
electrospinnability of HPC, the also mucoadhesive poly(vinyl alcohol) (PVA) was used. During the
experiments, the total polymer concentration was kept constantly at 15% (w/w), and only the ratio of
the two polymers was changed. Five different HPC:PVA ratios (5:5, 6:4, 7:3, 8:2, 9:1) were examined.
Combination of rheological measurements and molar reflectance characterization with scanning
electron microscopy was applied for the determination of the optimum composition of the gels for
fiber formation. The crystalline-amorphous transition of papaverine hydrochloride was also tracked by
Fourier transform infrared spectroscopy. A correlation was found between the macrostructural
properties of the polymer solutions and their electrospinnability and the consequent morphology of the
resultant samples. Along with the changes of the polymer ratio, the corresponding morphology of the
electrospun samples also varied. With decreasing HPC ratio of the system, a transition from the spray-
dried film-like structure through fibrous film to fibers was observed. Polymer solutions of the lowest
elasticity and smallest intermolecular interactions contributed to the best fiber characteristics of the
samples. The results enable the determination of the polymer ratio for the formation of applicable
quality of electrospun fibers. According to the results 5:5 and 6:4 polymer ratios enabled the best fiber
performance.
94
Optimization of dispersive liquid-phase microextraction based on solidified floating organic drop
combined with high-performance liquid chromatography for the analysis of glucocorticoid
residues in food
Yuan Huang, Zhiqun Zheng, Liying Huang, Hong Yao, Dandan Lin
ABSTRACT
A rapid, simple, cost-effective dispersive liquid-phase microextraction based on solidified floating
organic drop (SFOD-LPME) was developed in this study. Along with high-performance liquid
chromatography, we used the developed approach to determine and enrich trace amounts of four
glucocorticoids, namely, prednisone, betamethasone, dexamethasone, and cortisone acetate, in animal-
derived food. We also investigated and optimized several important parameters that influenced the
extraction efficiency of SFOD-LPME. These parameters include the extractant species, volumes of
extraction and dispersant solvents, sodium chloride addition, sample pH, extraction time and
temperature, and stirring rate. Under optimum experimental conditions, the calibration graph exhibited
linearity over the range of 1.2–200.0 ng/ml for the four analytes, with a reasonable linearity(r2:
0.9990–0.9999). The enrichment factor was 142–276, and the detection limits was 0.39–0.46 ng/ml
(0.078–0.23 μg/kg). This method was successfully applied to analyze actual food samples, and good
spiked recoveries of over 81.5%–114.3% were obtained.
A novel LCMSMS method for quantitative measurement of short-chain fatty acids in human
stool derivatized with 12C- and 13C-labelled aniline
James Chun Yip Chan, Dorinda Yan Qin Kioh, Gaik Chin Yap, Bee Wah Lee, Eric Chun Yong Chan
ABSTRACT
A novel liquid chromatography tandem mass spectrometry (LCMSMS) method for the quantitative
measurement of gut microbial-derived short-chain fatty acids (SCFAs) in human infant stool has been
developed and validated. Baseline chromatographic resolution was achieved for 12 SCFAs (acetic,
butyric, caproic, 2,2-dimethylbutyric, 2-ethylbutyric, isobutyric, isovaleric, 2-methylbutyric, 4-
methylvaleric, propionic, pivalic and valeric acids) within an analysis time of 15 min. A novel
sequential derivatization of endogenous and spiked SCFAs in stool via 12C- and 13C-aniline
respectively, facilitated the accurate quantitation of 12C-aniline derivatized endogenous SCFAs based
on calibration of exogenously 13C-derivatized SCFAs. Optimized quenching of derivatization agents
prior to LCMSMS analysis further reduced to negligible levels the confounding chromatographic peak
due to in-line derivatization of unquenched aniline with residual acetic acid present within the LCMS
system. The effect of residual acetic acid, a common LCMS modifier, in analysis of SCFAs has not
been addressed in previous SCFA assays. For the first time, a total of 9 SCFAs (acetic, butyric,
caproic, isobutyric, isovaleric, 2-methylbutyric, 4-methylvaleric, propionic and valeric acids) were
detected and quantitated in 107 healthy infant stool samples. The abundance and diversity of SCFAs in
infant stool vary temporally from 3 weeks onwards and stabilize towards the end of 12 months. This in
turn reflects the maturation of infant SCFA-producing gut microbiota community. In summary, this
novel method is applicable to future studies that investigate the biological roles of SCFAs in paediatric
health and diseases.
95
Mixed-mode reversed phase/positively charged repulsion chromatography for intact protein
separation
Ling Ding, Zhimou Guo, Zhuo Hu, Xinmiao Liang
ABSTRACT
A mixed-mode reversed phase/positively charged repulsion stationary phase C8PN composed of octyl
and amino group has been developed for separation of intact protein. Before the separation of proteins,
a set of probe compounds were employed to evaluate the chromatographic properties of C8PN,
demonstrating typical reversed phase/positively charged repulsion interaction on this stationary phase
as estimated. Then the new C8PN stationary phase was used to separate a standard protein mixture on
the reversed phase mode. Compared with a commercial C4 stationary phase, it showed different
selectivity for some proteins. In order to better understand the properties of C8PN, the effect of
acetonitrile content was investigated based on retention equation. Higher values of the equation
parameters on C8PN demonstrated that the protein retentions were more sensitive to the change of
acetonitrile content. Besides, the influences of buffer salt additives on the protein retentions were also
studied. The retention factors of the proteins got larger with the increase of buffer salt concentration,
which confirmed the positively charged repulsion interaction on the column. Finally, the C8PN was
further applied to separate oxidized- and reduced- forms of Recombinant Human Growth Hormone.
Our study indicated the advantages and application potential of mixed-mode reversed phase/positively
charged repulsion stationary phase for intact protein separation.
Quantitative determination of five metabolites of aspirin by UHPLC–MS/MS coupled with
enzymatic reaction and its application to evaluate the effects of aspirin dosage on the metabolic
profile
Jian-Ping Li, Jian-Ming Guo, Er-Xin Shang, Zhen-Hua Zhu, Jin-Ao Duan
ABSTRACT
Acetylsalicylic acid (Aspirin, ASA) is a famous drug for cardiovascular diseases in recent years.
Effects of ASA dosage on the metabolic profile have not been fully understood. The purpose of our
study is to establish a rapid and reliable method to quantify ASA metabolites in biological matrices,
especially for glucuronide metabolites whose standards are not commercially available. Then we
applied this method to evaluate the effects of ASA dosage on the metabolic and excretion profile of
ASA metabolites in rat urine. Salicylic acid (SA), gentisic acid (GA) and salicyluric acid (SUA) were
determined directly by UHPLC–MS/MS, while salicyl phenolic glucuronide (SAPG) and salicyluric
acid phenolic glucuronide (SUAPG) were quantified indirectly by measuring the released SA and SUA
from SAPG and SUAPG after β-glucuronidase digestion. SUA and SUAPG were the major
metabolites of ASA in rat urine 24 h after ASA administration, which accounted for 50% (SUA) and
26% (SUAPG). When ASA dosage was increased, the contributions dropped to 32% and 18%,
respectively. The excretion of other three metabolites (GA, SA and SAPG) however showed
remarkable increases by 16%, 6% and 4%, respectively. In addition, SUA and SUAPG were mainly
excreted in the time period of 12–24 h, while GA was excreted in the earlier time periods (0–4 h and
4–8 h). SA was mainly excreted in the time period of 0–4 h and 12–24 h. And the excretion of SAPG
was equally distributed in the four time periods. We went further to show that the excretion of five
metabolites in rat urine was delayed when ASA dosage was increased.
96
HPLC–MS/MS method for the simultaneous quantification of desmethylmebeverine acid,
mebeverine acid and mebeverine alcohol in human plasma along with its application to a
pharmacokinetics study
Natalia E. Moskaleva, Pavel A. Baranov, Natalia V. Mesonzhnik, Svetlana A. Appolonova
ABSTRACT
A new simple, rapid and sensitive high pressure liquid chromatography-tandem mass spectrometry
(HPLC–MS/MS) method was developed and validated for simultaneous analysis of mebeverine
metabolites as: mebeverine alcohol (MAL), mebeverine acid (MAC) and desmethylmebeverine acid
(DMAC) in human plasma. Sample preparation was performed by protein precipitation following the
separation of analytes using an Acquity UPLC BEN C8 column 1.7 mm 2.1 × 50 mm (Waters, USA).
2H5-desmethylmebeverine acid (2H5-DMAC) was used as the internal standard (IS). The proposed
method was validated with linear ranges of 0.1–10 ng/mL; 1–100 ng/mL and 5–1000 ng/mL for MAL,
MAC and DMAC, respectively. Accuracy for all analytes (%RE), given as deviation between nominal
and measured concentration and assay variability (CV) ranged from −4.04% to 4.60% and from 0.31%
to 6.43% respectively both for within- and between-run. The overall recoveries for all metabolites
were above 85%. The proposed method was used successfully for analysis of real samples from a
pharmacokinetics study.
The impact of ion-pairing reagents on the selectivity and sensitivity in the analysis of modified
oligonucleotides in serum samples by liquid chromatography coupled with tandem mass
spectrometry
Sylwia Studzińska, Rafał Rola, Bogusław Buszewski
ABSTRACT
Present study highlights the usage of various ion-pairing agents and their impact on the process of
separation and ionization of oligonucleotides in the fortified human serum samples. What is more,
retention studies involved different stationary phases, including: octadecyl, phenyl, pentafluorophenyl
groups and ligands with embedded polar groups. It was proved that retention of oligonucleotides
strongly depends on the alkyl chain branching in the structure of ion pairing reagent. Furthermore ion-
pairing agents build of straight alkyl chain are more easily adsorbed on the stationary phase modified
with octadecyl groups, while branching of alkyl chain caused more effective adsorption of studied
compounds at phenyl groups compared to octadecyl ones. The lowest limit of quantification values
were obtained for 5 mM N,N-dimethylbutylamine, while the highest values are characteristic for
hexylamine. Moreover it was shown that a 2-fold increase of ion-pairing agent concentration results in
higher LOQ. The greatest sensitivity was obtained for 2.5 mM N,N-dimethylbutylamine/150 mM
hexafluoroisopropanol. On the other hand Hypersil GOLD aQ column was the most appropriate in
terms of time and separation effectiveness. The developed method was successfully used for the
determination of studied oligonucleotides and their metabolites in human serum samples. The
compounds were separated in just 3.5 min with high sensitivity (0.09–0.16 ng).
97
A novel LC–MS/MS method for the simultaneous quantification of topiramate and its main
metabolites in human plasma
Daniela Milosheska, Robert Rońkar
ABSTRACT
The aim of the present report was to develop and validate simple, sensitive and reliable LC–MS/MS
method for quantification of topiramate (TPM) and its main metabolites: 2,3-desisopropylidene TPM,
4,5-desisopropylidene TPM, 10-OH TPM and 9-OH TPM in human plasma samples. The most
abundant metabolite 2,3-desisopropylidene TPM was isolated from patients urine, characterized and
afterwards used as an authentic standard for method development and validation. Sample preparation
method employs 100 μL of plasma sample and liquid–liquid extraction with a mixture of ethyl acetate
and diethyl ether as extraction solvent. Chromatographic separation was achieved on a 1290 Infinity
UHPLC coupled to 6460 Triple Quad Mass Spectrometer operated in negative MRM mode using
Kinetex C18 column (50 × 2.1 mm, 2.6 μm) by gradient elution using water and methanol as a mobile
phase and stable isotope labeled TPM as internal standard. The method showed to be selective,
accurate, precise and linear over the concentration ranges of 0.10–20 μg/mL for TPM, 0.01–2.0 μg/mL
for 2,3-desisopropylidene TPM, and 0.001–0.200 μg/mL for 4,5-desisopropylidene TPM, 10-OH TPM
and 9-OH TPM. The described method is the first fully validated method capable of simultaneous
determination of TPM and its main metabolites in plasma over the selected analytical range. The
suitability of the method was successfully demonstrated by the quantification of all analytes in plasma
samples of patients with epilepsy and can be considered as reliable analytical tool for future
investigations of the TPM metabolism.
UPLC–MS/MS method for the simultaneous quantification of three new antiretroviral drugs,
dolutegravir, elvitegravir and rilpivirine, and other thirteen antiretroviral agents plus cobicistat
and ritonavir boosters in human plasma
Marco Simiele, Alessandra Ariaudo, Amedeo De Nicolò, Fabio Favata, Antonio D‘Avolio
ABSTRACT
Rilpivirine (RPV), dolutegravir (DTG) and elvitegravir (EVG) are the latest antiretroviral drugs
approved for treatment of HIV infection. Currently, poor information is currently available concerning
their pharmacokinetic and pharmacodynamic properties, thus making the use of therapeutic drug
monitoring for these drugs not useful. This lack of information is partially due to the absence of a
high-throughput method for their simultaneous quantification together with other antiretroviral drugs.
In this work, we describe the development and validation of a new UPLC–MS/MS method to quantify
these drugs, together with other fourteen antiretroviral agents, in human plasma. One hundred
microliters of plasma samples were added with internal standard (6,7-Dimethyl- 2,3-di(2-pyridyl)
quinoxaline), underwent a simple protein precipitation with methanol:acetonitrile (50:50 v/v) followed
by sample dilution with water. Chromatographic separation was performed on a Acquity® UPLC HSS
T3 column (150 mm x 2.1 mm I.D) with a particle size of 1.8 μm and compounds were detected with a
tandem mass detector, monitoring two ion transitions for each drugs. The mean recovery of RPV, DTG
and EVG were 101%, 87% and 112.3% respectively.
98
Concentrations of antibodies against β-amyloid 40/42 monomer and oligomers in Chinese
intravenous immunoglobulins
Shengliang Ye, Renyong Zeng, Peng Jiang, Mingxia Hou, Changqing Li
ABSTRACT
Intravenous immunoglobulin (IVIg) preparations are being investigated as a potential agent for
treatment or prevention of Alzheimer‘s disease (AD). Antibodies towards soluble β-amyloid (Aβ)
contained in IVIg were considered to be the major component contributing to the beneficial effect of
the preparations in pilot studies. This study compared the antibody concentrations against Aβ in
Octagam® IVIg (Octapharma) and 9 IVIg preparations from different Chinese manufacturers by
ELISA, using Aβ40 monomer, Aβ40 soluble oligomers, Aβ42 monomer and Aβ42 soluble oligomers
as the antigens. The results showed that each preparation contained different antibody levels against
the four Aβ forms. The median values of the four antibody concentrations in Chinese IVIg
preparations were 16.53, 8.47, 24.36 and 33.25 μg/mL, which were remarkably higher than that in
Octagam® IVIg (1.66, 2.07, 4.61 and 4.64 μg/mL). Moreover, the anti-Aβ42 oligomer antibody levels
in almost all IVIg preparations were higher than the anti-Aβ42 monomer antibody, and the
concentrations of anti-Aβ42 antibodies in most of the IVIg preparations were significantly higher than
that of anti-Aβ40 antibodies. These findings will contribute to an increased understanding of the
uniqueness of Chinese IVIg preparations, and could provide support for a trial of a Chinese IVIg
product in AD patients.
Simultaneous quantification of estrogens, their precursors and conjugated metabolites in human
breast cancer cells by LC–HRMS without derivatization
Stefan Poschner, Martin Zehl, Alexandra Maier-Salamon, Walter Jäger
ABSTRACT
Liquid chromatography–mass spectrometry (LC–MS) is the state of the art technique for quantification
of steroid hormones. Currently used methods are typically limited by the need of pre-column
derivatization to increase ionization efficiency; however, this causes hydrolysis of conjugated
metabolites. Our newly established LC–HRMS method is able to simultaneously quantify conjugated
and unconjugated steroids without prior derivatization using deuterated internal standards and solid-
phase extraction. This assay was validated according to ICH Q2(R1) guidelines for the analysis of the
10 main steroids of the estrogenic pathway, namely 4-androstene-3,17-dione, dehydroepiandrosterone
(DHEA), DHEA-3-sulfate, estrone, 17β-estradiol, estriol (16α-OH-17β-estradiol), estrone-3-sulfate,
17β-estradiol-3-(β-d-glucuronide), 17β-estradiol-3-sulfate and testosterone. Assay performance
characteristics were excellent with results for accuracy (98.8–101.2%), precision (mean: 2.05%, all
≤2.80%), stability over five freeze–thaw-cycles (95.7–100.4%) and SPE accuracy (96.9–102.0%), as
well as suitable lower and upper limits of quantification for cell culture experiments (LLOQ 0.005–2
ng/ml, ULOQ 3–2000 ng/ml). Furthermore, we demonstrated the functionality of our method for the
monitoring of steroid levels in the human breast cancer cell line MCF-7. This sensitive assay allows
for the first time detailed investigations on estrogen metabolomics in breast cancer cells and may also
apply to other estrogen-dependent tumor entities.
99
Quantification and clinical application of carboplatin in plasma ultrafiltrate
Kim Downing, Berit Packert Jensen, Sue Grant, Matthew Strother, Peter George
ABSTRACT
Carboplatin is a chemotherapy drug used in a variety of cancers with the primary toxicity being
exposure-dependant myelosuppression. We present the development and validation of a simple, robust
inductively coupled plasma mass spectrometry (ICP-MS) method to measure carboplatin in plasma
ultrafiltrate. Plasma ultrafiltrates samples were prepared using Amicon Ultra 30,000 da cut-off filters
and then diluted with ammonia EDTA before ICP-MS analysis. The assay was validated in the range
0.19–47.5 mg/L carboplatin in ultrafiltrate. The assay was linear (r2 > 0.9999), accurate (<6% bias,
12% bias at LLOQ) and precise (intra- and inter-day precision of <3% coefficient of variation). No
matrix effects were observed between plasma ultrafiltrate and aqueous platinum calibrators and
recovery was complete. The assay was applied to 10 clinical samples from patients receiving
carboplatin. Incurred sample reanalysis showed reproducible values over 3 analysis days (<6% CV).
As plasma stability prior to ultrafiltration has been a major concern in previous clinical studies this was
studied extensively at room temperature (22 °C) over 24 h. Carboplatin was found to be stable in both
spiked plasma (n = 3) and real patient samples (n = 10) at room temperature for up to 8 h before
ultrafiltration. This makes routine measurement of carboplatin concentrations in clinical settings
feasible.
An LC–MS/MS method for the determination of digitoxigenin in skin samples and its
application to skin permeation and metabolic stability studies
Xinchi Feng, Joel Turley, Zijian Xie, Sandrine V. Pierre, Jinsong Hao
ABSTRACT
An LC–MS/MS method was developed for the determination of digitoxigenin in mice skin samples.
Chromatographic separation was achieved on an Agilent Poroshell 120 EC-C18 column. Mass
spectrometric detection was achieved by a triple-quadrupole mass spectrometer equipped with an ESI
interface operating in a positive ionization mode. Quantification was performed using selective
reaction monitoring of the precursor-product ion transitions of m/z 375.5 → 339 for digitoxigenin and
m/z 391.5 → 337 for internal standard (IS). The calibration curves were linear over the concentration
range of 1.00–500 ng/mL. The intra- and inter-batch precision was no more than 10.6% of the
coefficient of variation and the accuracy was within ±8.1% of the actual values. This validated method
has been successfully applied to skin permeation and skin metabolic stability studies of digitoxigenin
in mice. The steady-state flux and lag time of digitoxigenin permeated across the full-thickness mice
skin were 1.86 ± 0.45 μg/cm2/h and 0.46 ± 0.18 h, respectively. The metabolism of digitoxigenin in
the skin was not detected in our study.
100
Stress-induced changes of neurosteroid profiles in rat brain and plasma under immobilized
condition
Myeong Hyeon Park, Shaheed Ur Rehman, In Sook Kim, Min Sun Choi, Hye Hyun Yoo
ABSTRACT
In this study, various neurosteroids in brain and plasma were simultaneously determined using liquid
chromatography-tandem mass spectrometry and their profile changes in a stress-induced rat were
investigated. The investigated neurosteroids are as follows: progesterone (P4), 5α-dihydroprogesterone
(5α-DHP), 5β-dihydroprogesterone, estrone, androstenedione (AE), cortisol, cortisone, corticosterone
(CORT), dehydroepiandrosterone (DHEA), pregnanolone (3α,5β-THP), allopregnanolone (ALLO),
11-deoxycorticosterone (DOC), 11-deoxycortisol, pregnenolone (PREG), and 5α/5β-
tetrahydrodeoxycorticosterone (5α/5β-THDOC). Brain and plasma samples were processed using
solid-phase extraction with methanol and acetic acid (99:1), and derivatized with a hydroxylamine
reagent. Separation was achieved within 13 min at a flow rate of 0.4 mL/min with a C18 column (3.0 ×
50 mm, 2.7 μm). The triple quadrupole mass spectrometer was operated in the positive electrospray
ionization mode. Using this method, the neurosteroid level variation was quantitated and investigated
in the brain and plasma upon immobilization stress in rats. As a result, AE, CORT, DOC, P4, 5α-DHP,
5α/5β-THDOC, DHEA, 3α,5β-THP, ALLO, and PREG levels were significantly altered in both the
brain and plasma samples when stress was induced.
Identification, characterization and in silico ADMET prediction of Roflumilast degradation
products
Mariana S. Pinheiro, Gil M. Viana, Bárbara de A. Abrahim Vieira, Alessandra Mendonça Teles de
Souza, Valéria P. de Sousa
ABSTRACT
The present study reports the degradation behavior of roflumilast (RFL), a new drug developed for the
treatment of chronic obstructive pulmonary disease. The degradation of RFL was tested under various
stress conditions as per the guidelines of the International Conference on Harmonization. The
degradation products (DPs) of RFL were identified, characterized and in silico predictions were made
of their pharmacokinetic properties, absorption, distribution, metabolism, excretion and toxicity
(ADMET). RFL was subjected to various stress conditions including photodegradation, alkaline and
acidic hydrolysis, oxidative and metallic degradation. After analysis by HPLC-DAD, the DPs were
isolated by preparative TLC and characterized by high resolution mass spectrometry (HRMS), 1H
NMR, 13C NMR and infrared (IR) spectroscopy. RFL tablets were prepared by the addition of solid
stressing substances such as excipients and storage in an accelerated stability chamber (40 °C; 75%
r.h.) for sixteen months. Resulting DPs from the tablets were analyzed by UFLC-QTOF. The most
drastic degradation conditions for RFL were 5 M NaOH(aq), 6 M HCl(aq), 7.5% v/v peracetic acid,
which resulted in the isolation of four DPs. However, milder degradation conditions (1 M NaOH(aq)
and photolysis) generated six DPs (DP-1, 2, 3, 5, 7 and 8), and are more similar to the actual
conditions the drug will be exposed. For tablets containing RFL exposed to an alkaline reagent, two
DPs were formed: DP-1 and DP-11. Where as RFL-containing tablets exposed to acid and oxidizing
agents, formed one products DP-11. Forced degradation of RFL led to the formation of eleven DPs,
seven of which have never been previously reported. RFL is stable under metallic stress and it is
relatively stable during photodegradation testing.
101
A rapid and robust UHPLC-DAD method for the quantification of amphotericin B in human
plasma
Sebastiano Barco, Alessia Zunino, Antonio D‘Avolio, Laura Barbagallo, Giuliana Cangemi
ABSTRACT
Amphotericin B is an antifungal drug widely used in Intensive Care Units. Therapeutic drug
monitoring (TDM) of amphotericin B is recommended for the assessment of toxicity surveillance and
treatment optimization. In this paper we described the development and validation of a new Ultra High
Performance Liquid Chromatography coupled to Diode Array Detection (UHPLC-DAD) method for
the quantification of Amphotericin B in 200 μL human plasma over a wide range of concentrations
(0.125–10 mg/L). The new method has been validated following international guidelines on
bioanalytical method validation and showed high selectivity, high accuracy and precision and high
process efficiency. The new UHPLC-DAD method that we describe is robust, rapid, cost effective and
suitable for application to the routine TDM analyses.
Quantitative analysis of mycosporine-like amino acids in marine algae by capillary
electrophoresis with diode-array detection
Anja Hartmann, Adele Murauer, Markus Ganzera
ABSTRACT
Marine species have evolved a variety of physical or chemical strategies to diminish damage from
elevated environmental ultraviolet radiation. Mycosporine-like amino acids, a group of widely
distributed small water soluble compounds, are biologically relevant because of their photo-protective
potential. In addition, presumed antioxidant and skin protective strategies raise the interest for possible
medicinal and cosmetic applications. In this study the first CE method for the quantification of
mycosporine-like amino acids in marine species is presented. A borate buffer system consisting of 30
mM sodium tetraborate in water at a pH-value of 10.3 enabled the baseline separation of five MAAs,
namely palythine, mycosporine-serinol, asterina-330, shinorine and porphyra-334, in 27 min.
Separation voltage, temperature and detection wavelength were 25 kV, 25 °C and 320 nm,
respectively. The optimized method was fully validated and applied for the quantitative determination
of MAAs in the marine macroalgae Palmaria palmata, Porphyra umbilicalis, and Porphyra sp., as well
as the lichen Lichina pygmaea.
102
LC–MS/MS assay for the simultaneous quantitation of the ATM inhibitor AZ31 and the ATR
inhibitor AZD6738 in mouse plasma
Brian F. Kiesel, Jeffrey C. Shogan, Madani Rachid, Robert A. Parise, Jan H. Beumer
ABSTRACT
The ATM kinase inhibitor AZ31 and ATR kinase inhibitor AZD6738 are in various phases of
preclinical and clinical evaluation for their ability to potentiate chemoradiation. To support the
preclinical evaluation of their pharmacokinetics, we developed and validated an LC–MS/MS assay for
the simultaneous quantification of AZ31 and AZD6738 in mouse plasma. A ―dilute and shoot‖ method
was used to precipitate proteins from a sample volume of 50 μL. Chromatographic separation was
achieved using a Phenomenex Polar-RP column and a gradient mobile phase consisting of methanol–
water with 0.1% formic acid. Detection was accomplished using a Waters Quattro Micro mass
spectrometer in positive ionization mode. The assay utilizing 50 μL sample was linear from 10 to 5000
ng/mL and determined to be both accurate (−8.2 to 8.6%) and precise (<5.4% CV) and achieved the
criteria for U.S. FDA guidance for bioanalytical method validation. Quantification was achieved in
mouse tissue homogenate using a separate 200 μL sample preparation. This LC–MS/MS assay will be
essential for determining the tissue distribution and pharmacokinetics in future mouse studies.
The atypical excretion profile of meldonium: Comparison of urinary detection windows after
single- and multiple-dose application in healthy volunteers
Christian Görgens, Sven Guddat, Christina Bosse, Hans Geyer, Mario Thevis
ABSTRACT
Following a one-year monitoring program providing unequivocal analytical evidence for a high
prevalence in international elite sports, meldonium has been included in the World Anti-Doping
Agency‘s (WADA) list of prohibited substances that came into effect on 1 January 2016. Despite of
the polar and hydrophilic nature of the molecule, an unusual long detection window was observed in
pilot elimination studies. Consequently, in the present study, urinary excretion profiles after single-
dose (5 volunteers, 1 × 500 mg) and multiple-dose oral application (5 volunteers; 2 × 500 mg/day for 6
days) were determined in order to facilitate the result management concerning meldonium findings in
doping controls. Particularly the option to differentiate between recent use and tapering concentrations
was studied. Urinary meldonium concentrations were determined using an analytical approach based
on hydrophilic interaction liquid chromatography and high resolution tandem mass spectrometry. The
study corroborates the hypothesis of a non-linear, dose-depended and biphasic excretion profile after
oral application of meldonium and demonstrates that urinary detection windows are of considerable
extent with up to 65 and 117 days (concentrations > LOQ of 10 ng/mL) following single- and
multiple-dose applications, respectively.
103
Simultaneous quantitation of abiraterone, enzalutamide, N-desmethyl enzalutamide, and
bicalutamide in human plasma by LC–MS/MS
Kyu-pyo Kim, Robert A. Parise, Julianne L. Holleran, Lionel D. Lewis, Jan H. Beumer
ABSTRACT
Inhibiting the androgen receptor (AR) pathway is an important clinical strategy in metastatic prostate
cancer. Novel agents including abiraterone acetate and enzalutamide have been shown to prolong life
in men with metastatic, castration-resistant prostate cancer (mCRPC). To evaluate the
pharmacokinetics of AR-targeted agents, we developed and validated an LC–MS/MS assay for the
quantitation of enzalutamide, N-desmethyl enzalutamide, abiraterone and bicalutamide in 0.05 mL
human plasma. After protein precipitation, chromatographic separation was achieved with a
Phenomenex Synergi Polar-RP column and a linear gradient of 0.1% formic acid in methanol and
water. Detection with an ABI 4000Q mass spectrometer utilized electrospray ionization in positive
multiple reaction monitoring mode. The assay was linear over the ranges of 1–1000 ng/mL for
abiraterone and bicalutamide and 100–30,000 ng/mL for N-desmethyl enzalutamide and enzalutamide
and proved to be accurate (92.8–107.7%) and precise (largest was 15.3% CV at LLOQ for
bicalutamide), and fulfilled FDA criteria for bioanalytical method validation. We demonstrated the
suitability of this assay in plasma from patients who were administered enzalutamide 160 mg,
abiraterone 1000 mg and bicalutamide 50 mg once a day as monotherapy or in combination. The LC–
MS/MS assay that has been developed will be an essential tool that further defines the pharmacology
of the combinations of androgen synthesis or AR-receptor targeted agents.
New enantioselective LC method development and validation for the assay of modafinil
Marianna Harvanová, Taťána Gondová
ABSTRACT
For the first time, a new, fast and sensitive chromatographic method for the separation and
determination of modafinil enantiomers was developed on chiral stationary phase with macrocyclic
glycopeptide teicoplanin in the polar organic mode. The effect of the mobile phase composition and
column temperature on the retention and enantioseparation were studied. The separation was
performed using a Chirobiotic T column (250 × 4.6 mm, 5 μm) with methanol and triethylamine
(100/0.05, v/v) as the mobile phase at a flow rate of 1.0 mL/min, and UV detection at 225 nm. The
total analysis time is less than 6 min, which is faster than the previous chiral HPLC methods (total run
time of 12 min). Calibration curves were linear (R2 > 0.999) over a concentration range 5–150 μg/mL
for each modafinil enantiomer. Limit of detection (LOD) and limit of quantification (LOQ) for (R)-
modafinil were 15 and 45 ng/mL and for (S)-modafinil were 20 and 60 ng/mL, respectively. The
recoveries were in the range of 100.5-102.3% with the relative standard deviations ranging from 0.4%
to 1.0% for both enantiomers. It was demonstrated that the developed method was selective, precise
and robust. The validated method was successfully applied for the separation and determination of
modafinil enantiomers in the real samples.
104
Identification of a novel low-level impurity in fungicide pyraclostrobin by high-performance
liquid chromatography/tandem mass spectrometry
Kaimin Xia, ShanShan Shen, Qun Gao, Wei Shang, Jun Wu
ABSTRACT
Pyraclostrobin is one kind of new type methoxy acrylate fungicides that has been widely used in
agriculture at present, with a lot of advantages including broad spectrum, high efficiency and high
selectivity. In this work, a novel low-level impurity in the pyraclostrobin at about 0.2% was separated
and characterized by high-performance liquid chromatography/tandem mass spectrometry (HPLC–
MS). Firstly, the impurity was speculated to possess the same skeleton structure as the main product
pyraclostrobin while the methyl group on the methyl ester was substituted to be CH2CH2Cl on the
basis of the on-line multi-stage mass spectrometric behaviors compared with that of pyraclostrobin.
Then the accurate molecular weight and element composition of target impurity was verified to be
C20H19Cl2N3O4 by high resolution mass spectrometry. Finally, the proposed structure was further
confirmed by the 1H NMR data.
Paeonifiorin sulfonate as a characteristic marker for specifically inspecting Chinese patent
medicine Liu-Wei-Di-Huang-Wan contained sulfur-fumigated Moutan Cortex
Xiu-Yang Li, Fang Long, Jin-Di Xu, Hong Shen, Song-Lin Li
ABSTRACT
Sulfur fumigation can induce chemical transformation of bioactive components, consequently the
alteration of bioactivities or even toxicities of medicinal herbs. Inspecting Chinese patent medicines
(CPM) contained sulfur-fumigated constituent herbs is crucial for ensuring the safety and efficacy of
CPM. Paeonifiorin sulfonate is a sulfur-fumigation induced compound of Moutan Cortex (MC), one of
the main constituent herbs of a commonly used CPM Liu-Wei-Di-Huang-Wan (LWDHW). Herein, we
investigated the approach of paeonifiorin sulfonate as a characteristic marker for specifically
inspecting LWDHW potentially contained sulfur-fumigated MC (SFMC). First, mimic LWDHW
samples contained SFMC (SFMC-LWDHW) and non-fumigated MC (NFMC-LWDHW) were
prepared respectively. Second, an LC–MS method was developed and validated to qualitatively and
quantitatively determine paeonifiorin sulfonate in the mimic LWDHW samples. Third, the established
method was applied to analyze the commercial LWDHW samples. The results showed that
paeoniflorin sulfonate could only be detectable in SFMC-LWDHW, but not in NFMC-LWDHW
samples. The CPM matrix could enhance the response of paeoniflorin sulfonate in mass spectrometry
analysis. In addition, the LOQ, linearity, precision, accuracy and stability were also demonstrated to be
acceptable for quantifying paeoniflorin sulfonate in LWDHW. Commercial samples analysis indicated
that paeoniflorin sulfonate were detectable in 9 of 10 commercial LWDHW samples, with the content
varied between 105.53 μg/g and 438.61 μg/g. All the results suggested that paeoniflorin sulfonate
could be used as a characteristic and reliable chemical marker for specifically inspecting commercial
LWDHW contained SFMC. This study also provides a new strategy for the quality control of other
CPMs potentially produced from sulfur-fumigated constituent herbs.
105
Simple on-line pretreatment of column-switching coupled with ion chromatography for the
determination of lactic acid in lobaplatin
Zhendong Zhao, Jianzhong Zhu, Yanli Xie
ABSTRACT
In this study, a simple and sensitive on-line pretreatment of column-switching technique coupled with
ion chromatography method was achieved for the determination of key impurity of lactic acid in
lobaplatin. The first dimension of reverse phase liquid chromatography was used to reduce organic
matrices, whereas the second dimension of ion chromatography was applied for the separation and
detection of lactic acid. The results exhibited good linearity (R = 0.9994) ranging from 0.01 mg L−1 to
50 mg L−1. The limit of detection was below 0.0015 mg L−1. Also, a spiking study was performed
with satisfactory recoveries between 96% and 106%.
Discovery of discriminatory quality control markers for Chinese herbal medicines and related
processed products by combination of chromatographic analysis and chemometrics methods:
Radix Scutellariae as a case study
Fei Wang, Bo Wang, Long Wang, Zi-Yue Xiong, Hui-Jun Li
ABSTRACT
The processing procedure of traditional Chinese herbal medicines (CHMs) plays an essential role in
clinical applications. However, little progress has been made on the quality control of crude and
processed products. The present work, taking Radix Scutellariae (RS), wine-processed RS and
carbonized RS as a typical case, developed a comprehensive strategy integrating chromatographic
analysis and chemometric methods for quality evaluation and discrimination of crude RS and its
processed products. Chemical fingerprints were established by high-performance liquid
chromatography coupled with photodiode array detector and quadrupole time-of-flight mass
spectrometry, and similarity analyses were calculated based on eleven common characteristic peaks.
Subsequently, four chemical markers were discovered by back propagation-artificial neural network
(BP-ANN) modeling. The selected markers were quantified by the ‗single standard to determine multi-
components‘ (SSDMC) method, and then the quantitative data were subjected to principal component
analysis (PCA) and partial least-squares discriminant analysis (PLS-DA). Furthermore, support vector
machine (SVM) was employed to predict the different processed products of RS. Finally, a hotmap
visualization was conducted for clarifying the distribution of major flavonoids among different drugs.
Collectively, the proposed strategy might be well-acceptable for quality control of CHMs and their
related processed products from the processing mechanism-based perspective.
106
New and cost effective cell-based assay for Dialyzed Leukocyte Extract (DLE)-induced Jurkat
cells proliferation under azathioprine treatment
F.M. Cardoso, M. Tomkova, D. Petrovajova, M. Bubanova, T. Hornakova
ABSTRACT
The human Dialyzed Leukocyte Extract (DLE) is a heterogeneous mix of oligopeptides of <10 kDa,
extracted from leukocytes of healthy donors. There is significant clinical evidence of improvement
using DLE during treatment of allergies, cancer, immunodeficiencies, and in mycotic and viral
infections. Nevertheless, the DLE exact nature and mechanism of action have been elusive for more
than 50 years. DLE biological activity testing is necessary in DLE production and quality control. Both
in vitro and in vivo assays exist: E-rosette test, induction of delayed type hypersensitivity in mice,
leukocyte migration and IFN-γ secretion. The animal-origin materials and in vivo assays convey a
considerable logistic, ethic and economic burden, meanwhile the available in vitro assays have been
reported with limited reproducibility and sometimes contradictory results. Here we are reporting a new
DLE biological activity cell-based assay. The A20 and Jurkat cell lines were treated with (+Aza) or
without (−Aza) azathioprine, DLE (+DLE) or both (+Aza/+DLE). After 72 h, the cell proliferation was
analyzed by the MTT or BrdU incorporation assays. In +Aza/+DLE treated cells, we observed a
significant higher proliferation, when compared with +Aza/-DLE. In the absence of Aza, cells did not
present any proliferation difference between −DLE or +DLE treatments. Both assays, MTT and BrdU
showed similar results, being the MTT test more cost effective and we select it for validation as DLE
biological assay using Jurkat cells only. We tested three different lyophilized DLE batches and we
found consistent results with acceptable assay reproducibility and linearity. The DLE capacity for
rescuing Jurkat cell proliferation during +Aza treatment was consistent using different liquid and
lyophilized DLE batches, presenting also consistent chromatographic profiles. Finally, DLE treatment
in Jurkat cells did not result into significant IL-2 of IFN-γ secretion, and known lymphocyte
proliferative drugs failed to rescue Jurkat cells viability in presence of +Aza, as +DLE treatment did in
our MTT assay.
Spectroscopy analysis and molecular dynamics studies on the binding of penicillin V and
sulbactam to beta-lactamase II from Bacillus cereus
Penghui Shi, Pan Qiao, Yeli Zhang, Shuaihua Li, Liujiao Bian
ABSTRACT
The molecular recognition and interaction of beta-lactamase II from Bacillus cereus (Bc II) with
penicillin V (PV) and sulbactam (Sul) especially conformational changes of Bc II in the binding
process were studied through spectroscopy analysis in combination with molecular dynamics (MD)
simulation. The results show that in the binding process, a new coordination bond is observed between
the Zn2 of Bc II and the carboxyl-O of PV or Sul by replacing His204. Electrostatic interaction
between Zn2 and the ligand provide main driving force for the binding affinity. Compared with apo Bc
II, there are mainly four loops showing significant conformational changes in ligand-bound Bc II. A
weak conformational transformation from β-sheets to random coils is observed in the loop2 of ligand-
bound Bc II. The conformational transformation may depend on the functional group and binding pose
of the ligand, giving the binding pocket greater flexibility and accordingly allowing for an induced fit
of the enzyme-ligand binding site around the newly introduced ligand. The change in the loop2 of
ligand-bound Bc II may lead to the opening of the binding pocket of Bc II.
107
Variations in gut microbiota and fecal metabolic phenotype associated with depression by 16S
rRNA gene sequencing and LC/MS-based metabolomics
Meng Yu, Hongmei Jia, Chao Zhou, Yong Yang, Zhongmei Zou
ABSTRACT
As a prevalent, life-threatening and highly recurrent psychiatric illness, depression is characterized by
a wide range of pathological changes; however, its etiology remains incompletely understood.
Accumulating evidence supports that gut microbiota affects not only gastrointestinal physiology but
also central nervous system (CNS) function and behavior through the microbiota-gut-brain axis. To
assess the impact of gut microbiota on fecal metabolic phenotype in depressive conditions, an
integrated approach of 16S rRNA gene sequencing combined with ultra high-performance liquid
chromatography-mass spectrometry (UHPLC–MS) based metabolomics was performed in chronic
variable stress (CVS)-induced depression rat model. Interestingly, depression led to significant gut
microbiota changes, at the phylum and genus levels in rats treated with CVS compared to controls. The
relative abundances of the bacterial genera Marvinbryantia, Corynebacterium, Psychrobacter,
Christensenella, Lactobacillus, Peptostreptococcaceae incertae sedis, Anaerovorax, Clostridiales
incertae sedis and Coprococcus were significantly decreased, whereas Candidatus Arthromitus and
Oscillibacter were markedly increased in model rats compared with normal controls. Meanwhile,
distinct changes in fecal metabolic phenotype of depressive rats were also found, including lower
levels of amino acids, and fatty acids, and higher amounts of bile acids, hypoxanthine and stercobilins.
Moreover, there were substantial associations of perturbed gut microbiota genera with the altered fecal
metabolites, especially compounds involved in the metabolism of tryptophan and bile acids. These
results showed that the gut microbiota was altered in association with fecal metabolism in depressive
conditions. These findings suggest that the 16S rRNA gene sequencing and LC–MS based
metabolomics approach can be further applied to assess pathogenesis of depression.
Chemical profiling of Fufang-Xialian-Capsule by UHPLC-Q-TOF-MS and its antioxidant
activity evaluated by in vitro method
Shizhe Li, Shu Liu, Zifeng Pi, Fengrui Song, Zhiqiang Liu
ABSTRACT
Fufang Xialian (FXL) capsule, a traditional Chinese medicine (TCM) formula, has been used for a
long time to treat Chronic Atrophic Gastritis, but the pharmacodynamic material basis and mechanism
of action are unclear. In this research, ferric-reducing antioxidant power (FRAP) method and DPPH
scavenging method were utilized to evaluate the antioxidant activity of FXL in vitro. An ultra high-
performance liquid chromatography coupled with Linear Ion Trap Mass Spectrometer (UHPLC-IT-
MS) method and an ultra high-performance liquid chromatography coupled with quadrupole time-of-
flight mass spectrometry (UHPLC-Q-TOF-MS) method were utilized to acquire the constituents
information of FXL. Mass spectra were acquired in both positive and negative ion modes. As the
results, FXL showed a moderate DPPH scavenging activity and ferric-reducing antioxidant power. A
total of 73 components were identified or tentatively characterized in FXL, which included alkaloids,
flavones, flavone glycosides, ginsenoside, lignins, triterpene saponins, flavanones, chalcones and
coumarin. The results in this research could provide a basis for further study in bioactivity and
metabolites.
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Volume 139 May 2017
Development of an UPLC–MS/MS method for quantification of Avitinib (AC0010) and its five
metabolites in human cerebrospinal fluid: Application to a study of the blood-brain barrier
penetration rate of non-small cell lung cancer patients
Weicong Wang, Xin Zheng, Hanping Wang, Lu Wang, Pei Hu
ABSTRACT
Avitinib (AC0010) is a mutant-selective epidermal growth factor receptor tyrosine kinase inhibitors
(EGFR-TKI), designed to be a targeted therapeutic agent for non-small cell lung cancer (NSCLC)
patients harboring EGFR active and T790M resistant mutations. A rapid and sensitive ultra-
performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) method was
developed and validated for the determination of Avitinib and its five metabolites (M1, M2, M4, M7,
MII-6) in human cerebrospinal fluid (CSF). The samples were purified by protein precipitation and
separated on a BEH C18 column (2.1 × 50 mm, 1.7 μm). Electrospray ionization (ESI) in positive ion
mode and multiple reaction monitoring (MRM) were used to monitor the ion transitions at m/z
488/257, 474/403, 504/487, 434/377, 490/405, 476/391. The results indicated that the method had
excellent sensitivity and specificity. The linear range covered from 0.05 to 50 ng/mL for Avitinib, M1,
M4, M7, and MII-6, and from 0.01 to 10 ng/mL for M2. Intra-day and inter-day precisions (in terms
of% RSD) were all <15% and the accuracies (in terms of% RE) were within the range of ±15%. The
lower limit of quantification (LLOQ), matrix effect, extraction recovery, stability and dilution integrity
were also validated and satisfied with the criteria of validation. Finally, the method was successfully
applied to a blood-brain barrier (BBB) penetration rate research of NSCLC patients after an oral
administration of Avitinib.
The sodium salt of the enantiomers of ricobendazole: Preparation, solubility and chiroptical
properties
Roberto Cirilli, Paolo Guglielmi, Francesca Romana Formica, Adriano Casulli, Simone Carradori
ABSTRACT
Albendazole (ABZ) is a sulfanyl-benzimidazole anthelmintic drug used worldwide in the treatment
and prevention of parasitic diseases in animals and humans. Following oral administration, ABZ is
rapidly oxidized into the pharmacologically active chiral sulfoxide metabolite known as ricobendazole
(RBZ). As its achiral precursor, RBZ shows very low intestinal absorption due to its poor solubility in
water (0.06 mg mL−1). To the best of our knowledge, there is no known example in human medicine
of a water-soluble salt form of racemic or enantiomerically pure RBZ. In the present study, we
describe in detail the preparation of the sodium (Na) salt of the enantiomers of RBZ through a two-step
process: i) the multi-milligram resolution of RBZ by HPLC on the amylose-based Chiralpak IG chiral
stationary phase under polar organic mode; ii) the salification of the isolated enantiomers of RBZ by
reaction with sodium hydroxide solution. The spectroscopic and chiroptical properties of the RBZ-Na
enantiomers were determined. Due to their unique solubility in 0.01 M phosphate buffer at
physiological pH (14.49 mg mL−1) and the high sample throughput obtained on semipreparative
separation of the non-salified form, it is potentially possible to develop new anthelmintic enantiopure
formulations with improved pharmacokinetic properties and lower toxicity.
109
Application of design space optimization strategy to the development of LC methods for
simultaneous analysis of 18 antiretroviral medicines and 4 major excipients used in various
pharmaceutical formulations
Védaste Habyalimana, Jérémie Kindenge Mbinze, Achille Loconon Yemoa, Christelle Waffo, Roland
Djang'eing'a Marini
ABSTRACT
As one of the world‘s most significant public health challenges in low- and middle-income countries,
HIV/AIDS deserves to be treated with appropriate medicines, however which are not spared from
counterfeiting. For that, we developed screening and specific HPLC methods that can analyze 18
antiretroviral medicines (ARV) and 4 major excipients. Design of experiments and design space
methodology were initially applied for 15 ARV and the 4 excipients with prediction thanks to Monte
Carlo simulations and focusing on rapidity and affordability thus using short column and low cost
organic solvent (methanol) in gradient mode with 10 mM buffer solutions of ammonium hydrogen
carbonate. Two other specific methods dedicated to ARV in liquid and in solid dosage formulations
were also predicted and optimized. We checked the ability of one method for the analysis of a fixed-
dose combination composed by emtricitabine/tenofovir/efavirenz in tablet formulations. Satisfying
validation results were obtained by applying the total error approach taking into account the accuracy
profile as decision tool. Then, the validated method was applied to test two samples coded A and B,
and claimed to contain the tested ARV. Assay results were satisfying only for sample B.
Modeling and optimizing inhibitory activities of Nelumbinis folium extract on xanthine oxidase
using response surface methodology
Mangmang Sang, Guangyan Du, Jia Hao, Linlin Wang, Lifeng Han
ABSTRACT
Xanthine oxidase (XOD), which could oxidize hypoxanthine to xanthine and then to uric acid, is a key
enzyme in the pathogenesis of hyperuricemia and also a well-known target for the drug development
to treat gout. In our study, the total alkaloids of Nelumbinis folium markedly inhibited XOD activity,
with IC50 value being 3.313 μg/mL. UHPLC-Q-TOF-MS and 3D docking analysis indicated that
roemerine was a potential active ingredient. A response surface methodology combined with central
composite design experiment was further developed and validated for the optimization of the reaction
conditions between the total alkaloids of Nelumbinis folium and XOD, which could be considered as a
meaningful research for the development of XOD inhibitor rapidly and sensitively.
110
Comparative preclinical evaluation of 68Ga-NODAGA and 68Ga-HBED-CC conjugated
procainamide in melanoma imaging
György Trencsényi, Noémi Dénes, Gábor Nagy, Adrienn Kis, István Kertész
ABSTRACT
Malignant melanoma is the most aggressive form of skin cancer. The early detection of primary
melanoma tumors and metastases using non-invasive PET imaging determines the outcome of this
disease. Previous studies have shown that benzamide derivatives (e.g. procainamide) conjugated with
PET radionuclides specifically bind to melanin pigment of melanoma tumors. 68Ga chelating agents
can have high influence on physiological properties of 68Ga labeled bioactive molecules, as was
experienced during the application of HBED-CC on PSMA ligand. The aim of this study was to assess
this concept in the case of the melanin specific procaindamide (PCA) and to compare the melanin
specificity of 68Ga-labeled PCA using HBED-CC and NODAGA chelators under in vitro and in vivo
conditions. Procainamide (PCA) was conjugated with HBED-CC and NODAGA chelators and was
labeled with Ga-68. The melanin specificity of 68Ga-HBED-CC-PCA and 68Ga-NODAGA-PCA was
investigated in vitro and in vivo using amelanotic (MELUR and A375) and melanin containing (B16-
F10) melanoma cell lines. Tumor-bearing mice were prepared by subcutaneous injection of B16-F10,
MELUR and A375 melanoma cells into C57BL/6 and SCID mice. 21 ± 2 days after tumor cell
inoculation and 90 min after intravenous injection of the 68Ga-labelledlabeled radiopharmacons whole
body PET/MRI scans were performed. 68Ga-NODAGA-PCA and 68Ga-HBED-CC-PCA were
produced with excellent radiochemical purity (98%). In vitro experiments demonstrated that after 30
and 90 min incubation time 68Ga-NODAGA-PCA uptake of B16-F10 cells was significantly (p ≤
0.01) higher than the 68Ga-HBED-CC-conjugated PCA accumulation in the same cell line.
Furthermore, significant difference (p ≤ 0.01 and 0.05) was found between the uptake of melanin
negative and positive cell lines using 68Ga-NODAGA-PCA and 68Ga-HBED-CC-PCA. In vivo
PET/MRI studies using tumor models revealed significantly (p ≤ 0.01) higher 68Ga-NODAGA-PCA
uptake (SUVmean: 0.46 ± 0.05, SUVmax: 1.96 ± 0.25, T/M ratio: 40.7 ± 4.23) in B16-F10 tumors in
contrast to 68Ga-HBED-CC-PCA where the SUVmean, SUVmax and T/M ratio were 0.13 ± 0.01,
0.56 ± 0.11 and 11.43 ± 1.24, respectively.
Development and validation of a HILIC-ELSD method for simultaneous analysis of non-
substituted and acetylated xylo-oligosaccharides
Jianghua Pu, Xia Zhao, Lin Xiao, Haitao Zhao
ABSTRACT
A new HILIC-ELSD method was developed for compositional analysis of both xylo-oligosaccharides
(XOS) with degree of polymerization (DP) from 2 to 8 and acetylated XOS with DP from 3 to 8. The
method was carried out on a zwitterionic HILIC column using ELSD as a detector. The influences of
mobile phase composition, column temperature and flow rate on the retention time and resolution of
XOS were investigated. An excellent separation result was achieved with a linear gradient elution of
75%−50% acetonitrile in 30 min, at a flow rate of 1 mL/min and the column temperature at 35 °C. In
addition, LC–ESI–MS was employed to determine the structural information of X7, X8 and acetylated
XOS. The proposed method was simple, reliable, and no derivatization procedure was needed. It is
suitable for compositional analysis and quality control of XOS.
111
Enantioselective recognition of radezolid by cyclodextrin modified capillary electrokinetic
chromatography and electronic circular dichroism
Katarzyna Michalska, Ewa Gruba, Wojciech Bocian, Judyta Cielecka-Piontek
ABSTRACT
A method for the enantioseparation of radezolid (RAD), an analogue of a truly new class of
antibacterial agents, oxazolidinones, was developed based on capillary electrokinetic chromatography
using a cyclodextrin as a chiral pseudophase (CD-cEKC). The mechanism of RAD separation, together
with its precursor, were investigated to directly define the relationship between the oxazolidinone
structure and the complexation process. During the development of the method, anionic single isomer
cyclodextrins were tested. They were ranked in order from hydrophilic to hydrophobic as follows:
heptakis-(2,3-dihydroxy-6-sulfo)-β-cyclodextrin (HS-β-CD), heptakis-(2,3-diacetyl-6-sulfo)-β-
cyclodextrin (HDAS-β-CD) and heptakis-(2,3-dimethyl-6-sulfo)-β-cyclodextrin (HDMS-β-CD).
Experiments were performed at pH values of 2.5, 6.6, 8.2 and 9.6. The cyclodextrins that had an acetyl
or methyl group at the C2 and C3 positions, referred to as HDAS-β-CD and HDMS-β-CD,
respectively, exhibited partial and baseline separation of enantiomers in a low pH buffer. However,
higher temperatures were required for HDAS-β-CD and acetonitrile addition was required for HDMS-
β-CD. During the experiments, different organic solvents, varying in their amphiprotic or aprotic
nature, were tested. The best results for the separation of enantiomers using the CD-cEKC method
were obtained with 40 mM HDMS-β-CD dissolved in a 50 mM phosphate buffer (pH 2.5) with the
addition of acetonitrile (65:35, v/v) at 27 °C, reversed polarity and a voltage equal to 28 kV. The
apparent binding constants for each enantiomer to HDAS-β-CD or HDMS-β-CD were calculated.
Finally, the stereochemistry of (S) and (R)-RAD and the behaviour of selected complex formations
were established using electronic circular dichroism.
Identification of leachables observed in the size exclusion chromatograms of a low concentration
product stored in prefilled syringes
Joseph J. Valente, Michael B. Peddicord, Frank A. Rinaldi, Kathleen A. Kelly, Mark S. Bolgar
ABSTRACT
Requisite leachables testing of pharmaceutical products is commonly conducted with pre-defined
analytical methods on a subset of materials intended to be representative of the marketed product.
Throughout product development, leachables may occasionally be detected in other methods not
specifically intended for monitoring such impurities. We have identified two leachables, ethyl 4-
ethoxybenzoate (E4E) and 2,6-di(t-butyl)-4-hydroxy-4-methyl-2,5-cyclohexadien-1-one (BHT-OH) in
a low concentration product stored in prefilled syringes (PFS). The leachables were initially detected
by size exclusion chromatography (SEC) as late-eluting impurity peaks. Syringe component extraction
studies indicated that the impurities were related to the syringe stoppers. Positive identification of E4E
was accomplished by reversed phase liquid chromatography- tandem mass spectrometry (RPLC–
MS/MS). Positive identification of BHT-OH required RPLC-solid phase extraction-cryoflow NMR
(RPLC-SPE-NMR), as initial RPLC–MS/MS investigations were unsuccessful in elucidating the
structure. We focus specifically on the efforts required to identify the leachables, and the fortuitous
mixed mode separation mechanism and low concentration nature of the product, which were the main
factors contributing to the unlikely detection of the leachables by SEC.
112
Isolation and characterization of bioactive polyacetylenes Panax ginseng Meyer roots
Chia-Rou Yeo, Jin-Jie Yong, David G. Popovich
ABSTRACT
Panax ginseng has been studied for its chemo-preventive properties and pharmaceutical potential.
Polyacetylenic compounds isolated from Panax ginseng root typically comprised of non-polar C17
compound have been reported to exhibit bioactive properties. The objective of this project is to extract,
isolate, and characterize bioactive polyacetylenes from Panax ginseng root using various extraction
and separation methods Ginseng was extracted by reflux using methanol, ethanol, hexane, ethyl
acetate, methanolic ultrasonication. The extracts were partitioned with hexane to obtain water-soluble
portion and hexane-soluble portion. Hexane was subsequently removed under vacuum, and formed a
crude polyacetylenes extract (crude PA). Silica gel chromatography and semi-preparative HPLC were
utilized to prepare 5 fractions and the polyacetylenes were measure by HPLC and molecular weights
confirm my APCI–MS and MNR. The bioactive effect was measured by MTT viability assay using
murine 3T3-L1 cells. Extraction with methanol under reflux produced significantly larger amount of
polyacetylenes (p < 0.05). Liquid-liquid extraction and column chromatography were used to separate
polyacetylenic compounds into five different fractions. Major polyacetylenes, panaxynol and
panaxydol were found in fraction 1 and 2 respectively. Dose-response relationships were observed in
3T3-L1 cells and LC50 were 13.52 ± 3.05 μg/mL (fraction 1), 3.69 ± 1.09 μg/mL (fraction 2), 52.88 ±
11.16 μg/mL (fraction 3), 85.91 ± 27.37 μg/mL (fraction 4) and 135.52 ± 32.91 μg/mL (fraction 5).
Fraction 2 containing panaxydol was found to have exhibited the greatest anti-proliferative effects on
3T3-L1 preadipocytes.
Purity assessment of ginsenoside Rg1 using quantitative 1H nuclear magnetic resonance
Bao-Ming Huang, Sheng-Yuan Xiao, Ting-Bo Chen, Ying Xie, Hua Zhou
ABSTRACT
Ginseng herbs comprise a group of the most popular herbs, including Panax ginseng, P. notoginseng
and P. quinquefolius (Family Araliaceae), which are used as traditional Chinese medicine (TCM) and
are some of the best-selling natural products in the world. The accurate quantification of ginsenoside
Rg1 is one of the major aspects of its quality control. However, the purity of the commercial Rg1
chemical reference substance (CRS) is often measured with high-performance chromatography
coupled with an ultraviolet detector (HPLC–UV), which is a selective detector with unequal responses
to different compounds; thus, this detector introduces probable error to purity assessments. In the
present study, quantitative nuclear magnetic resonance (qNMR), due to its absolute quantification
ability, was applied to accurately assess the purity of Rg1 CRS. Phenylmethyl phthalate was used as
the internal standard (IS) to calibrate the purity of Rg1 CRS. The proton signal of Rg1 CRS in
methanol-d4 at 4.37 ppm was selected to avoid interfering signals, enabling accurate quantitative
analysis. The relaxation delay, number of scans, and NMR windowing were optimized for data
acquisition. For post-processing, the Lorentz/Gauss deconvolution method was employed to increase
the signal accuracy by separating the impurities and noise in the integrated region of the quantitative
proton. The method validation showed that the developed method has acceptable sensitivity, linearity,
precision, and accuracy. The purity of the commercial Rg1 CRS examined with the method developed
in this research was 90.34 ± 0.21%, which was obviously lower than that reported by the manufacturer
(>98.0%, HPLC–UV).
113
Identification of forced degradation products of tedizolid phosphate by liquid
chromatography/electrospray ionization tandem mass spectrometry
Yinping Lei, Bo Jin, Chen Ma, Tingting Zhang, Tong Li
ABSTRACT
In this study, stress degradation behavior of tedizolid phosphate was investigated and structural
characterization of its degradation products were performed with the use of the UPLC–MSn and LC-
HRMS. The toxicity prediction of the degradation products was performed with web-based prediction
system. Tedizolid phosphate was subjected to forced degradation under hydrolytic (acid, base and
neutral), oxidative, photolytic and thermal conditions in accordance with ICH guidelines Q1A(R2).
The drug was degraded significantly under acid, base and oxidative conditions, while it was relatively
stable to neutral, thermal and photolytic conditions. A total of four degradation products were formed.
All of them have been identified and characterized based on QTRAP MSn and accurate mass
measurements. To the best of our knowledge, three of these impurities were identified for the first time
and two of them further synthesized and characterized by NMR spectroscopy.
Evaluation of automated Wes system as an analytical and characterization tool to support
monoclonal antibody drug product development
Jinyu Wang, Anulfo Valdez, Yingchen Chen
ABSTRACT
Monitoring and evaluation of critical quality attributes (cQA) in monoclonal antibodies (mAb) are a
regulatory requirement in pharmaceutical industry. High molecular weight (HMW) species are of
critical importance due to the potential risk associated with immunogenicity. HMW species are
typically monitored by size exclusion chromatography (SEC). Although low molecular weight (LMW)
species are also detected by SEC, low-resolution separation of LMW limits its capability to monitor
mAb fragmentation. Recently, we have developed new methods for LMW characterization and
evaluation based on the Wes instrument from ProteinSimple. The capillary western blot is based upon
size-based separation in a capillary system, and detection by specific immunoprobing, following the
separation. The capability of this method for characterization of mAb fragments were demonstrated.
The characterization was achieved by probing two antibodies targeted to specific regions (Fc region or
Fab region) of IgG1 protein. The specificity of these two antibodies was evaluated against F (ab‘) 2
and Fc/2 fragments generated from Ides enzyme treated IgG1 protein. The results showed the selected
antibodies provide high specificity to F (ab‘) 2 and Fc/2 fragments. Fractions collected from SEC were
used to evaluate this method. The detected fragments from SEC fractions were identified based on
their estimated molecular weight and antibody detection. The result proved the capability of the
capillary western blot as a characterization method for IgG1 fragments. In addition, with the specific
detection to IgG1 and IgG4, the power of the capillary western blot to specifically characterize and
evaluate individual IgG fragmentations in an IgG1 and IgG4 mixture was also demonstrated. When
heat stressed samples were used, results showed method capability as stability indicating in IgG1 and
IgG4 mixture samples. The stressed mixture samples were also evaluated by the total protein assay in
which protein samples were biotinylated after separation and were labeled with HRP linked
streptavidin to provide chemiluminescence detection. The results indicated total protein assay can be a
useful complementary method to capillary western blot immunoassay.
114
Pharmacokinetics and tissue distribution of 4, 5-dimethoxycanthin-6-one and its major
metabolites in rats
Xiaolei Miao, Junjun Wang, Yong Chen
ABSTRACT
4,5-Dimethoxycanthin-6-one and 5-hydroxy-4-methoxycanthin-6-one are the active ingredients of P.
quassiodes. In the present work, a LC–MS/MS method was developed for the determination of 4,5-
dimethoxycanthin-6-one and its major metabolites 5-hydroxy-4-methoxycanthin-6-one (M1) and 4-
hydroxy-5-methoxycanthin-6-one (M2) in rat plasma and tissues, and applied to study their
pharmacokinetics and tissue distribution after intramuscular administration of 4,5-dimethoxycanthin-6-
one to rats. By protein precipitation with methanol for plasma samples and liquid–liquid extraction
with ethyl acetate for tissue samples, the analytes were separated on an ODS C18 column with a
mobile phase consisted of methanol and water (0.1% formic acid), and quantified by a MS detector in
positive multiple reaction monitoring (MRM) mode. MS transitions were m/z 281.0 → 167.1 for 4,5-
dimethoxycanthin-6-one, m/z 267.0 → 168.1 for M1 and M2, m/z 251.0 → 195.1 for 3-methylcanthin-
2,6-dione (IS). The pharmacokinetic results indicate that 4,5-dimethoxycanthin-6-one is absorbed
rapidly (Tmax = 5.4–6.4 min), distributed rapidly and widely in the order of liver > kidney ≈ lung ≈
large intestine ≈ small intestine, and eliminated quickly (t1/2z = 64.9–77.7 min) following the
intramuscular administration. Furthermore, M1 and M2 were detected only in rat plasma and liver at
the indicated times after the intramuscular administration.
Development and validation of an ELISA method for the quantification of nivolumab in plasma
from non-small-cell lung cancer patients
Alicja Puszkiel, Gaëlle Noé, Pascaline Boudou-Rouquette, Chloé Le- Cossec, Benoit Blanchet
ABSTRACT
Nivolumab, an anti PD-1 monoclonal antibody, has been approved for the treatment of previously
treated advanced or metastatic non-small-cell lung cancer (NSCLC). The aim of this study was to
develop and validate an ELISA method for the quantification of nivolumab in plasma from patients
with NSCLC in order to perform future pharmacokinetic/pharmacodynamic (PK/PD) studies. A home-
made ELISA was developed and validated according to the general recommendations for the
immunoassays. Then, the ELISA method was applied to quantify plasma trough levels (Cmin) of
nivolumab (3 mg/kg every two weeks) in 27 NSCLC patients at days 14, 28 and 42 after start of
treatment. Blood samples were collected just before the infusion on days 0 (baseline), 14, 28 and 42
after start of treatment. The dynamic calibration range for nivolumab assay was 5–100 μg/mL. Within-
and between-day imprecision for quality controls (5, 20 and 75 μg/mL) were less than 5 and 12%,
respectively. The mean (± standard deviation) nivolumab Cmin was 17.3 ± 4.8 μg/mL (coefficient of
variation, CV = 27.8%), 25.0 ± 9.7 μg/mL (CV = 38.8%) and 33.0 ± 12.9 μg/mL (CV = 39.1%) on
days 14, 28 and 42, respectively. IgG (p = 0.002) and ALT (p = 0.041) were independently associated
with plasma nivolumab Cmin at day 42.
115
Bioanalysis of Pseudomonas aeruginosa alkyl quinolone signalling molecules in infected mouse
tissue using LC–MS/MS; and its application to a pharmacodynamic evaluation of MvfR
inhibition
Paul Turnpenny, Anthony Padfield, Patrick Barton, Joanne Teague, Aileen Rubio
ABSTRACT
Alkyl quinolone molecules 2-heptyl-4-quinolone (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone
(PQS) are important quorum sensing signals, which play a mediatory role in the pathogenesis of acute
and chronic Pseudomonas aeruginosa infection. A targeted approach inhibiting the bacterial ‗multiple
virulence factor regulon‘ (MvfR) protein complex, offers the possibility to block the synthesis of
MvfR-dependant signal molecules. Here, a high throughput bioanalytical method was developed using
LC–MS/MS detection for the selective determination of HHQ and PQS in mouse tissue homogenate,
over a sensitive range of 1–5000 and 10–5000 pg/mL, respectively. Chromatographic peak distortion
of the iron chelator PQS was overcome with the applied use of a bidentate chelator mobile phase
additive 2-Picolinic acid at 0.2 mM concentration, giving an improved separation and response for the
analyte, whilst maintaining overall MS system robustness. Following thigh infection with P.
aeruginosa strain 2-PA14 in mice, the concentration and time course of HHQ and PQS (4-hydroxy-2-
alkyl-quinolone (HAQ) biomarkers) residing in the biophase were evaluated, and exhibited a low level
combined with a substantial inter-individual variability. Quantifiable levels could be obtained from
approximately 15 h post infection, to the study termination at 21–22 h. A dose dependant reduction in
HAQ tissue concentrations at selected time points were obtained following MvfR inhibitor
administration versus drug vehicle (p < 0.01, Kruskal–Wallis—one way ANOVA) and meta -analyses
of several studies enabled an inhibitory concentration (IC50) of 80 nM free drug to be determined.
Metabolites identification of berberine in rats using ultra-high performance liquid
chromatography/quadrupole time-of-flight mass spectrometry
Kun Wang, Liwei Chai, Xinchi Feng, Zhongbo Liu, Feng Qiu
ABSTRACT
Berberine (BBR), the principle component for many medicinal plants such as Coptis chinensis Franch,
Phellodendron chinense Schneid, and Mahonia bealei (Fort.) Carr, possesses diverse pharmacological
activities, including anti-bacterial, anti-inflammatory, antitumor, hypolipidemic and antidiabetic
activities. In this study, a rapid and reliable method using a five-step strategy based on the ultra-
performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry
(UPLC/Q-TOF-MS), and metabolynx™ software with mass defect filter (MDF) technique was
developed to investigate the metabolism of BBR. Plasma, bile, urine and feces samples were collected
from rats after oral administration of BBR with a dose of 100 mg/kg/day for three consecutive days
and analyzed to characterize the metabolic profile of BBR. By comparing the molecular weights and
MS fragmentations of the metabolites with those of the parent drug and reference standards, a total of
97 metabolites were identified, including 68 metabolites in urine, 45 metabolites in plasma, 44
metabolites in bile and 41 metabolites in feces. Demethylation, demethylenation, reduction,
hydroxylation, and subsequent glucuronidation, sulfation and methylation were the major metabolic
pathways of BBR in vivo.
116
Quantitative chiral and achiral determination of ketamine and its metabolites by LC–MS/MS in
human serum, urine and fecal samples
Mahmoud Hasan, Robert Hofstetter, Georg M. Fassauer, Andreas Link, Stefan Oswald
ABSTRACT
Ketamine (KET) is a widely used anesthetic drug which is metabolized by CYP450 enzymes to
norketamine (n-KET), dehydronorketamine (DHNK), hydroxynorketamine (HNK) and
hydroxyketamine (HK). Ketamine is a chiral compound and S-ketamine is known to be the more
potent enantiomer. Here, we present the development and validation of three LC–MS/MS assays; the
first for the quantification of racemic KET, n-KET and DHNK in human serum, urine and feces; the
second for the separation and quantification of the S- and R-enantiomers of KET, n-KET and DHNK,
and the third for separation and quantification of 2S,6S-hydroxynorketamine (2S,6S-HNK) and 2R,6R-
hydroxynorketamine (2R,6R-HNK) in serum and urine with the ability to separate and detect 10
additional hydroxylated norketamine metabolites of racemic ketamine. Sample preparation was done
by liquid-liquid extraction using methyl tert-butyl ether. For achiral determination of KET and its
metabolites, an isocratic elution with ammonium acetate (pH 3.8; 5 mM) and acetonitrile on a C18
column was performed. For the separation of S- and R-enantiomers of KET, n-KET and DHNK, a
gradient elution was applied using a mobile phase of ammonium acetate (pH 7.5; 10 mM) and
isopropanol on the CHIRAL-AGP® column. The enantioselective separation of the HNK metabolites
was done on the chiral column Lux®-Amylose-2 with a gradient method using ammonium acetate (pH
9; 5 mM) and a mixture of isopropanol and acetonitrile (4:1). The mass spectrometric detection
monitored for each analyte 2–3 mass/charge transitions. D4-ketamine and D4-n-KET were used as
internal standards. The assays were successfully validated according to current bioanalytical guidelines
and applied to a pilot study in one healthy volunteer. Compared to previously published methods, our
assays have superior analytical features such as a lower amount of required matrix, faster sample
preparation, shorter analytical run time and higher sensitivity (LLOQ up to 0.1 ng/ml).
Irinotecan binds to the internal cavity of beta-lactoglobulin: A multi-spectroscopic and
computational investigation
N. Bijari, S. Ghobadi, K. Derakhshandeh
ABSTRACT
The binding of irinotecan, a potent anti cancer drug, to bovine beta-lactoglobulin (β-LG) was
investigated by various spectroscopic techniques including fluorimetry, circular dichroism (CD), UV–
vis, and Fourier transform infrared (FT-IR), in 10 mM phosphate buffer, pH 7.75, in combination with
a molecular docking study. Analysis of the fluorescence quenching data showed that combined static
and dynamic quenching occurs, with the predominant contribution of the static mode. Molecular
docking results were in full agreement with the results obtained from thermodynamic analysis of the
fluorescence data indicating the existence of one binding site for irinotecan in β-LG structure and
revealed the hydrophobic nature of the interaction between irinotecan and the protein. The binding
distance between β-LG and irinotecan, r, was estimated to be 5.74 nm based on the Förster‘s theory of
non-radiative energy transfer. The obtained results of near-UV CD and FT-IR experiments suggested
the occurrence of partial compactness of the protein structure upon irinotecan binding. Based on the
experimental data and the possible binding mode revealed by molecular docking study, we concluded
that irinotecan binds to the hydrophobic calyx of β-LG with induction of some alterations in the
secondary and tertiary structure of the protein.
117
Determination of drugs in plasma samples by disposable pipette extraction with C18-BSA phase
and liquid chromatography–tandem mass spectrometry
Mônia Ap. Lemos Pinto, Israel D. de Souza, Maria Eugênia C. Queiroz
ABSTRACT
This work describes restricted access material (RAM) constituted of porous octadecylsilane particles
with the outer surface covered with bovine serum albumin (C18-BSA) as a stationary phase to extract
drugs from plasma samples by disposable pipette extraction (DPX) for further analysis by liquid
chromatography–tandem mass spectrometry (LC–MS/MS). The C18-BSA phase simultaneously
excluded macromolecules by chemical diffusion barrier (BSA network) and enrichment of the interior
phase (C18) with drug traces by sorption. The hydrophilic barrier of the C18-BSA allows small
molecules (drugs) to permeate through the hydrophobic part (C18), while at the same time it excludes
the macromolecules by chemical diffusion barrier (BSA network). Optimization of the DPX variables
(sorption equilibration time, exclusion of endogenous compounds, and elution step) improved the
sensitivity and selectivity of the method, which presented a linear range from the lower limit of
quantification (0.5–20.0 ng mL−1) to the upper limit of quantification (32.5–10,500 ng mL−1), inter-
and intra-assay precision with coefficients of variation (CV) lower than 15%, and relative standard
error (RSE) of the accuracy ranging from −12% to 11%. The developed method was successfully used
to determine five antipsychotics (olanzapine, quetiapine, clozapine, haloperidol, and chlorpromazine)
in combination with seven antidepressants (mirtazapine, paroxetine, citalopram, sertraline, imipramine,
clomipramine, and fluoxetine), two anticonvulsants (carbamazepine and lamotrigine), and two
anxiolytics (diazepam and clonazepam) in plasma samples from schizophrenic patients for therapeutic
drug monitoring.
Differentiation of protein secondary structure in clear and opaque human lenses: AFM – IR
studies
C. Paluszkiewicz, N. Piergies, P. Chaniecki, M. Rękas, W.M. Kwiatek
ABSTRACT
Here, we present the first approach to human lenses investigations with and without cataract
development changes in nanoscale resolution using AFM – IR spectroscopy. We proved that the
application of this technique allowed us to better understand of structural changes connected with
advancing disease process in studied lenses. The obtained results show the impact of the disease
development on the secondary structure of proteins in these human tissues. The domination of the β–
turn protein secondary structure is observed in the clear (non-affected by cataract) lens. While, in the
case of the opaque (cataractous) samples the different degree of the degradation due to development of
cataract, was recognized. Briefly, this process is associated with the protein secondary changes from
β–turn/β–sheet parallel for less altered part of the lens to stable anti-parallel β–sheet for the more
degraded part. Interestingly, the AFM – IR technique provided estimation of the protein secondary
structure without the need for using deconvolution procedure.
118
Introduction of a carbon paste electrode based on nickel carbide for investigation of interaction
between warfarin and vitamin K1
Maryam Torkashvand, Mohammad Bagher Gholivand, Avat (Arman) Taherpour, Arash Boochani,
Arsalan Akhtar
ABSTRACT
In this paper a novel electrochemical sensor based on nickel carbide (Ni3C) nanoparticles as a new
modifier was constructed. Ni3C nanoparticle was synthesized and characterized by scanning electron
microscopy, X-ray diffraction and first-principles study. Electrochemical impedance spectroscopy
(EIS) and cyclic voltammetry (CV) studies confirmed the electrode modification. Afterwards, the new
electrode for the first time was used for interaction study between vitamin K1 and warfarin as an
anticoagulant drug by differential pulse voltammetry. The adduct formation between the drug and
vitamin K1 was improved by decreasing in anodic peak current of warfarin in the presence of different
amounts of vitamin K1. The binding constant between warfarin and vitamin K1 was obtained by
voltammetric and UV–vis and fluorescence spectroscopic methods. The molecular modeling method
was also performed to explore the structural features and binding mechanism of warfarin to vitamin
K1. The different aspects of modeling of vitamin K1 and warfarin and their adduct structures
confirmed the adduct formation by hydrogen bonding.
An integrated strategy using UPLC–QTOF-MSE and UPLC–QTOF-MRM (enhanced target)
for pharmacokinetics study of wine processed Schisandra Chinensis fructus in rats
Kuangyi Liu, Yonggui Song, Yali Liu, Mi Peng, Dan Su
ABSTRACT
Currently the pharmacokinetic (PK) research of herbal medicines is still limited and facing critical
technical challenges on quantitative analysis of multi-components from biological matrices which
often accompanied by lacking of authentic standards and low concentration. This present work
contributes to the development of an integrated strategy for extensive pharmacokinetics assessments,
and a selective and sensitive method independent of authentic standards for multi-components analysis
based on the use of ultra-performance liquid chromatography/quadrupole-time-of-flight/MSE (UPLC-
TOF-MSE) and UPLC-TOF-MRM (rnhanced target). Initially, phytochemicals were identified by
UPLC-TOF-MSE analysis, subsequently the identified components were matched with authentic
standards and pre-classified, and UPLC–QTOF-MRM method optimized and developed. To guarantee
reliable results, three rules are necessary: (1) detection with a mass error of less than 5 ppm; (2) same
class chemical compositions with structural high similarity between analytes with and without
authentic reference substance; (3) a matching retention time between TOF-MRM mode and TOF-MSE
within 0.2 min. The developed and validated method was applied for the simultaneous determination
of 12 lignans in rat plasma after administered with wine processed Schisandra Chinensis fructus
(WPSCF) extract. Such an approach was found capable of providing extensive pharmacokinetic
profiles of multi-components absorbed into blood after oral administrated with WPSCF extract. The
results also indicated that significant difference in pharmacokinetics parameters of
dibenzocyclooctadiene lignans was observed between schizandrin and gomisin compounds. For
lignans, the absorption via gastrointestinal tract were all rapid and maintained relatively long retention
time, especially for schisantherin A and schisantherin B with higher plasma exposure.
119
Simultaneous determination of glipizide and its four hydroxylated metabolites in human urine
using LC–MS/MS and its application in urinary phenotype study
Bo Tan, Aidong Yang, Weian Yuan, Yue Li, Furong Qiu
ABSTRACT
Cytochrome P450 (CYP) 2C9 and CYP2C19 genetic mutant could influence the plasma concentration
of glipizide in human subjects, which refers to glipizide safety and adverse effects in clinic practice. A
further study to investigate the relationship of the concentrations between glipizide and its metabolites
in human with different CYP mutants was valuable. We firstly develop a validated liquid
chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous quantification of
glipizide and its hydroxylated metabolites in human urine. After simple protein precipitation with
methanol including 4′-OH-tolbutamide and gliclazide (both are internal standards), the analytes were
chromatographed on a reversed-phased column with a mobile phase of 0.1% formic acid in acetonitrile
and 0.1% formic acid in water by a gradient elution. The ion transitions of the precursor to the product
ion were principally protonated ions [M + H]+ at m/z 446.4 → m/z 321.1 for glipizide, m/z 462.2 →
m/z 321.1 for the four hydroxylated forms of glipizide, m/z 287.2 → m/z 188.0 for 4′-OH-tolbutamide,
and m/z 324.1 → m/z 127.1 for gliclazide. The method was linear over a concentration range of 0.02–
20.0 ng/mL. The intraday and inter-day variances were less than 9.9%, and accuracy was within
±6.8%. The method was successfully applied to the urinary phenotyping study in volunteers after a
single oral administration of 5-mg glipizide tablet, and two new hydroxycyclohexyl metabolites of
glipizide (OH-gp), 4-cis-OH-gp and 3-trans-OH-gp, were found in this study.
Plasma pharmacokinetics and bioavailability of verticillin A following different routes of
administration in mice using liquid chromatography tandem mass spectrometry
Jiang Wang, Xiaohua Zhu, Shamala Kolli, Hongyan Wang, Mitch A. Phelps
ABSTRACT
Verticillin A is a natural product isolated from fungal cultures and has displayed potent antibiotic,
antiviral, nematocidal, and anticancer properties in vitro. While in vivo studies have been limited due
to sparse supply, the in vivo efficacy data that does exist demonstrates potent anti-tumor activity in
murine cancer models. The current study aims to investigate the pharmacokinetics and bioavailability
of verticillin A in mice to provide guidance for further efficacy assessment in mouse models. A
sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and
validated for the quantification of verticillin A in mouse plasma. Sample preparation was
accomplished through protein precipitation, and chromatographic separation was achieved on an
Agilent Zorbax Extend C18 column with a security guard cartridge C8 using a binary gradient with
mobile phase A (water/0.1% formic acid) and B (ACN/0.1% formic acid) at a flow rate of 400 μl/min.
Elution of verticillin A and internal standard, hesperetin, occurred at 4.87 and 2.06 min, respectively.
The total chromatographic run time was 8 min, and the assay was linear in the concentration range of
1–1000 nM. The within- and between day precisions and accuracy were in the range of 2.58–8.71 and
90–105%, respectively. The assay was applied to determine plasma drug concentration in a mouse
pharmacokinetic study. It was found that intraperitoneal dosing of 3 mg/kg resulted in high systemic
exposure and achieved Cmax of 110 nM with plasma concentrations sustained above 10 nM for the
24-h duration of the study.
120
Robust quantitation of basic-protein higher-order aggregates using size-exclusion
chromatography
David Gervais, Andrew Downer, Darryl King, Patrick Kanda, Stuart Smith
ABSTRACT
Detection of higher-order aggregates (HOA) using size-exclusion chromatography (SEC) was found to
be variable for a basic protein, using exposed-silanol or diol-silica-based SEC columns. Preparations
of the tetrameric biopharmaceutical enzyme Erwinia chrysanthemi l-asparaginase (ErA), which has an
isoelectric point of 8.6, were analysed using a diol-silica SEC column. Although the proportions of
ErA main peak and octamer species were unaffected, HOA recovery and detection were extremely
variable and had poor agreement with an orthogonal measurement technique, analytical
ultracentrifugation (AUC). The observation that only HOA was selectively affected by non-specific
silanol interactions was unexpected, so alternatives were sought. Coated-silica SEC columns improved
the resolution and reproducibility of HOA detection for this alkaline-pI protein, and improved the
agreement of HOA with the AUC method. Basic proteins, such as ErA, should be thoroughly
evaluated in SEC method development, to ensure that resolution of larger aggregate species is not
compromised.
A metabolomic approach shows sphingosine 1-phosphate and lysophospholipids as mediators of
the therapeutic effect of liver growth factor in emphysema
A. Navarrete, F.J. Rupérez, T.O. Mendes, S. Pérez-Rial, A. García
ABSTRACT
Tobacco smoke exposure is the principal cause of lung tissue destruction, which in turn results in
emphysema that leads into shortness of breath. Liver growth factor (LGF, a cell and tissue
regenerating factor with therapeutic activity in several organs) has antifibrotic and antioxidant
properties that could be useful to promote lung tissue regenerating capacity in damaged lungs. The
current study has examined differences in metabolite profiles (fingerprints) of plasma from mice
(strain C57BL/6J, susceptible to develop emphysema) exposed to tobacco smoke during six months.
One group of mice received a treatment with Liver Growth Factor (LGF) after emphysema was
established, whereas the other group did not receive the treatment. Age and sex-matched mice not
exposed to smoke were also maintained with or without treatment as controls. Metabolic fingerprints
(untargeted analysis) of plasma after protein precipitation were obtained by LC-QTOF-MS. The
signals were processed and a large number of possible metabolites were found (23944). Multivariate
data analysis provided models that highlighted the differences between control and smoke exposed
mice in both conditions. Accurate masses of features (possible compounds) representing significant
differences were searched using online public databases. Lipid mediators, related to intracellular
signaling in inflammation, were found among the metabolites putatively identified as markers of the
different conditions and among them, sphingosine, sphingosine 1-phosphate and lysophospholipids
point at the relevance of such metabolites in the regulation of the processes related to tissue
regeneration mediated by LGF. These results also suggest that metabolomic fingerprinting could
potentially guide the characterization of relevant metabolites leading the regeneration of lungs in
emphysema disease.
121
Hepatic and renal metabolism of genistein: An individual-based model to predict
glucuronidation behavior of genistein in different organs
Junjin Liu, Xiaoming Yu, Shilong Zhong, Weichao Han, Lan Tang
ABSTRACT
The present study aims to develop an individual-based model to predict the glucuronidation
characteristics of genistein in 45 human livers. The model was validated in 18 human kidneys. Ten
UDP-glucuronosyltransferases (UGTs) were expressed in liver abundantly. Among them, UGT1A1,
UGT1A3, UGT1A6, UGT1A9 and UGT2B7 contributed 95.6% to genistein glucuronidation in human
liver, with significant correlation between gene expression of the five UGTs and the glucuronidation of
genistein. The genistein glucuronidation differences between individuals were varied enormously;
meanwhile the expression variations can explain 24.7% total metabolic variation in livers. In kidney,
UGT1A6, UGT1A9 and UGT2B7 were amply expressed. Their gene expressions and glucuronidation
rates of genistein had high correlations. Expression variations of UGT1A9/2B7 can explain up to
40.9% of the total variation of genistein glucuronidation in kidney. The model was therefore
established based on UGT expression between individuals taking into account an important assessment
which is the ratio of metabolic rate of each recombinant UGT isoform. Excellent linear correlations
between predicted and observed glucuronidation rate revealed that the model could predict metabolic
activity of genistein using quite a small size of tissue. In conclusion, the individual-based model can be
employed for predicting individual glucuronidation behavior of genistein in different organs.
Determination of free polysaccharide in Vi glycoconjugate vaccine against typhoid fever
C. Giannelli, E. Cappelletti, R. Di Benedetto, F. Pippi, F. Micoli
ABSTRACT
Glycoconjugate vaccines based on the Vi capsular polysaccharide directed against Salmonella enterica
serovar Typhi are licensed or in development against typhoid fever, an important cause of morbidity
and mortality in developing countries. Quantification of free polysaccharide in conjugate vaccines is
an important quality control for release, to monitor vaccine stability and to ensure appropriate immune
response. However, we found that existing separation methods based on size are not appropriate as
free Vi non-specifically binds to unconjugated and conjugated protein. We developed a method based
on free Vi separation by Capto Adhere resin and quantification by HPAEC-PAD. The method has been
tested for conjugates of Vi derived from Citrobacter freundii with different carrier proteins such as
CRM197, Tetanus Toxoid and Diphtheria Toxoid.
122
HPLC–MS/MS method for quantification of paclitaxel from keratin containing samples
Emily A. Turner, Alexandra C. Stenson, Saami K. Yazdani
ABSTRACT
Local drug delivery of paclitaxel is becoming ever more prevalent. As complex drug/excipient
combinations are being developed and tested, new high performance liquid chromatography-mass
spectrometry (HPLC–MS) techniques capable of quantifying paclitaxel from such formulations are
needed. Here a method for quantifying paclitaxel from aqueous, protein and oil containing samples
was developed and validated. Keratin, derived from human hair, is the protein component/paclitaxel
excipient in the development and validation of said method. The novelty of this method is described by
its ability to overcome water solubility issues and address clean-up of residual solvents in clinical
grade paclitaxel injection composition. The method evaluates tert-butyl methyl ether and ethanol as
extraction solvents with an extraction efficiency of 31.9 ± 2.3% and 86.4 ± 4.5% respectively. Upon
evaporation and rehydration, samples were evaluated by HPLC–MS and a method was developed for
paclitaxel quantification. The method developed had an inter-day precision of 9.1% relative standard
deviation and an intra-day precision of 4.3% relative standard deviation normalized to a docetaxel
internal standard. The described method is applicable to any aqueous paclitaxel sample containing
protein and/or oils.
Development of an enzyme-linked immunosorbent assay for detection of CDCP1 shed from the
cell surface and present in colorectal cancer serum specimens
Yang Chen, Brittney S. Harrington, Kevin C.N. Lau, Lez J. Burke, John D. Hooper
ABSTRACT
CUB domain containing protein 1 (CDCP1) is a transmembrane protein involved in progression of
several cancers. When located on the plasma membrane, full-length 135 kDa CDCP1 can undergo
proteolysis mediated by serine proteases that cleave after two adjacent amino acids (arginine 368 and
lysine 369). This releases from the cell surface two 65 kDa fragments, collectively termed ShE-
CDCP1, that differ by one carboxyl terminal residue. To evaluate the function of CDCP1 and its
potential utility as a cancer biomarker, in this study we developed an enzyme-linked immunosorbent
assay (ELISA) to reliably and easily measure the concentration of ShE-CDCP1 in biological samples.
Using a reference standard we demonstrate that the developed ELISA has a working range of 0.68–
26.5 ng/ml, and the limit of detection is 0.25 ng/ml. It displays high intra-assay (repeatability) and high
inter-assay (reproducibility) precision with all coefficients of variation ≤7%. The ELISA also displays
high accuracy detecting ShE-CDCP1 levels at ≥94.8% of actual concentration using quality control
samples. We employed the ELISA to measure the concentration of ShE-CDCP1 in human serum
samples with our results suggesting that levels are significantly higher in serum of colorectal cancer
patients compared with serum from individuals with benign conditions (p < 0.05). Our data also
suggest that colorectal cancer patients with stage II–IV disease have at least 50% higher serum levels
of ShE-CDCP1 compared with stage I cases (p < 0.05). We conclude that the developed ELISA is a
suitable method to quantify ShE-CDCP1 concentration in human serum.
123
Volume 140 June 2017
Capillary blood collected on volumetric absorptive microsampling (VAMS) device for
monitoring hydroxychloroquine in rheumatoid arthritis patients
Ying Qu, Kelley Brady, Robert Apilado, Tyler O‘Malley, Thierry Dervieux
ABSTRACT
A novel technique for collection of capillary blood, termed volumetric absorptive microsampling
(VAMS), has been recently cleared by the FDA for collection of human blood. VAMS absorbs a fixed
volume of blood (10 μl) and overcomes area bias and homogeneity issues associated with dried blood
spot (DBS). This study is the application of VAMS for therapeutic drug monitoring (TDM) in human
capillary blood. A liquid chromatography–tandem mass spectrometry (LC–MS/MS) workflow for
analysis of VAMS samples was developed and validated. Concentrations of hydroxychloroquine
(HCQ) and its metabolites, desethylhydroxychloroquine (DHCQ), desethylchloroquine (DCQ), and
bisdesethylchloroquine (BDCQ), in capillary blood on VAMS samplers were compared to those in
venous blood in rheumatoid arthritis patients. Feasibility of capillary blood collected on both VAMS
and DBS cards were evaluated on patients. Stability of dried capillary blood on VAMS was also
examined. Our results established that VAMS is a simple and accurate sampling technique that
delivers the benefits of DBS sampling while overcoming the issues associated with hematocrit and
homogeneity. It requires a small blood volume, simplifies sample logistics management, and may
allow sample collection in the patient‘s home setting.
Isolation and characterization of novel degradation products of Doxofylline using HPLC, FTIR,
LCMS and NMR
Ch. Krishnam Raju, Avadhesh K. Pandey, Gururaj S., Kaushik Ghosh, Sameer G. Navalgund
ABSTRACT
Forced degradation of Doxofylline (DFL) in different stress (base and peroxide) conditions gave rise to
two potential unknown impurities. These unknown degradation products DFL DEG-I and DFL DEG-II
were evaluated using a new-reverse-phase high performance liquid chromatography (HPLC), where it
was eluted at 0.44 and 1.09 relative retention times to DFL peak. DFL DEG-I and DFL DEG-II were
isolated using preparative HPLC from degradation mixtures. The structure of DFL DEG-I and DFL
DEG-II were elucidated using high resolution MS, multi-dimensional NMR and FTIR spectroscopic
techniques, and characterized. The stereochemistry of the enantiomers in DFL DEG-II has further been
investigated using computational techniques. To the best of our knowledge, DFL DEG-I and DFL
DEG-II are novel impurities and not reported elsewhere.
124
Enantiomers of triclabendazole sulfoxide: Analytical and semipreparative HPLC separation,
absolute configuration assignment, and transformation into sodium salt
Rosella Ferretti, Simone Carradori, Paolo Guglielmi, Marco Pierini, Roberto Cirilli
ABSTRACT
Direct HPLC separation of the enantiomers of triclabendazole sulfoxide (TCBZ-SO), which is the
main metabolite of the anthelmintic drug triclabendazole, was carried out using the polysaccharide-
based Chiralpak AS-H and Chiralpak IF-3 chiral stationary phases (CSPs). The chromatographic
behaviour of both CSPs was evaluated and compared using normal-phase and reversed-phase eluents
at different column temperatures. The eluent mixture of n-hexane-2-propanol-trifluoroacetic acid
70:30:0.1 (v/v/v) and a column temperature of 40 °C were identified as the best operational conditions
to carry out semipreparative enantioseparations on a 1-cm I.D. AS-H column. Under these conditions,
12.5 mg of racemic sample were resolved in a single chromatographic run within 15 min. Comparison
of calculated and experimental chiroptical properties provided the absolute configuration assignment at
the sulfur atom. The salification of the isolated enantiomers of TCBZ-SO by reaction with sodium
hydroxide solution produced water-soluble Na salts which are potentially useful in the development of
new anthelmintic enantiomerically pure formulations.
Separation and characterization of unknown impurities and isomers in flomoxef sodium by LC-
IT-TOF MS and study of their negative-ion fragmentation regularities
Xu Yu, Fan Wang, Jiani Li, Weiguang Shan, Jian Wang
ABSTRACT
Thirteen unknown impurities in flomoxef sodium were separated and characterized by liquid
chromatography coupled with high resolution ion trap/time-of-flight mass spectrometry (LC-IT-TOF
MS)with positive and negative modes of electrospray ionization method for further improvement of
official monographs in pharmacopoeias. The fragmentation patterns of impurities in flomoxef in the
negative ion mode were studied in detail, and new negative-ion fragmentation regularities were
discovered. Chromatographic separation was performed on a Kromasil C18 column (250 mm × 4.6
mm, 5 μm). The mobile phase consisted of (A) ammonium formate aqueous solution (10 mM)–
methanol (84:16, v/v) and (B) ammonium formate aqueous solution (10 mM)–methanol (47:53, v/v).
In order to determine the m/z values of the molecular ions and formulas of all detected impurities, full
scan LC–MS in both positive and negative ion modes was firstly executed to obtain the m/z value of
the molecules. Then LC–MS2 and LC–MS3 were carried out on target compounds to obtain as much
structural information as possible. Complete fragmentation patterns of impurities were studied and
used to obtain information about the structures of these impurities. Structures of thirteen unknown
degradation products in flomoxef sodium were deduced based on the high resolution MSn data with
both positive and negative modes. The forming mechanisms of degradation products in flomoxef
sodium were also studied.
125
Heparin and homogeneous model heparin oligosaccharides form distinct complexes with
protamine: Light scattering and zeta potential analysis
Cynthia D. Sommers, Hongping Ye, Jian Liu, Robert J. Linhardt, David A. Keire
ABSTRACT
Large multimolecular complexes of heparin with positively charged proteins such as platelet factor 4
(PF4) or protamine can initiate immune responses associated with heparin use in patients, including
the most significant adverse event, heparin-induced thrombocytopenia (HIT). Current evidence
suggests that platelet-activating antibodies that recognize large multi-molecular complexes (300–700
kDa) of PF4 bound to heparin cause HIT [1] and in very rare cases anti-protamine-heparin antibodies
can induce thrombocytopenia [2]. Heparin is administered as a mixture of sulfated
glycosaminoglycans of variable lengths and sulfation levels. To date the potential impact of chain
length, sulfation level and impurities on the formation, size and immunogenicity of heparin-protamine
complexes has not been addressed due to the lack of purified, homogenous heparin chains for testing
purposes. Here, a set of well-characterized model heparin oligosaccharides was used with protamine
sulfate to evaluate the physicochemical properties of the resulting complexes. Hydrodynamic radii and
zeta potential profiles of heparin-protamine complexes were observed to be dependent upon the
sulfation location, size and concentration of the model heparin oligosaccharides. The well-
characterized oligosaccharide-protamine complexes analyzed in this work will be useful for
establishing links between heparin-protamine complex physiochemical attributes to their potential to
illicit cellular immunogenicity.
Quantitative analysis of binary polymorphs mixtures of fusidic acid by diffuse reflectance FTIR
spectroscopy, diffuse reflectance FT-NIR spectroscopy, Raman spectroscopy and multivariate
calibration
Canyong Guo, Xuefang Luo, Xiaohua Zhou, Beijia Shi, Xiaoxia Zhang
ABSTRACT
Vibrational spectroscopic techniques such as infrared, near-infrared and Raman spectroscopy have
become popular in detecting and quantifying polymorphism of pharmaceutics since they are fast and
non-destructive. This study assessed the ability of three vibrational spectroscopy combined with
multivariate analysis to quantify a low-content undesired polymorph within a binary polymorphic
mixture. Partial least squares (PLS) regression and support vector machine (SVM) regression were
employed to build quantitative models. Fusidic acid, a steroidal antibiotic, was used as the model
compound. It was found that PLS regression performed slightly better than SVM regression in all the
three spectroscopic techniques. Root mean square errors of prediction (RMSEP) were ranging from
0.48% to 1.17% for diffuse reflectance FTIR spectroscopy and 1.60–1.93% for diffuse reflectance FT-
NIR spectroscopy and 1.62–2.31% for Raman spectroscopy. The results indicate that diffuse
reflectance FTIR spectroscopy offers significant advantages in providing accurate measurement of
polymorphic content in the fusidic acid binary mixtures, while Raman spectroscopy is the least
accurate technique for quantitative analysis of polymorphs.
126
Mesoporous silica nanoparticles incorporated hybrid monolithic stationary phase immobilized
with pepsin for enantioseparation by capillary electrochromatography
Shujuan Xu, Rongzhen Mo, Can Jin, Xiaoqin Cui, Yibing Ji
ABSTRACT
In this study, a novel mesoporous silica nanoparticles incorporated chiral hybrid monolithic stationary
phase was developed. The stationary phase was firstly prepared by an in situ copolymerization of
amino-modified mesoporous silica nanoparticles (NH2-MSN), glycidyl methacrylate (GMA), and
ethylene dimethacrylate (EDMA) and then functionalized with pepsin as chiral selector. The
incorporated mesoporous silica nanoparticles provided additional interactions sites, and in turn yielded
different enantioselectivity thus enhancing the overall separation. The column was successfully
employed for enantioseparation of fifteen basic chiral drugs in capillary electrochromatography.
Effects of nanoparticles percentage, pepsin concentration, the pH of running buffer and the applied
voltage were investigated. All the analytes could be eluted in less than ten minutes and nine of them
could achieve baseline separation. Satisfactory repeatabilities with relative standard deviations less
than 4.2% were achieved through intraday, interday, column-to-column and batch-to-batch
investigations. These results indicated that the simultaneous utilization of the unique properties of
mesoporous silica nanoparticles and versatile features of monoliths could be a promising strategy for
enantioseparation.
Host-guest kinetic interactions between HP-β-cyclodextrin and drugs for prediction of bitter
taste masking
Zhen Guo, Fei Wu, Vikramjeet Singh, Tao Guo, Jiwen Zhang
ABSTRACT
Cyclodextrins (CD) are widely used bitter taste masking agents, for which the binding equilibrium
constant (K) for the drug-CD complex is a conventional parameter for quantitating the taste masking
effects. However, some exceptions have been reported to the expected relationship between K and
bitterness reduction and the relationship between kinetic parameters of a drug-CD interaction,
including association rate constant (Ka) and disassociation rate constant (Kd), and taste masking
remains unexplored. In this study, based upon a database of kinetic parameters of drugs-HP-β-CD
generated by Surface Plasmon Resonance Imaging for 485 drugs, the host-guest kinetic interactions
between drugs and HP-β-CD for prediction of taste masking effects have been investigated. The taste
masking effects of HP-β-CD for 13 bitter drugs were quantitatively determined using an electronic
gustatory system (α-Astree e-Tongue). Statistical software was used to establish a model based on
Euclidean distance measurements, Ka and Kd of the bitter drugs/HP-β-CD-complexes (R2 = 0.96 and
P < 0.05). Optimized parameters, Ka3, Kd, KaKd, Kd3, Ka2 and Ka/Kd with notable influence, were
obtained by stepwise regression from 12 parameters derived from Ka, Kd and K (Ka/Kd). 10-fold
cross-validation was used to verify the reliability of the model (correlation coefficient of 0.84, P <
0.05). The established model indicated a relationship between Ka, Kd, K and taste masking by HP-β-
CD and was successful in predicting the extent of taste masking by HP-β-CD of 44 bitter drugs, which
was in accordance with the literature reported. In conclusion, the relationship between kinetics of drug-
CD interactions and taste masking was established and providing a new strategy for predicting the
cyclodextrin mediated bitter taste masking.
127
Crystal structures and physicochemical properties of amisulpride polymorphs
Wen-Peng Zhang, Dong-Ying Chen
ABSTRACT
The purpose of this work was to investigate the crystal structures and physicochemical properties of
amisulpride polymorphs. Except of the previously reported polymorph (named as Form I), a new
polymorphic form (named as Form II) was discovered through comprehensive solid-state screening
experiments. Both polymorphic forms were characterized by single crystal X-ray structure analysis
(SXRD), powder X-ray diffraction (PXRD), dynamic vapor sorption (DVS) and thermal analysis
(TGA and DSC) as well. It has been found that the Forms I and II are of conformational polymorph
with the main conformational difference around ethylsulfonyl group. Form II possesses lower
hygroscopicity and better solubility compared with that of Form I, indicating Form II could be an
alternate solid form for formulation development.
A UHPLC method for the rapid separation and quantification of phytosterols using tandem
UV/Charged aerosol detection – A comparison of both detection techniques
Jakub Fibigr, Dalibor Ńatínský, Petr Solich
ABSTRACT
The presented work describes the development and validation of a rapid UHPLC-UV/CAD method
using a core–shell particle column for the separation and quantitative analysis of seven plant sterols
and stanols. The phytosterols (ergosterol, brassicasterol, campesterol, fucosterol, stigmasterol, and β-
sitosterol) and the phytostanol stigmastanol were separated and analyzed in 8.5 min. The sample pre-
treatment procedure was optimized to be less time-consuming than any other published method,
especially due to no need of derivatization, evaporation and even reconstitution step. The
chromatographic separation was performed on the Kinetex 1.7 μ Phenyl-hexyl column (100 × 2.1 mm)
with a mobile phase acetonitrile/water according to the gradient program at a flow rate of 0.9 mL
min−1 and a temperature of 60 °C. A tandem connection of PDA and CAD (Corona Charged Aerosol
Detector) was used and both detection techniques were compared. The method was validated using
saponification as a first step in sample pre-treatment and an universal CAD as the detector. Recoveries
for all analyzed compounds were between 95.4% and 103.4% and relative standard deviation ranged
from 1.0% to 5.8% for within-day and from 1.4% to 6.7% for between-day repeatability. The limits of
detection were in the range of 0.4–0.6 μg mL−1 for standard solutions and 0.3–1.2 μg mL−1 for
phytosterols in real samples. Although several gradient programs and different stationary phases were
tested, two compounds, campesterol and campestanol, were not separated. Their peak was quantified
as a sum of both analytes.
128
Analysis of chemical constituents in an herbal formula Jitong Ning Tablet
Di Gao, Baojun Wang, Zhipeng Huo, Yi He, Lian-Wen Qi
ABSTRACT
Jitong Ning Tablet (JTNT), a traditional Chinese herbal formula, consists of Eucommia ulmodies oliv,
Angelicae pubescentis radix, Aconiti radix cocta, Corydalis yanhusuo w.t. wang, Glycyrrhizae radix et
rhizoma, Paeoniae radix rubra and Radix puerariae. It has been demonstrated to show protective
effects on ankylosing spondylitis and anti-inflammatory effects. The chemical compositions of JTNT,
playing a key role in quality control, remain unknown. In this study, an ultra-performance liquid
chromatography combined with quadrupole time of flight mass spectrometry (UPLC-Q–TOF–MS)
method in both positive and negative ion mode was established to investigate the chemical constituents
of JTNT formula. In total, 162 compounds including flavonoids, triterpenoids, coumarins, alkaloids,
phenylpropionic acids, lignans, terpenoids, and organic acids were detected, 152 of which were
unambiguously or tentatively identified by comparing their retention times and accurate mass
measurement with reference compounds and data in literatures. Our results would benefit quality
control and chemical basis for JTNT.
Molecular recognition of pseudodistamine isomeric precursor trans-3(4)-aminopiperidin-4(3)-ols
by EI mass spectrometry
D.M. Mazur, G.V. Grishina, A.T. Lebedev
ABSTRACT
Synthesis of drugs, biologically active compounds or their derivatives always requires precise and
reliable method of their identification, including differentiation of the possible isomers.
Pseudodistamines and their precursors became a matter of elevated attention due to their different
enzymatic inhibition. This paper deals with one of the groups of the pseudodistamine precursors −
trans-3(4)-aminopiperidin-4(3)-ols. Their synthesis brings to a mixture of 2 regioisomers, resulting in
the necessity of their reliable recognition. NMR spectroscopy commonly used by organic chemists
requires advance knowledge and experience to analyse the spectra of these regioisomers. Therefore,
we herein proposed a simpler way to recognize trans-3(4)-aminopiperidin-4(3)-ols using mass
spectrometry with electron ionization. Fragmentation of 4 pairs of aminopiperidinol regioisomers with
variation of amine moiety was studied. The obtained results allowed defining a group of 3 ions ([M-
18]+., [M-19]+, [M-43]+) related only to the structure of trans-4-aminopiperidin-3-ols and 1 ion (m/z
100) related to the structure of trans-3-aminopiperidin-4-ols. Besides, interrogation of intensity of ions
common for spectra of both regioisomers allows making differentiation as well.
129
Development and validation of a general derivatization HPLC method for the trace analysis of
acyl chlorides in lipophilic drug substances
Xiangyuan Zheng, Lan Luo, Jie Zhou, Xiaoling Ruan, Feng Zheng
ABSTRACTs
Acyl chlorides are important acylating agents in the synthesis of active pharmaceutical ingredients.
Determining the residual acyl chlorides in drug substances is a challenge due to their high reactivity
and the matrix interferences from drug substances and their related impurities. This paper describes a
general derivatization HPLC method for the determination of aromatic and aliphatic acyl chlorides in
lipophilic drug substances. Since most drug substances have weak absorptions in the visible range
(above 380 nm), the nitro-substituted anilines and nitro-substituted phenylhydrazines were selected as
the derivatization reagents due to their weak basicity and red-shift of UV absorption spectra. The
maximum wavelength and absorption intensity of nitro-substituted anilines decreased after
derivatization with acyl chlorides, whereas the derivatization products of nitro-substituted
phenylhydrazines showed the slight increases of maximum wavelength and absorbance intensity.
Hence, 2-nitrophenylhydrazine was selected as the suitable derivatization reagent because the
derivatives have the maximum UV wavelength absorbance at 395 nm, which could largely minimize
the matrix interferences. The optimization of the concentration of 2-nitrophenylhydrazine is important
for the sensitivity and stability of derivatives. Other reaction conditions including reaction temperature,
time and the influence of three competitive solvents (water, methanol and ethanol) on the reaction
efficiency were also studied. After derivatization with 100 μg mL−1 2-nitrophenylhydrazine at room
temperature for 30 min, the method was validated for high specificity and sensitivity with the detection
limits in the range of 0.01–0.03 μg mL−1. The proposed method was applied as a generic method to
determine the residual acyl chlorides in lipophilic drug substances.
An isocratic hydrophilic interaction liquid chromatographic method for simultaneous
determination of iodixanol and its related impurities in drug substance
Bruno D. Rondon, Neila M. Cassiano, Quezia B. Cass
ABSTRACT
This work reports a simple isocratic hydrophilic interaction liquid chromatographic (HILIC) method
for the simultaneous quantification of iodixanol and of its related impurities C, D and E in drug
substance. The chromatographic separation was carried out with a Kinetex™ HILIC column, using
acetonitrile and formic acid aqueous solution (1.0 mmol/L, pH 3.2) (92:08, v/v) as eluent at a flow rate
of 0.8 mL/min. The autosampler and column temperature were maintained at 20 °C and UV detection
was set at 243 nm. The method was validated in accordance to the ICH guideline and employed for the
analysis of two different lots of iodixanol drug substance. The developed method is presented as a
valuable alternative to the current methods described in the USP monograph.
130
Electrochemical and optical study of metallothionein interactions with prion proteins
Alzbeta Cardova, Pavlina Adam, Stefano Mariani, Lukas Richtera, Vojtech Adam
ABSTRACT
The prion protein (PrPC) can be structurally shifted to its PrPSc isoform causing a wide range of
neurodegenerative diseases, which are currently incurable. There is an evidence that metallothioneins
(MTs), and especially MT-3, are associated with neurodegenerative diseases. PrPC and MTs play
pivotal roles in maintaining metal homeostasis; therefore, it is conceivable that each of them has its
own significance in prion diseases. In this paper, we study the nature of interactions between PrPC,
MT, and copper ions, Cu(II), using the method of differential pulse voltammetry (DPV) coupled with
adsorptive transfer stripping technique (AdTS). Electrochemical properties of PrP itself and its
interactions with both the Cu(II) ions and MTs have been found. Based on the results obtained, we
hypothesised the formation of the complex in molar ratio 2:1 (PrPC:MT). Surface plasmon resonance
imaging (SPRi) was used as a control reference assay to further confirm results obtained by the
electrochemical approach, such as the specific interactions between PrPC and MT-3.
Development of matrix effect-free MISPE-UHPLC–MS/MS method for determination of
lovastatin in Pu-erh tea, oyster mushroom, and red yeast rice
Pavel Svoboda, Daniel Sander, Kateřina Plachká, Lucie Nováková
ABSTRACT
Matrix effect-free UHPLC–MS/MS method was developed and validated for the determination of
cholesterol-lowering lovastatin in food samples represented by Pu-erh tea, oyster mushroom, and red
yeast rice. The resulting method was fully validated in terms of intra-day and inter-day precision,
accuracy, linearity, range, LOD, LOQ, and matrix effects. The matrix effect phenomenon evaluated by
comparison of slopes of calibration curves was completely eliminated by solid-phase extraction based
on the technique of molecularly imprinted polymers (MIPs). Comparison of elution profiles obtained
on the MIP and corresponding control non-imprinted polymer (NIP) showed selectivity of the
extraction procedure. In addition, selectivity of the MIP material and the molecularly imprinted solid-
phase extraction (MISPE) was also proved by experiments evaluating retention of analytes physico-
chemically similar to the target molecule. Extraction recoveries of these analytes represented by
estrogen derivatives (estrone, estriol, 17α‐ethinylestradiol, and β-estradiol) were very low or even null.
Synthesis and preparation of the resulting MIP sorbent was characterized by excellent repeatability
expressed as RSD 7.7% (n = 9) of extraction recoveries. The determined capacity of the MIP material
reaching 375 ng/mg is sufficient for analysis of the evaluated statin in its natural sources. Suitability of
the resulting MISPE-UHPLC–MS/MS procedure for real sample analysis was verified by the
determination of lovastatin in one dietary supplement based on the red yeast rice with a given amount
of the target analyte. Finally, three mushroom and fifteen tea samples obtained in Czech food stores
and tearooms were subjected to analysis. Low or null amount of lovastatin was found in these samples.
131
Facile preparation of fibrin coated open tubular column for characterization of monoclonal
antibody variants by capillary electrochromatography
Xue Xiao, Wentao Wang, Yamin Zhang, Li Jia
ABSTRACT
Monoclonal antibodies (mAbs) are one of the most promising classes of therapeutic protein
biopharmaceuticals. However, the complexity of mAbs poses a daunting analytical challenge for
heterogeneity characterization of mAbs. In this study, inspired by blood coagulation, we adopted a
fibrin coating as a novel stationary phase in open tubular (OT) column for the separation of the mAbs
variants by capillary electrochromatography. The fibrin coating was prepared by in situ polymerization
of fibrin in the presence of thrombin as a catalyst inside a fused-silica capillary. Scanning electron
microscopy and electroosmotic flow measurement were carried out to characterize the fibrin coated
OT columns. The average thickness of the fibrin coating was about 1.13 μm. And the EOF of the
column was pH-dependent. The electrochromatographic performance of the prepared columns was
evaluated by characterization of the variants of three mAbs (cetuximab, trastuzumab and rituximab).
The columns demonstrated good repeatability with the run-to-run, day-to-day and column-to-column
relative standard deviations of migration times less than 2.42%. The study highlighted the potential of
adsorbed proteins as stationary phases for the separation of mAbs variants. Furthermore, the study
provided a new platform for characterization of heterogeneity of mAbs in pharmaceutical industry.
Development and validation of a fast SFC method for the analysis of flavonoids in plant extracts
Yang Huang, Ying Feng, Guangyun Tang, Minyi Li, Zhengjin Jiang
ABSTRACT
Flavonoids from plants always show a wide range of biological activities. In the present study, a rapid
and highly efficient supercritical fluid chromatography (SFC) method was developed for the separation
of 12 flavonoids. After careful optimization, the 12 flavonoids were baseline separated on a ZORBAX
RX-SIL column using gradient elution. A 0.1% phosphoric acid solution in methanol was found to be
the most suitable polar mobile phase component for the separation of flavonoids. From the viewpoint
of retention and resolution, a backpressure of 200 bar and a temperature of 40 °C were shown to give
the best results. Compared with a previously developed reverse phase liquid chromatography method,
the SFC method could provide flavonoid separations that were about three times faster, while
maintaining good peak shape and comparable peak efficiency. This SFC method was validated and
applied to the analysis of five flavonoids (kaempferol, luteolin, quercetin, luteoloside, buddleoside)
present in Chrysanthemum morifolium Ramat. from different cultivars (Chuju, Gongju, Hangju, Boju).
The results indicated a good repeatability and sensitivity for the quantification of the five analytes with
RSDs for overall precision lower than 3%. The limits of detection ranged from 0.73 to 2.34 μg/mL,
while the limits of quantification were between 2.19 and 5.86 μg/mL. The method showed that SFC
could be employed as a useful tool for the quality assessment of Traditional Chinese medicines
(TCMs) containing flavonoids as active components.
132
A multi-matrix HILIC-MS/MS method for the quantitation of endogenous small molecule
neurological biomarker N-acetyl aspartic acid (NAA)
Dewakar Sangaraju, Sheerin K. Shahidi-Latham, Braydon L. Burgess, Brian Dean, Xiao Ding
ABSTRACT
A multi-matrix hydrophilic interaction liquid chromatography tandem mass spectrometric method
(HILIC-MS/MS) was developed for the quantitation of N-Acetyl Aspartic acid (NAA) using stable
isotope labeled internal standard, D3-NAA in various biological matrices such as human plasma,
human CSF, mouse plasma, brain and spinal cord. A high throughput 96-well plate format supported
liquid extraction (SLE) procedure was developed and used for sample preparation. Mass spectrometric
analysis of NAA was performed using selected reaction monitoring transitions in positive electrospray
ionization mode. As NAA is endogenously present, a surrogate matrix approach was used for
quantitation of NAA and the method was qualified over linear calibration curve range of 0.01–10
μg/mL. Intra and inter assay precision indicated by percent relative standard deviation (%RSD) was
less than 7.1% for low, medium, medium high and high QCs. The accuracy of the method ranged from
92.6-107.0% of nominal concentration for within-run and between-run for the same QCs. Extraction
recovery of NAA and D3-NAA was greater than 76%. Stability of NAA was established in the above
biological matrices under bench top (RT, 5 h), freeze thaw (–20 ± 10 °C, 3 cycles) and moues/human
plasma sample collection (Wet ice, RT) conditions. HILIC-MS/MS method was then used to quantify
and compare the NAA levels in human plasma and CSF of ALS patients versus control human
subjects. NAA CSF levels in control human subjects (73.3 ± 31.0 ng/mL, N = 10) were found to be
slightly higher than ALS patients (46.1 ± 22.6 ng/mL, N = 10) (P = 0.04). No differences were
observed in NAA plasma levels in human control subjects (49.7 ± 13.8 ng/mL, N = 9) as compared to
ALS patients (49.6 ± 8.1 ng/mL, N = 10) (P = 0.983). NAA endogenous concentrations in mouse
plasma, brain and spinal cord were found to be 243.8 ± 56.8 ng/mL (N = 6), 1029.8 ± 115.2 μg/g tissue
weight (N = 5) and 487.6 ± 178.4 μg/g tissue weight (N = 5) respectively.
Chemotaxonomic studies of nine Paris species from China based on ultra-high performance
liquid chromatography tandem mass spectrometry and Fourier transform infrared spectroscopy
Yuanzhong Wang, Ehu Liu, Ping Li
ABSTRACT
Paris species, which contain steroid saponins, have been used as herb folk medicines in Asia. In the
present study, a comprehensive strategy based on liquid chromatography-tandem mass spectrometry
(LC–MS/MS) and Fourier transform infrared (FT-IR) spectroscopy was firstly proposed to evaluate
the chemotaxonomic relationships of nine Paris species sampled from different geographical regions in
China. Principle component analysis (PCA) based on FT-IR data revealed chemical similarities in term
of the nine species and geographical regions, indicating the accumulation of metabolites affected by
the combination of geographical factors and species. The chemotaxonomic relationships of four
species supported the morphological taxonomy and implied ancestry from P. polyphylla. After high-
efficiency chromatographic separation, ions trap/time-of-flight mass spectrometry (IT-TOFMS) and
triple quadrupole mass spectrometry (QQQ-MS) were used to identify unknown metabolites and
simultaneously determine six key compounds (polyphyllin I, II, V, VI, VII and gracillin) in Paris
species, respectively. The tentative identification of 22 steroid saponins was indicative of a common
biosynthetic pathway in Paris species. Phytoecdysones, gracillin and open-chain steroid saponins were
considered as key precursors.
133
Rapid profiling and pharmacokinetic studies of major compounds in crude extract from
Polygonum multiflorum by UHPLC-Q-TOF-MS and UPLC–MS/MS
Linlin Wang, Mangmang Sang, Erwei Liu, Prince Osei Banahene, Xiumei Gao
ABSTRACT
A reliable, rapid analytical method was established for characterization of constituents in the ethanol
extract of Polygonum multiflorum by combining an ultra-high performance liquid chromatography
with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). 131 constituents which
including phenolic acids, stilbenes, flavones, anthraquinones, naphthalenes and their derivatives were
identified or tentatively identified by using characteristic diagnostic fragment ions and references. The
established method was further applied to analyze blood samples, and successfully identified 41
compounds which were absorbed through the gastrointestine in rats after administration the extract of
P. multiflorum. Moreover, the pharmacokinetic studies of some major compounds in blood were
investigated by using ultra performance liquid chromatography with tandem mass spectrometry
(UPLC–MS/MS) method. This study showed a comprehensive research of P. multiflorum, which
could provide a meaningful basis for further quality control, pharmacological as well as toxicological
researches.
Metabolic profiling of nuciferine in rat urine, plasma, bile and feces after oral administration
using ultra-high performance liquid chromatography-diode array detection-quadrupole time-of-
flight mass spectrometry
Xiao-Lei Wu, Ming-Jiang Wu, Xin-Ze Chen, Hao-Ling Ma, De-Qin Zhang
ABSTRACT
Nuciferine, a major alkaloid found in Nelumbinis Folium, exhibits a broad spectrum of bioactivities,
such as antiobesity, anti-diabetes and anti-inflammatory. However, many research regarding nuciferine
focused on the extraction, isolation and biological activity, the metabolism is not comprehensively
explained in vivo. Thence, the present of this paper is to establish a simple method for speculating
metabolites of nuciferine. A total of 15 metabolites were detected and tentatively identified through
ultra high performance liquid chromatography-diode array detection-quadrupole time-of-flight mass
spectrometry (UHPLC-DAD-QTOF-MS), including 7 new metabolites. Among them, we also
discovered a previously unmentioned metabolically active site at the C1-OCH3 position. These
metabolites suggested that demethylation, oxidation, glucuronidation and sulfation were major
metabolic pathways. This study provided significant experiment basis for its safety estimate and
valuable information about the metabolism of nuciferine, which will be advantageous for new drug
development.
134
Development and validation of liquid chromatography tandem mass spectrometry method
quantitative determination of polymyxin B1, polymyxin B2, polymyxin B3 and isoleucine-
polymyxin B1 in human plasma and its application in clinical studies
Kim H. Hee, Yee K.J. Leaw, Jun L. Ong, Lawrence S. Lee
ABSTRACT
Polymyxin B (PB) is an antibiotic consisting of a cyclic heptapeptide and a tripeptide side chain used
in treatment of infections caused by Gram-negative bacteria. Commercial formulations of PB contain
multiple structurally related components with major constituents of PB1, PB2, PB3 and ile-PB1. To
understand the pharmacokinetics of these major components, we have developed and validated a LC–
MS/MS method to quantify PB1, PB2, PB3 and ile-PB1 in human plasma. PB was extracted from
plasma by protein precipitation using trichloroacetic acid followed by chromatographic separation on
Zorbax Bonus-RP column (100 mm × 2.1 mm, 1.8 μm) using stepwise gradient elution of water
containing 0.1% of formic acid and 0.1% of trichloroacetic acid (mobile phase A) and 90% acetonitrile
with 0.1% formic acid (mobile phase B). Despite of structural similarities, these PBs were completely
resolved in the analytical run time of 6.5 min. Detection and quantification of PBs were performed by
selected reaction monitoring (SRM) under positive ionization mode in the mass spectrometer.
Separation of PB1 and ile-PB1, as well as PB2 and PB3, before quantification is crucial because they
are structural isomers detected based the same SRM. Excellent linearity was achieved (r2 > 0.99) in
the calibration curves of PB. The developed method was accurate (95.3–111.7%) and precise (CV <
5.1%). Recovery of PB from the plasma extraction was between 53 and 76% and reproducible (CV <
4.5%). Matrix effect was not observed by post-column infusion of PB in the mass spectrometer. This
methodology has been successfully applied to clinical study of patients dosed with intravenous
infusions of PB.
Human exposure to Bisphenol A and liver health status: Quantification of urinary and
circulating levels by LC–MS/MS
Carla Nicolucci, Sonia Errico, Alessandro Federico, Marcello Dallio, Nadia Diano
ABSTRACT
A selective and highly sensitive analytical methodology for determination of Bisphenol A in human
plasma was developed and validated. The method was based on selective liquid/solid extraction,
combined with liquid chromatography–electrospray ionization tandem mass spectrometry in the
multiple reaction monitoring mode and negative ionization. The linearity of the detector response was
verified in human plasma over the concentration range 0.100–200 ng mL−1. The detection limit was
0.03 ng mL−1 and the quantification limit was 0.100 ng mL−1. The analytical features of the proposed
in-house validated method were satisfactory: precision was <10% and recoveries were around 84–
104%. The matrix effect was studied and compensated using deuterated labeled standard. The
applicability of the proposed method was demonstrated analyzing human plasma samples from
individuals affected by non-alcoholic fatty liver disease. Bisphenol A was detected above the detection
limit in all samples. The data show a persistence of unconjugated Bisphenol A levels in plasma and
indicate a chronic Bisphenol A exposure of the target organ, suggesting an association between liver
health status and Bisphenol A exposure. The results from our study are valuable for further
investigation with large sample size and longitudinal study designs, necessary to confirm the observed
association.
135
Determination of prodrug treosulfan and its biologically active monoepoxide in rat plasma, liver,
lungs, kidneys, muscle, and brain by HPLC–ESI–MS/MS method
Michał Romański, Anna Kasprzyk, Artur Teżyk, Agnieszka Widerowska, Franciszek Główka
ABSTRACT
A prodrug treosulfan (TREO) is currently investigated in clinical trials for conditioning prior to
hematopoietic stem cell transplantation. Bioanalysis of TREO and its active derivatives, monoepoxide
(S,S-EBDM) and diepoxide, in plasma and urine underlay the pharmacokinetic studies of these
compounds but cannot explain an organ pharmacological action or toxicity. Recently, distribution of
TREO and S,S-EBDM into brain, cerebrospinal fluid, and aqueous humor of the eye has been
investigated in animal models and the obtained results presented clinical relevance. In this paper, a
selective and rapid HPLC–ESI–MS/MS method was elaborated and validated for the studies of
disposition of TREO and S,S-EBDM in rat plasma, liver, lungs, kidneys, muscle, and brain. The two
analytes and codeine, internal standard (IS), were isolated from 50 μL of plasma and 100 μL of
supernatants of the tissues homogenates using ultrafiltration Amicon vials. Chromatographic
resolution was accomplished on C18 column with isocratic elution. The limits of quantitation of TREO
and S,S-EBDM in the studied matrices ranged from 0.11 to 0.93 μM. The HPLC–MS/MS method was
adequately precise and accurate within and between runs. The IS-normalized matrix effect differed
among the tissues and was the most pronounced in a liver homogenate supernatant (approximately
0.55 for TREO and 0.35 for S,S-EBDM).
Profiles of amino acids and biogenic amines in the plasma of Cri-du-Chat patients
Danielle Zildeana Sousa Furtado, Fernando Brunale Vilela de Moura Leite, Cleber Nunes Barreto,
Bernadete Faria, Nilson Antonio Assunção
ABSTRACT
Cri-du-chat syndrome (CDCS) is a rare innate disease attributed to chromosome 5p deletion
characterized by a cat-like cry, craniofacial malformation, and altered behavior of affected children.
Metabolomic analysis and a chemometric approach allow description of the metabolic profile of
CDCS as compared to normal subjects. In the present work, UHPLC/MS was employed to analyze
blood samples withdrawn from CDCS carriers (n = 18) and normal parental subjects (n = 18), all aged
0–34 years, aiming to set up a representative CDCS profile constructed from 33 targeted amino acids
and biogenic amines. Methionine sulfoxide (MetO) was of particular concern with respect to CDCS
redox balance. Increased serotonin (3-fold), methionine sulfoxide (2-fold), and Asp levels, and a little
lower Orn, citrulline, Leu, Val, Ile, Asn, Gln, Trp, Thr, His, Phe, Met, and creatinine levels were found
in the plasma of CDCS patients. Nitrotyrosine and Trp did not differ in normal and CDCS
individuals.The accumulated metabolites may reflect, respectively, disturbances in the redox balance,
deficient purine biosynthesis, and altered behavior, whereas the amino acid abatement in the latter
group may affect the homeostasis of the urea cycle, citric acid cycle, branched chain amino acid
synthesis, Tyr and Trp metabolism and amino acid biosynthesis. The identification of enzymatic
deficiencies leading to the amino acid burden in CDCS is further required for elucidating its molecular
bases and eventually propose specific or mixed amino acid supplementation to newborn patients
aiming to balance their metabolism.
136
Online microdialysis-ultra performance liquid chromatography–mass spectrometry method for
comparative pharmacokinetic investigation on iridoids from Gardenia jasminoides Ellis in rats
with different progressions of type 2 diabetic complications
Xueju Zhang, Lu Wang, Zhong Zheng, Zifeng Pi, Fengrui Song
ABSTRACT
Iridoid glycosides consist the main bioactive constituents of Gardenia jasminoides Ellis (G.
jasminoides), which is used alone or in combination with other medicine herbs for treatment of
diabetes mellitus in China. This paper investigated for the first time comparative pharmacokinetics
(PKs) of four unbound iridoids (including genipin, geniposide, gardenoside and geniposidic acid) in rat
blood between healthy and type 2 diabetic groups with different disease progressions (9 or 13 weeks).
Online microdialysis-ultra performance liquid chromatography-mass spectrometry (MD- UPLC–
MS/MS) method was used after oral administration of iridoid extracts obtained from the fruits of G.
jasminoides. Student's t-test was used for statistical comparison of PK parameters. Results showed that
genipin, geniposide and geniposidic acid feature higher Cmax, larger area under the curve (AUC),
lower clearance (CL), longer Tmax and mean residence time (MRT) in type 2 diabetic rats than those
in healthy rats (p < 0.05). However, no significant difference was observed in PK parameters between
the two diabetic groups with different complication progressions (p > 0.05). Type 2 diabetic rats
showed significantly altered PK behaviors of genipin, geniposide and geniposidic acid (especially
systemic exposure, AUCs of genipin, geniposide and geniposidic acid). Online MD-UPLC–MS/MS
provides real-time sampling and monitoring, these functions can be applied to PKs of iridoids from G.
jasminoides.
Optimization of a new methodology for trace determination of elements in biological fluids:
Application for speciation of inorganic selenium in children’s blood
Reza Akramipour, Mitra Hemati, Nazir Fattahi, Meghdad Pirsaheb, Toraj Ahmadi-Jouibari
ABSTRACT
The continuous sample drop flow microextraction (CSDFME) joined with the iridium-modified tube
graphite furnace atomic absorption spectrometry (GFAAS) has been developed as a highly sensitive
technique for the speciation of selenium in blood samples. In this method 32.0 μl carbon tetrachloride
is transferred to the bottom of a conical sample cup. Then the 5.0 ml of aqueous solution transforms to
fine droplets while passing through the organic solvent. At this stage, Se(IV)-APDC hydrophobic
complex is extracted into the organic solvent. After extraction, the conical sample cup is transferred to
the GFAAS and 20 μl of extraction solvent was injected into the graphite tube by the aim of
autosampler. Under the optimum conditions, the calibration graph was linear in the range of 0.06–3.0
μg l−1 with detection limit of 0.02 μg l−1. The enrichment factor and enhancement factor were 106
and 91, respectively. Repeatability (intra–day) and reproducibility (inter–day) of method based on
seven replicate measurements of 2.5 μg l−1 of selenium were 3.7% and 4.2%, respectively. Total
inorganic Se(IV, VΙ) was measured after reduction of Se(VΙ) with gentle boiling in 5 M HCl medium
for 50 min and adjusting pH to 3, and the concentration of Se(VΙ) was calculated by subtracting the
Se(IV) concentration from the total selenium concentration.
137
Detailed analysis of cortisol, cortisone and their tetrahydro- and allo-tetrahydrometabolites in
human urine by LC–MS/MS
Katarzyna Kosicka, Anna Siemiątkowska, Dąbrówka Pałka, Agata Szpera-Goździewicz, Franciszek K.
Główka
ABSTRACT
Cortisol (F) and cortisone (E) are metabolized to A-ring reduced metabolites in the reactions catalyzed
by 5α- and 5β-reductase. 5α-tetrahydrocortisol (alloTHF) and 5β-tetrahydrocortisol (THF) are
produced from F. The metabolism of E takes place in analogy to form alloTHE and THE. Up to now,
the analysis of endogenous glucocorticoids did not consider alloTHE, limiting the metabolism of E to
THE only. Nevertheless, such simplification can generate inaccuracy in the assessment of the function
of enzymes crucial for glucocorticoids metabolism: 11β-hydroxysteroid dehydrogenase type 1 and
type 2 (11β-HSD1 and 11β-HSD2), as well as 5α- and 5β-reductase. The paper presents the new LC–
MS/MS method for the simultaneous analysis of F and E with their tetrahydro- (THF and THE) and
allo-tetrahydrometabolites (alloTHF and alloTHE) in urine. The method was fully validated and allows
determining both the unconjugated and total concentrations of urinary glucocorticoids. The method
meets the EMA‘s recommendations and was proved to be useful in the analysis of clinical samples.
The LLOQ of 1 ng/mL allows the determination of free urinary F, E, THF and THE, but not alloTHF
and alloTHE, in samples obtained from pregnant women.
Urinary metabonomics study of the hepatoprotective effects of total alkaloids from Corydalis
saxicola Bunting on carbon tetrachloride-induced chronic hepatotoxicity in rats using 1H NMR
analysis
Fang Wu, Hua Zheng, Zheng-Teng Yang, Bang Cheng, Zhi-Heng Su
ABSTRACT
Chronic liver injury has been shown to cause liver fibrosis due to the sustained pathophysiological
wound healing response of the liver, and eventually progresses to cirrhosis. The total alkaloids of
Corydalis saxicola Bunting (TACS), a collection of important bioactive ingredients derived from the
traditional Chinese folk medicine Corydalis saxicola Bunting (CS), have been reported to have
protective effects on the liver. However, the underlying molecular mechanisms need further
elucidation. In this study, the urinary metabonomics and the biochemical changes in rats with carbon
tetrachloride (CCl4)-induced chronic liver injury due to treatment TACS or administration of the
positive control drug-bifendate were studied via proton nuclear magnetic resonance (1H NMR)
analysis. Partial least squares-discriminate analysis (PLS-DA) suggested that metabolic perturbation
caused by CCl4 damage was recovered with TACS and bifendate treatment. A total of seven
metabolites including 2-oxoglutarate, citrate, dimethylamine, taurine, phenylacetylglycine, creatinine
and hippurate were considered as potential biomarkers involved in the development of CCl4-induced
chronic liver injury. According to pathway analysis using identified metabolites and correlation
network construction, the tricarboxylic acid (TCA) cycle, gut microbiota metabolism and taurine and
hypotaurine metabolism were recognized as the most affected metabolic pathways associated with
CCl4 chronic hepatotoxicity. Notably, the changes in 2-oxoglutarate, citrate, taurine and hippurate
during the process of CCl4-induced chronic liver injury were significantly restored by TACS
treatment, which suggested that TACS synergistically mediated the regulation of multiple metabolic
pathways including the TCA cycle, gut microbiota metabolism and taurine and hypotaurine
metabolism.
138
Pharmacokinetic properties of the synthetic cannabinoid JWH-018 and of its metabolites in
serum after inhalation
Stefan W. Toennes, Anna Geraths, Werner Pogoda, Alexander Paulke, Johannes G. Ramaekers
ABSTRACT
Each year, synthetic cannabinoids are occurring in high numbers in the illicit drug market, but data on
their pharmacology and toxicology are scarcely available. Therefore, a pilot study was performed to
assess adverse effects of JWH-018, which is one of the oldest and best known synthetic cannabinoids.
Six subjects inhaled smoke from 2 and 3 mg JWH-018. The drug and nine of its metabolites were
analyzed in their blood samples taken during the following 12 h by liquid chromatography–mass
spectrometry (LC–MSMS). The maximum concentration of JWH-018 reached 2.9–9.9 ng/ml after
inhalation and markedly decreased during the next 1.5 h, followed by a multiexponential decline (t1/2
in median 1.3 h and 5.7 h). The concentration of the pentanoic acid metabolite was slightly higher than
that of the 3-, 4- and 5-hydroxypentyl metabolites and of the 6-hydroxyindol metabolite. The data also
suggest a multiexponential decline and slow terminal elimination of JWH-018 and all metabolites. The
detection of JWH-018 and of its metabolites in serum requires high analytical sensitivity. The
pharmacokinetic properties of inhaled JWH-018 are similar to that of THC. A slow terminal
elimination of drug and metabolites may lead to accumulation in chronic users.
Charged derivatization and on-line solid phase extraction to measure extremely low cortisol and
cortisone levels in human saliva with liquid chromatography–tandem mass spectrometry
Balázs Magda, Zoltán Dobi, Katalin Mészáros, Éva Szabó, Pál T. Szabó
ABSTRACT
The aim of this study was to develop a sensitive, reliable and high-throughput liquid chromatography –
electrospray ionization – mass spectrometric (LC-ESI–MS/MS) method for the simultaneous
quantitation of cortisol and cortisone in human saliva. Derivatization with 2-hydrazino-1-
methylpyridine (HMP) was one of the most challenging aspects of the method development. The
reagent was reacting with cortisol and cortisone at 60 °C within 1 h, giving mono- and bis-hydrazone
derivatives. Investigation of derivatization reaction and sample preparation was detailed and discussed.
Improvement of method sensitivity was achieved with charged derivatization and use of on-line solid
phase extraction (on-line SPE). The lower limit of quantitation (LLOQ) was 5 and 10 pg/ml for
cortisol and cortisone, respectively. The developed method was subsequently applied to clinical
laboratory measurement of cortisol and cortisone in human saliva.
139
Comparability study of Rituximab originator and follow-on biopharmaceutical
Othman Montacir, Houda Montacir, Murat Eravci, Andreas Springer, Maria Kristina Parr
ABSTRACT
Immunglobolin G (IgG)-based biopharmaceuticals are emerging on the pharmaceuticals market due to
their high target selectivity in different diseases. In parallel, a growing interest by other companies to
produce similar or highly similar follow-on biologics exits, once the patent of blockbuster
biotherapeutics is about to expire. In correlation to their complex structure, an analytical challenge is
facing the approval of these biosimilars. Health authorities (e.g. FDA and EMA) have issued several
guidelines to define critical quality attributes during manufacturing process changes. In the current
study, physicochemical characterization using state-of-the-art analytics was applied to analyse intact
mass, post-translational modifications (PTMs) and higher order structure of Rituximab and one of its
biosimilars. Intact mass analysis, middle-up approach as well as subunit analysis revealed similar
glycoforms but additional lysine variants in the biosimilar. The N-glycosylation site was confirmed for
both, the originator and the biosimilar. PTMs and higher order structure were confirmed to be similar.
A special focus was given to N-glycosylation due to its potential to monitor the batch-to-batch
consistency and alteration during the production bioprocess. Comparison of the N-glycosylation
profiles obtained from three batches of the biosimilar and the reference product showed quantitative
variations, although the N-glycans were qualitatively similar. Furthermore, a head-to-head
comparability of functional properties was performed to investigate the impact of glycosylation
alteration and PTMs on potency within the biosimilar batches and between originator and follow-on
biodrug. The data affirm that the difference is still in the acceptable range for biosimilarity.
Whole blood microsampling for the quantitation of estetrol without derivatization by liquid
chromatography-tandem mass spectrometry
Gwenaël Nys, Anne Gallez, Miranda G.M. Kok, Gaël Cobraiville, Marianne Fillet
ABSTRACT
Quantitative bioanalysis and especially pharmacokinetic studies are challenging since only low
volumes of biological material are available and low concentrations (ng/ml) are often expected. In this
context, volumetric absorptive microsampling (VAMS) devices were developed to accurately collect
10 or 20 μl of whole blood from tested subjects. In this study, we present the development and
validation of ultra-high performance liquid chromatography coupled to tandem mass spectrometry
method after VAMS sampling for the quantitation of estetrol (E4), a potentially new medicine for
hormone replacement, contraception and osteoporosis therapies. Interestingly, a very simple sample
preparation procedure was developed without any derivatization step. Even if lack of sensitivity is a
common consideration when using negative ionization mode, we demonstrated in this work that an
excellent sensitivity could be reached by carefully optimizing the nature and concentration of the
mobile phase additive. After the optimization of every experimental parameter, the stability,
selectivity, trueness, precision and accuracy of the final method were successfully demonstrated. In
addition, the excellent performances of the method were confirmed by two independent proof-of-
concept pharmacokinetic studies of E4 after VAMS collection in a murine model.
140
Meropenem, levofloxacin and linezolid in human plasma of critical care patients: A fast semi-
automated micro-extraction by packed sorbent UHPLC-PDA method for their simultaneous
determination
Vincenzo Ferrone, Roberto Cotellese, Lorenzo Di Marco, Simona Bacchi, Giuseppe Carlucci
ABSTRACT
An ultra high-performance liquid chromatographic (UHPLC) method with PDA detection was
developed and validated for the simultaneous quantification of meropenem, linezolid, and levofloxacin
in human plasma and applied in human plasma of critical care patients. A semi-automated
microextraction by packed sorbent (MEPS) for sample preparation was used. All parameters in the
extraction step (pH, sample volume, sample dilution and number of aspiration – ejection cycles) and in
the desorption step (percentage of acetonitrile in the solvent of elution and number of aspirations of
elution solvent through the device) were statistically significant when the recovery was used as
response. The method showed good linearity with correlation coefficients, r2 > 0.9991 for the three
drugs, as well as high precision (RSD%< 10.83% in each case). Accuracy ranged from −7.8% to
+6.7%. The limit of quantification of the three drugs was established at 0.01 μg/mL for linezolid and
levofloxacin and 0.02 μg/mL for meropenem. Linezolid, meropenem, levofloxacin and the internal
standard were extracted from human plasma with a mean recovery ranged from 92.4% to 97.4%.
During validation, the concentration of meropenem, linezolid and levofloxacin was found to be stable
after 3 freeze-thaw cycles and for at least 24 h after extraction. This method will be subsequently used
to quantify the drugs in patients to establish if the dosage regimen given is sufficient to eradicate the
infection at the target site.
Investigation on the combined effect of cocaine and ethanol administration through a liquid
chromatography–mass spectrometry metabolomics approach
Elena Sánchez-López, Alberto Marcos, Emilio Ambrosio, Oleg A. Mayboroda, Antonio L. Crego
ABSTRACT
Alcohol is the most widely consumed legal drug, whereas cocaine is the illicit psychostimulant most
commonly used in Europe. The combined use of alcohol and cocaine is frequent among drug-abuse
consumers and leads to further exacerbation of health consequences compared to individual
consumption. The pharmacokinetic and metabolic interactions leading to an increase in their combined
toxicity still remains poorly understood. Here, the first metabolomics study of combined cocaine and
ethanol chronic exposure effects is reported. A Liquid Chromatography strategy based on sample
derivatization with 9-fluorenylmethyloxycarbonyl chloride and using a C18 column coupled to high
resolution Mass Spectrometry (time of flight analyzer) was employed to analyze plasma from rats
exposed intravenously to these drugs in a 52-min analysis. Using a combination of non-supervised and
supervised multivariate analysis the metabolic differences between our experimental groups were
explored and unraveled. A comparative analysis of the individual models and their variable importance
in the projection values have shown that every experiment intervention includes a subset of specific
metabolites. Eleven of these metabolites were annotated, where eight were unequivocally identified
using standards and three were tentatively identified by matching the MS/MS spectra to libraries. The
results demonstrated that the affected metabolic pathways were mainly those related to the metabolism
of different amino acids.
141
Validation of a dried blood spot method for therapeutic drug monitoring of citalopram,
mirtazapine and risperidone and its active metabolite 9-hydroxyrisperidone using HPLC–MS
Johanna Weber, Stefanie Oberfeld, Andrea Bonse, Klaus Telger, Georg Hempel
ABSTRACT
Citalopram, mirtazapine and risperidone are frequently prescribed for psychiatric illnesses such as
depression and psychosis or for aggressive behavior in elderly patients with dementia. The plasma
concentrations vary greatly between patients, especially in elderly patients. Thus, therapeutic drug
monitoring (TDM) increases the safety of antipsychotic treatment and a more rapid response to
treatment may be achieved. To facilitate TDM, the objectives of this study were to develop and
validate a reliable dried blood spot method to simultaneously quantify citalopram, mirtazapine and
risperidone including its active metabolite 9-hydroxyrisperidone. The blood punches were extracted by
methanol using an ultrasonic bath, purified by liquid–liquid extraction and analyzed by liquid
chromatography/mass spectrometry (LC–MS). All acceptance criteria of the EMA and FDA guidelines
for method validation were fulfilled. Linearity was shown over the range of 2.5–300 μg/L for all
substances. The analytes were stable for at least one month at all investigated storage conditions,
including storing at room temperature exposed to light. Retrieving capillary blood by finger-pricking
the assay was successfully applied in elderly patients. Venous serum samples were drawn
simultaneously to compare capillary blood with serum concentrations. Given the validated results and
the calculated capillary blood:serum ratio, the studied dried blood spot method offers an excellent
application in TDM and can be applied in ambulatory care.
Characterization of an unknown impurity in doxofylline using LC–MS and NMR
Peixi Zhu, Jingxian Lu, Liya Hong, Weike Su, Erwin Adams
ABSTRACT
During quality control of doxofylline, a novel impurity was detected, which was above the
identification threshold defined by ICH. First, a liquid chromatographic method compatible with mass
spectrometric (MS) detection was developed. Based on tandem multistage MS and high resolution MS
data, the unknown impurity was found to consist of two theophylline groups connected by a methylene
group. The structure was further confirmed by 1D and 2D nuclear magnetic resonance (NMR)
experiments after semi-preparative isolation. In addition, the formation of the impurity was also
discussed.
142
Determination of AB-CHMINACA and its metabolites in human hair and their deposition in
hair of abusers
Juhyun Sim, Han Soo Cho, Jaesin Lee, Sangwhan In, Eunmi Kim
ABSTRACT
Despite global efforts to control the abuse of synthetic cannabinoids, the high-level of turnover from
the market impedes regulation, endangering public health. N-[(1S)-1-(aminocarbonyl)-2-
methylpropyl]-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide (AB-CHMINACA) is the most
popular synthetic cannabinoid in South Korea since its introduction in 2014. Nonetheless, few studies
have been carried out on AB-CHMINACA and its metabolites, and its deposition in human hair. The
purpose of this study was to develop and validate an analytical method for detection of AB-
CHMINACA and its six metabolites in hair using a liquid chromatography tandem mass spectrometry
(LC–MS/MS) system, for forensic applications. The methanol extracts of hair samples were
evaporated, filtered, and analyzed by LC–MS/MS with electrospray ionization in positive ion mode.
The limits of detection and quantification ranged from 0.5 to 10 pg/mg and 2 to 50 pg/mg,
respectively. Good linearity was achieved within the range of 5–1000 pg/mg or 10–1000 pg/mg
depending on the analyte. Intra- and inter-assay precision and accuracy values were below 15%. No
significant variation was observed using different sources of hair matrices. These validation results
proved the selectivity, accuracy and reproducibility of the method. The established method was
applied to 37 authentic samples from suspected synthetic cannabinoid users. AB-CHMINACA and its
two metabolites, AB-CHMINACA M2 and AB-CHMINACA M4, were detected. The concentration of
the parent drug was much higher than those of its metabolites, and the amount of AB-CHMINACA
M2 was greater than that of AB-CHMINACA M4 in all samples. No other metabolites were detected
in the samples.
Development of a new chlorogenic acid certified reference material for food and drug analysis
Dezhi Yang, LingTai Jiao, Baoxi Zhang, Guanhua Du, Yang Lu
ABSTRACT
This paper reports the preparation and characterization of a new chlorogenic acid (CHA) certified
reference material (CRM), which is unavailable commercially. CHA is an active ingredient found in
many geo-authentic Chinese medicinal materials and developed as an anti-cancer drug. In this work,
trace impurities were isolated and identified through various techniques. CHA CRM was quantified
with two analytical methods, and their results were in good agreement with each other. The certified
value and corresponding expanded uncertainty of CHA CRM reached 99.4% ± 0.2%, which was
calculated by multiplying the combined standard uncertainty by the coverage factor (k = 2), at a
confidence level of 95%. This CRM can be used to calibrate measurement system, evaluate or validate
measurement procedures, assign traceable property values to non-CRMs, and conduct quality control
assays.
143
Automation of plasma protein binding assay using rapid equilibrium dialysis device and Tecan
workstation
Zhengqi Ye, Craig Zetterberg, Hong Gao
ABSTRACT
Binding of drug molecules to plasma proteins is an important parameter in assessing drug ADME
properties. Plasma protein binding (PPB) assays are routinely performed during drug discovery and
development. A fully automated PPB assay was developed using rapid equilibrium dialysis (RED)
device and Tecan workstation coupled to an automated incubator. The PPB assay was carried out in
unsealed RED plates which allowed the assay to be fully automated. The plasma pH was maintained at
7.4 during the 6-h dialysis under 2% CO2 condition. The samples were extracted with acetonitrile and
analyzed by liquid chromatography tandem mass spectrometry. The percent bound results of 10
commercial drugs in plasma protein binding were very similar between the automated and manual
assays, and were comparable to literature values. The automated assay increases laboratory
productivity and is applicable to high-throughput screening of drug protein binding in drug discovery.
―Ghost peaks‖ of ezetimibe: Solution degradation products of ezetimibe in acetonitrile induced
by alkaline impurities from glass HPLC vials
Jianyang Jin, Zhiying Wang, Jinsheng Lin, Wenquan Zhu, Min Li
ABSTRACT
Unpredictable degradation of Ezetimibe solutions in pure acetonitrile occurs when they are stored in
glass HPLC vials. The occurrence of the two main degradation peaks and one minor peak was
unpredictable at the time of each sample preparation and over time, it appeared that approximately
15% of the sample solutions in glass HPLC vials would eventually show the degradation peaks. Once
the degradation peaks occurred in a particular vial, typically within 24 h, they would keep growing
until reaching a total yield of about 4–5%. Through a comprehensive investigation, it is determined
that the solution degradation is caused by a base-catalyzed process, during which ezetimibe undergoes
(1) dimerization to form two dimeric impurities, which have not been reported in the literature, and (2)
to a less degree, isomerization to produce an isomeric impurity that has been reported before.
144
Metabolite quantification by NMR and LC-MS/MS reveals differences between unstimulated,
stimulated, and pure parotid saliva
João Figueira, Sandra Gouveia-Figueira, Carina Öhman, Pernilla Lif Holgerson, Anders Öhman
ABSTRACT
Saliva is a readily available biofluid that is sensitive to metabolic changes and can be collected through
rapid and non-invasive collection procedures, and it shows great promise for clinical metabolomic
studies. This work studied the metabolite composition of, and the differences between, saliva samples
collected by unstimulated spitting/drooling, paraffin chewing-stimulated spitting, and parotid gland
suction using targeted nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography
coupled to tandem mass spectrometry (LC-MS/MS) for metabolite quantification. As applied here,
these two analytical techniques provide complementary metabolite information and together extend the
metabolome coverage with robust NMR quantification of soluble metabolites and sensitive targeted
LC-MS/MS analysis of bioactive lipids in specific metabolic pathways. The NMR analysis was
performed on ultrafiltrated (3 kDa cutoff) saliva samples and resulted in a total of 45 quantified
metabolites. The LC-MS/MS analysis was performed on both filtered and unfiltered samples and
resulted in the quantification of two endocannabinoids (AEA and PEA) and 22 oxylipins, which at
present is the most comprehensive targeted analysis of bioactive lipids in human saliva. Important
differences in the metabolite composition were observed between the three saliva sample collection
methods, which should be taken into consideration when designing metabolomic studies of saliva.
Furthermore, the combined use of the two metabolomics platforms (NMR and LC-MS/MS) proved to
be viable for research and clinical studies of the salivary metabolome.
Determination of AZD3759 in rat plasma and brain tissue by LC–MS/MS and its application in
pharmacokinetic and brain distribution studies
Shan Xiong, Mingxing Xue, Yanling Mu, Zhipeng Deng, Ruican Zhou
ABSTRACT
A simple and sensitive high performance liquid chromatography with tandem mass spectrometry (LC–
MS/MS) method for determination of AZD3759, a novel epidermal growth factor receptor tyrosine
kinase inhibitor, in rat plasma and brain homogenate was developed and validated over the range of
1.0–1000 ng/mL. Chromatographic separation was carried out on a C18 column with acetonitrile and
0.1% formic acid in water as mobile phase with gradient elution at a flow rate of 0.4 mL/min. The
lower limits of quantification (LLOQs) were 1.0 ng/mL for AZD3759 in both rat plasma and brain
homogenate. The intra-day and inter-day precision and accuracy of AZD3759 were well within the
acceptable limits of variation. The simple and sensitive LC–MS/MS method was successfully applied
to the pharmacokinetic and brain distribution studies following an oral administration of AZD3759 to
rats.
145
Studying the effects of natural extracts with metabolomics: A longitudinal study on the
supplementation of healthy rats with Polygonum cuspidatum Sieb. et Zucc.
Gregorio Peron, Jalal Uddin, Matteo Stocchero, Stefano Mammi, Stefano Dall‘Acqua
ABSTRACT
Background: A longitudinal study was performed to evaluate the effects of Polygonum cuspidatum
extract (standardized at 20% resveratrol) supplementation on healthy rats. The effects were explored
by monitoring urinary metabolome changes using UPLC-HRMS and 1H NMR-based approaches. The
aim of the study was to explore the effects of P. cuspidatum supplementation on a healthy animal
model using metabolomics, in order to determine possible modes of action and obtain information on
bioactivity.
Methods: Healthy Sprague-Dawley rats were orally supplemented with 100 mg/kg of dried P.
cuspidatum extract for 49 days and 24-h urinary outputs were collected. Samples were analysed by
untargeted UPLC-HRMS and 1H NMR approaches and the obtained data sets were modelled by an
adaptation of post-transformation of PLS2 to longitudinal studies. Putative markers were discovered
by a stability selection procedure and specific oxidative stress markers were monitored by a targeted
HPLC–MS/MS analysis to assess the in vivo antioxidant activity of P. cuspidatum extract.
Results: UPLC-HRMS and 1H NMR platforms showed two different but complementary patterns of
metabolites describing the changes ascribable to P. cuspidatum supplementation and using both
approaches, a comprehensive resveratrol metabolism and urinary excretion could be observed.
Markers of P. cuspidatum supplementation effects identified by UPLC-HRMS were mainly related to
its antioxidant activity and to a possible ―adaptogenic‖ activity. Urinary changes observed by 1H NMR
were mainly related to energy metabolism. UPLC-HRMS and 1H NMR metabolomics approaches
allowed the effects of a prolonged supplementation with P.
Identification and characterization of a thermally cleaved fragment of monoclonal antibody-A
detected by sodium dodecyl sulfate-capillary gel electrophoresis
Kei Kubota, Naoki Kobayashi, Masayuki Yabuta, Motomu Ohara, Koji Otsuka
ABSTRACT
This report describes a novel, comprehensive approach to identifying a fragment peak of monoclonal
antibody-A (mAb-A), detected by sodium dodecyl sulfate-capillary gel electrophoresis (SDS-cGE).
The fragment migrated close to the internal standard (10 kDa marker) of SDS-cGE and increased
about 0.5% under a 25 °C condition for 6 months. Generally, identification of fragments observed in
SDS-cGE is challenging to carry out due to the difficulty of collecting analytical amounts of
fractionations from the capillary. In this study, in-gel digestion peptide mapping and reversed phase
liquid chromatography-mass spectrometry (RPLC–MS) were employed to elucidate the structure of
the fragment. In addition, a Gelfree 8100 fractionation system was newly introduced to collect the
fragment and the fraction was applied to the structural analysis of a mAb for the first time. These three
analytical methods showed comparable results, proving that the fragment was a fraction of heavy chain
HC1-104. The fragment contained complementarity determining regions (CDRs), which are significant
to antigen binding, and thus would affect the efficacy of mAb-A. In addition, SDS-cGE without the 10
kDa marker was demonstrated to clarify the increased amount of the fragment, and the experiment
revealed that the fragment increases 0.2% per year in storage at 5 °C.
146
Synchronous determination with double-wavelength by RP-HPLC-UV and optimization of
ultrasound-assisted extraction of phenolic acids from Caragana species using response surface
methodology
Zhi Zeng, Zhongyin Ji, Na Hu, Shasha Chen, Yourui Suo
ABSTRACT
The utilization of Caragana korshinskii Kom (CK) is currently concentrated on its ecological and fuel
functions. Little attention has been devoted to the analysis of their phenolic acid (PA) components. To
obtain more data for further utilization of CK, a new analysis protocol was tested to determine PAs
synchronously by RP-HPLC-UV with double-wavelength (280 nm and 320 nm) detection.
Specifically, separation of PA components was performed on a Hypersil Gold C18 reverse phase
column with gradient elution. A four-factor-three-level Box-Behnken design was implemented for
optimization of PA extraction. The results demonstrated that CK were rich primarily in chlorogenic
acid, vanillic acid, caffeic acid and rosmarinic acid. The total content of PAs in CK leaves was the
highest compared with its other parts. The distribution of total flavonoid content of CK was leaves >
flowers > bark, while that of the total phenolic content of CK was flowers > leaves > bark.
Untargeted metabolite analysis-based UHPLC-Q-TOF-MS reveals significant enrichment of p-
hydroxybenzyl dimers of citric acids in fresh beige-scape Gastrodia elata (Wutianma)
Chang-Jiang-Sheng Lai, Yuan Yuan, Da-Hui Liu, Chuan-Zhi Kang, Lu-Qi Huang
ABSTRACT
In order to comprehensively elucidate the chemical biosynthesis process of the beige-scape Gastrodia
elata Blume (Wutianma) as a traditional herbal medicines, the untargeted analysis–based UHPLC-
PDA-ESI-Q-TOF-MS reveals the metabolites ranging from the skeletons to novel dimers of citric
acids in fresh and dried immature/mature stem tubers. Interestingly, two novel types of dimers for
citric acids with the anhydride groups at sn-1 and/or sn-5 were discovered in fresh samples. Moreover,
the classical mono- versus novel di-mers, and the aglycons versus the glycosides could be easily
discriminated by signature fragmentation patterns and some novel adduct ions. The heat map of
contents demonstrated more p-hydroxybenzyl metabolites than gastroxyl ones were determined in
fresh Wutianma revealing a significant specificity with the lack of the sufficient gastrodin and
gastroxyl products in biosynthetic pathway.
147
Volume 141 July 2017
Matrix-assisted laser-desorption/ionization mass spectrometric imaging of olanzapine in a single
hair using esculetin as a matrix
Hang Wang, Ying Wang, Ge Wang, Lizhi Hong
ABSTRACT
Matrix-assisted laser desorption/ionization-mass spectrometric imaging (MALDI-MSI) for the analysis
of intact hair is a powerful tool for monitoring changes in drug consumption. The embedding of a low
drug concentration in the hydrophobic hair matrix makes it difficult to extract and detect, and requires
an improved method to increase detection sensitivity. In this study, an MSI method using MALDI-
Fourier transform ion cyclotron resonance was developed for direct identification and imaging of
olanzapine in hair samples using the positive ion mode. Following decontamination, scalp hair samples
from an olanzapine user were scraped from the proximal to the distal end three times, and 5 mm hair
sections were fixed onto an Indium-Tin-Oxide (ITO)-coated microscopic glass slide. Esculetin (6,7-
dihydroxy-2H-chromen-2-one) was used as a new hydrophobic matrix to increase the affinity,
extraction and ionization efficiency of olanzapine in the hair samples. The spatial distribution of
olanzapine was observed using five single hairs from the same drug user. This matrix improves the
affinity of olanzapine in hair for molecular imaging with mass spectrometry. This method may provide
a detection power for olanzapine to the nanogram level per 5 mm hair. Time course changes in the
MSI results were also compared with quantitative HPLC–MS/MS for each 5 mm segment of single
hair shafts selected from the MALDI target. MALDI imaging intensities in single hairs showed good
semi-quantitative correlation with the results from conventional HPLC–MS/MS. MALDI-MSI is
suitable for monitoring drug intake with a high time resolution.
A novel liquid chromatography/tandem mass spectrometry (LC–MS/MS) based bioanalytical
method for quantification of ethyl esters of Eicosapentaenoic acid (EPA) and Docosahexaenoic
acid (DHA) and its application in pharmacokinetic study
Sekarbabu Viswanathan, P.R.P. Verma, Muniyandithevar Ganesan, Jeganathan Manivannan
ABSTRACT
Omega-3 fatty acids are clinically useful and the two marine omega-3 fatty acids eicosapentaenoic acid
(EPA) and docosahexaenoic acid (DHA) are prevalent in fish and fish oils. Omega-3 fatty acid
formulations should undergo a rigorous regulatory step in order to obtain United States Food and Drug
Administration (USFDA) approval as prescription drug. In connection with that, despite quantifying
EPA and DHA fatty acids, there is a need for quantifying the level of ethyl esters of them in biological
samples. In this study, we make use of reverse phase high performance liquid chromatography coupled
with mass spectrometry (RP-HPLC–MS)technique for the method development. Here, we have
developed a novel multiple reaction monitoring method along with optimized parameters for
quantification of EPA and DHA as ethyl esters. Additionally, we attempted to validate the bio-
analytical method by conducting the sensitivity, selectivity, precision accuracy batch, carryover test
and matrix stability experiments. Furthermore, we also implemented our validated method for
evaluation of pharmacokinetics of omega fatty acid ethyl ester formulations.
148
Identification of a host cell protein impurity in therapeutic protein, P1
Deepti Ahluwalia, Harbhajan Dhillon, Thomas Slaney, Hangtian Song, Girija Krishnamurthy
ABSTRACT
Residual host cell proteins (HCPs) are process-related impurities present in biotherapeutics that can
pose safety health risks to patients. An adequate control of HCP levels in the final product, and
demonstration of HCP clearance throughout a product manufacturing process is critical for all
biotherapeutic products. Developing effective downstream purification processes can be challenging as
HCPs and product proteins may possess an affinity for each other or have similar physicochemical
properties, resulting in co-purification. In the current study, we identified the presence of CHO-
catalase subunit protein as an impurity present in purified P1 protein. This previously unreported HCP
impurity, was detected in P1 protein generated in Chinese hamster ovary (CHO) cells. Purified drug
substance samples contained elevated CHO HCP levels when measured using a commercial anti-CHO
HCP Enzyme-Linked Immunosorbent Assay (ELISA) kit. This finding, prompted further
characterization of the HCP profile using 1D and 2D gels/ western blots using an anti-human IgG
antibody as well as a commercial anti-CHO HCP antibody (Cygnus 813) for the detection of host cell
proteins. The CHO-catalase protein has been characterized using a combination approach of one-
dimensional (1D) and two-dimensional (2D) gels and western blotting techniques, and the identity
confirmed using liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Western blot
analyses using the anti-CHO HCP antibody detected a potential HCP band at ∼60 kDa and a pI of ∼8
in the purified P1 sample. The 60 kDa HCP band was excised from 1D SDS-PAGE gels and LC-
MS/MS analysis identified it to be CHO-catalase subunit. The identity of catalase monomer was
further confirmed by western blot analysis using a specific anti-catalase antibody.
Simultaneous separation and determination of four uncaria alkaloids by capillary
electrophoresis using dual cyclodextrin system
Lou Li, Liying Xu, Meng Chen, Guangbin Zhang, Anjia Chen
ABSTRACT
The purpose of this study was to develop a simple, quick and precise capillary zone electrophoresis
method (CZE) for the separation and determination of uncaria alkaloids using dual cyclodextrins as
additives for the separation. The four analytes were baseline separated within 15 min at the applied
voltage of 15 kV with a running buffer (pH 5.7) consisting of 40.0 mM phosphate buffer, 161.7 mM 2-
hydroxypropyl-β-cyclodextrin (HP-β-CD) and 2.21 mM mono-(6-ethylenediamine-6-deoxy)-β-
cyclodextrin (ED-β-CD). Under the optimum conditions, a good linearity was achieved with
correlation coefficients from 0.9989 to 0.9992. The detection limits and the quantitation limits ranged
from 0.63 to 0.98 μg/mL and from 2.08 to 3.28 μg/mL, respectively. Excellent accuracy and precision
were obtained. Recoveries of the analytes varied from 97.1 to 103.2%. This method was suitable for
the quantitative determination of these alkaloids in the stem with hook of Uncaria rhynchophylla and
the formulations of Uncaria rhynchophylla.
149
CE method for the in-process control of the synthesis of active substances conjugated with gold
nanoparticles
Wioleta Maruszak, Elżbieta U. Stolarczyk, Krzysztof Stolarczyk
ABSTRACT
A fast capillary electrophoresis method was developed and validated for the in-process control (IPC)
of the synthesis of active substances (APIs) with gold nanoparticles (AuNPs). The capillary
electrophoresis method was key to ensure that the reaction step conducted in order to obtain AuNP and
API conjugates will produce the expected product without the presence of free APIs, which is a critical
parameter determining the quality of the synthetic material. Capillary electrophoresis was performed
using uncoated fused-silica capillaries with the effective length of 40 cm, 50 μm i.d. and the
background electrolyte consisted of 20 mM borate buffer (pH 8.5) with the application of
hydrodynamic injection 50 mbar/5s, voltage 20 kV, temperature of the capillary cassette 25 °C and UV
detection at 261 nm for GE, 541 nm for AuNP-GE, 227 nm for PE and 535 nm for AuNP-PE. During
validation the specificity, linearity, accuracy, precision, range, and stability of the sample solution
were confirmed. The linear regression (R2 = 0.999) between the corrected peak areas of the analytes
and their amount was fulfilled in the range from 2.4 μg/mL to 0.3 mg/mL for genistein and from 4.6
μg/mL to 0.6 mg/mL for pemetrexed. Within this range the method was proved to be accurate (99.0%
for genistein and 99.9% for pemetrexed) and precise for both analytes with the intra-day RSD values
of 0.77% and 0.97% for the migration time of genistein and pemetrexed, respectively. The inter-day
RSD values were 1.90% and 2.27% for the migration time of genistein and pemetrexed, respectively.
The LOD and LOQ values for pemetrexed were 1.4 μg/mL and 4.6 μg/mL, respectively, and for
genistein 0.72 μg/mL and 2.4 μg/mL, respectively. The results obtained during the validation indicate
that the method is sufficient to be applied for the IPC of the synthesis of APIs with gold nanoparticles.
Achievable separation performance and analysis time in current liquid chromatographic
practice for monoclonal antibody separations
Szabolcs Fekete, Jean-Luc Veuthey, Davy Guillarme
ABSTRACT
The separation performance of a chromatographic system is often described in terms of column
efficiency and peak capacity. Thanks to the new developments in column technology over the past few
years, the achievable peak capacity drastically improved and the analysis time can be significantly
shortened. Indeed, highly efficient wide-pore reversed-phase (RPLC) materials packed with small fully
porous and superficially porous particles can be successfully used for the analytical characterization of
therapeutic proteins. For non denaturating chromatographic approaches, such as ion exchange (IEX)
and size-exclusion chromatography (SEC), non-porous ion-exchanger as well as sub −3 μm size
exclusion supports are commercially available and open new avenues in protein separations. In this
study, the current possibilities offered by chromatography for the characterization of monoclonal
antibody (mAb) are discussed. For this purpose, recently published data have been reviewed and
calculations were performed to compare the maximum achievable peak capacity and related analysis
times using typical samples under RPLC, IEX and SEC conditions. Carefully chosen realistic column
pressure, mobile phase temperature, flow rate and column dimensions were considered for the case
studies discussed through the paper.
150
Identification and characterization of process-related substances and degradation products in
apremilast: Process optimization and degradation pathway elucidation
Yuting Lu, Xiaoyue Shen, Taijun Hang, Min Song
ABSTRACT
This study aims at investigating the separation, identification and characterization of related substances
in apremilast by LC–MS hyphenated techniques, as well as the synthesis optimization and the
degradation pathways elucidation. Forced degradation studies were conducted under the ICH
prescribed stress conditions. The chromatographic separation was achieved on XBridge C18 column
(4.6mm × 150 mm, 3.5 μm) using a mobile phase consisting of water adjusted to pH 3.0 with formic
acid as solvent A and acetonitrile as solvent B in linear gradient elution program. Twelve related
substances were detected all together in apremilast and its stress samples. Their structures were
identified mainly through positive ESI high-resolution TOF-MS analysis of the parent ions' accurate
masses and elemental compositions, and the corresponding MS/MS spectra elucidation. There were
three process-related substances and nine degradation products, seven of them were first reported. Two
degradation products and one process-related substance were further verified by semi-preparation and
NMR determination. Their origins and formation mechanisms were also discussed, based on which
effective approaches for the synthesis optimization were conducted. Therefore, the related substances
investigation are valuable for apremilast manufacturing process optimization and quality control.
Antiproliferative hydroxy-fatty acids from the fodder legume Stylosanthes guianensis
Marco Clericuzio, Bruno Burlando, Barbara Borghesi, Annalisa Salis, Laura Cornara
ABSTRACT
Stylosanthes guianensis is a fodder legume native from South America and widely grown worldwide.
Dried plant material was purchased on the web and taxonomically identified by light and SEM
microscopy, and morphological analysis of plants germinated from seeds. The plant was extracted with
dichloromethane:2-propanol (9:1). Bioguided fractionation using calcein-AM cytotoxicity assay on
HeLa and A431 tumor cells allowed to isolate a lipophilic fraction, endowed with strong cytotoxicity.
By means of 1- and 2-D NMR, HPLC–MS, and HR-ESIMS it could be seen that the fraction was an
inseparable mixture of complex lipids, mainly consisting of esterified 3-hydroxy fatty acids. Acidic
methanolysis of the mixture yielded 3-OH C10 and C12 carboxylic acids, together with palmitic,
stearic, and arachidonic acids. Mass values indicate the presence of dimeric and trimeric combinations
of 3-hydroxy, C10/C12 acids, and C16/C18/C20 acids, linked via ester bond. Monomeric hydroxyl-
fatty acids were also observed, in particular derivatives of mono-hydroxy and di-hydroxy linolenic,
linoleic, and oleic acids. 3-O-acylated, esterified fatty acids are unusual in higher plants, and recall
motifs of Gram-negative endotoxin lipid A. These oxylipins are likely to be responsible for the
antiproliferative activity of S. guianensis, suggesting possible use of the plant in the development of
antitumor drugs.
151
Development and validation of a liquid chromatographic method for the analysis of squaric acid
dibutyl ester and its impurities
Marwa F. Mansour, Peixi Zhu, Ann Van Schepdael, Erwin Adams
ABSTRACT
A simple, fast and selective stability indicating liquid chromatographic method has been described for
the simultaneous determination of squaric acid dibutyl ester and its impurities. The chromatographic
separation was achieved on a C2 column (250 mm × 4.6 mm i.d., 5 μm) using a mobile phase
consisting of 0.15% phosphoric acid – acetonitrile – methanol (30:60:10, v/v/v). Isocratic elution was
performed at a flow rate of 1.0 mL min−1. The analytes were detected by UV at 252 nm. The method
was validated according to the ICH guidelines and satisfactory results were obtained. The specificity
of the developed method was tested using forced degradation solutions of the drug substance.
Characterization of squaric acid dibutyl ester and its forced degradation products was achieved by
coupling mass spectrometry (MS) to the liquid chromatographic (LC) system. The method was
successfully applied for quality control purposes including assay and determination of related
compounds as required by regulatory guidelines to ensure its safety and efficacy since no monograph
is available in official compendia.
Macro-Raman spectroscopy for bulk composition and homogeneity analysis of multi-component
pharmaceutical powders
Hui Wang, David Barona, Sulayman Oladepo, Lisa Williams, Reinhard Vehring
ABSTRACT
A new macro-Raman system equipped with a motorized translational sample stage and low-frequency
shift capabilities was developed for bulk composition and homogeneity analysis of multi-component
pharmaceutical powders. Different sampling methods including single spot and scanning measurement
were compared. It was found that increasing sample volumes significantly improved the precision of
quantitative composition analysis, especially for poorly mixed powders. The multi-pass cavity of the
macro-Raman system increased effective sample volumes by 20 times from the sample volume
defined by the collection optics, i.e., from 0.02 μL to about 0.4 μL. A stochastic model simulating the
random sampling process of polydisperse microparticles was used to predict the sampling errors for a
specific sample volume. Comparison of fluticasone propionate mass fractions of the commercial
products Flixotide® 250 and Seretide® 500 simulated for different sampling volumes with
experimentally measured compositions verified that the effective sample volume of a single point
macro-Raman measurement in the multi-pass cavity of this instrument was between 0.3 μL and 0.5 μL.
The macro-Raman system was also successfully used for blend uniformity analysis. It was concluded
that demixing occurred in the binary mixture of l-leucine and d-mannitol from the observation that the
sampling errors indicated by the standard deviations of measured leucine mass fractions increased
during mixing, and the standard deviation values were all larger than the theoretical lower limit
determined by the simulation. Since sample volume was shown to have a significant impact on
measured homogeneity characteristics, it was concluded that powder homogeneity analysis results, i.e.,
the mean of individual test results and absolute and relative standard deviations, must be presented
together with the effective sample volumes of the applied testing techniques for any measurement of
powder homogeneity to be fully meaningful.
152
Isolation, identification and characterization of potential impurities of anidulafungin
Lanning Zhao, Qilong Wang, Yi Bie, Xiaoxia Lu
ABSTRACT
Eight impurities ranging from 0.03 to 0.97% in anidulafungin bulk drug were detected by HPLC. Four
impurities (Imp-I, Imp-II, Imp-III and Imp-VIII) among impurities were isolated from the self-
prepared or marketed samples of anidulafungin bulk drug by means of preparative HPLC. A thorough
study was undertaken to characterize these impurities and based on 1D (1H, 13C, H-D, DEPT 90 and
135) and 2D (COSY, TOCSY, HSQC, HMBC) NMR and ESI–MS spectral data. Based on the
characterization data, Imp-I was found to be known open-chain hydrolysis product formed during the
synthesis and degradation. Imp-II and Imp-III was lacked a methyl group at the C-4 and C-8 in
anidulafungin, respectively, whereas Imp-VIII contained a methoxy group at the C-23. The latter three
new impurities were identified as process-related substances.
Dialkyl anionic surfactant in field-amplified sample injection and sweeping-micellar
electrokinetic chromatography for determination of eight leanness-promoting β-agonists in
animal feeds
Sung-Yu Hsieh, Chun-Chi Wang, Hwang-Shang Kou, Shou-Mei Wu
ABSTRACT
The beta-adrenergic agonists (β-agonists) working as repartitioning agents that make the carcass leaner
and enhance the feeding efficiency in animals have been banned in the European Union, China and
Taiwan. Here, traditional anionic surfactants, such as sodium dodecyl sulfate (SDS) were replaced
with sodium di-(2-ethylhexyl)-sulfosuccinate (AOT) in field-amplified sample injection and sweeping-
micellar electrokinetic chromatography (FASI-sweeping MEKC) for simultaneous analysis of eight β-
agonists in animal feeds. The AOT vesicles provided a better resolution of β-agonists than micelles of
SDS. The detection limits of the eight β-agonists were above 5 ng/mL by using this stacking capillary
electrophoresis (CE) method. In comparison of traditional MEKC method (sample injection, 1 psi for 5
s), the stacking strategy provided 400–2000 fold sensitivity enhancement. After method validation, this
method was successfully applied for analyzing four animal feeds, and none β-agonist was detected.
This strategy possessing good resolution of eight β-agonists was suitable for serving as a tool for
routine analysis of animal feeds.
153
Isolation and structural characterization of glucosylceramides from Ethiopian plants by
LC/APCI-MS/MS
Efrem N. Tessema, Tsige Gebre-Mariam, Christian E.H. Schmelzer, Reinhard H.H. Neubert
ABSTRACT
Chronic skin conditions such as atopic dermatitis, psoriasis and aged skin are characterized by
defective skin barrier and dryness which are associated with reduced levels of skin ceramides (CERs).
The beneficial effects of plant-derived CERs for skin hydration and skin barrier recovery have been
shown in several studies. Although plenty of glucosylceramide (GlcCER)-based dietary supplements
meant for skin barrier improvement have been marketed, there are limited commercial sources of plant
GlcCERs. In an attempt to explore alternative GlcCER sources, a reversed phase LC–MS/MS method
with atmospheric pressure chemical ionization (APCI) interface was developed for separation and
structural identification of GlcCERs isolated from three plants. The GlcCERs were extracted from the
seeds of grass pea (Lathyrus sativus L.), Ethiopian mustard (Brassica carinata) and haricot bean
(Phaseolus vulgaris) and purified by column chromatography and preparative LC–MS. The individual
GlcCER species were further separated and qualitatively analyzed by LC/APCI-MS/MS. The amount
of GlcCERs in each plant was quantified by HPTLC. All GlcCER species detected in the three plants
consisted of C18 di/trihydroxy sphingoid bases amide linked with hydroxy fatty acids (C14-C24). The
trihydroxy SBs were acylated with very long chain FAs (C22-C24). The major GlcCERs derived from
grass pea, Ethiopian mustard and haricot bean are composed of sphingenine (d18:1) linked to
hydroxypalmitic acid (h16:0), 4-hydroxy-8-sphingenine (t18:1) coupled with hydroxynervonic acid
(h24:1) and sphingadienine (d18:2) joined with h16:0, respectively. The GlcCERs contents in haricot
bean (161.2 mg/kg) and grass pea (130.0 mg/kg) were found to be higher compared to Ethiopian
mustard (71.8 mg/kg).
Drug-protein binding mechanism of juglone for early pharmacokinetic profiling: Insights from
ultrafiltration, multi-spectroscopic and molecular docking methods
Pan Zhao, Guihua Gao, Lianjun Zhang, Qian Cai, Xiaohong Hou
ABSTRACT
Juglone (JL), as one of the major bioactive components present in the bark of Juglans mandshruica
Maxim, exhibits versatile bioactivities, especially anti-cancer activity. To better understand the
pharmacokinetic properties of juglone, the protein binding rate of juglone was determined by
ultrafiltration method, and the binding affinity and mechanism between JL and human serum albumin
(HSA) was investigated in vitro through multi-spectroscopic, thermodynamic, and molecular modeling
methods. The binding degree of JL was measured more than 99.0% which suggested that JL had high
binding ability to serum albumin. Fluorescence data showed that juglone quench the intrinsic
fluorescence of HSA upon forming the JL–HSA nonfluorescent complex at 1:1 stoichiometric
proportion, and the complex formation had a high affinity of 104 L·mol−1. Meanwhile, the site marker
competitive experiments and the thermodynamic parameters (ΔG = -26.08 kJ·mol−1, ΔH = –16.34
kJ·mol−1, ΔS = 32.69 J·mol−1·K−1) indicated that juglone could spontaneously bound to the site I
(subdomain IIA) of HAS through hydrophobic and hydrogen bonding interactions. As further revealed
by the synchronous fluorescence, three-dimensional fluorescence, Fourier transform infrared (FT-IR)
and circular dichroism (CD) spectroscopy, JL could cause conformational and structural alterations of
HSA. Additionally, molecular docking was employed to further define the specific binding site and the
result was in accordance with the conclusion of experimental analysis.
154
Quantification of alprenolol and propranolol in human plasma using a two-dimensional liquid
chromatography (2D-LC)
Virgínia M.F. Gonçalves, Patrícia Rodrigues, Cláudia Ribeiro, Maria E. Tiritan
ABSTRACT
A new two-dimensional liquid chromatography (2D-LC) method using a column switching valve, with
a restricted-access media (RAM) column in the first dimension was developed and validated for the
quantification of two β-blockers in human plasma. Several parameters, such as sample collection,
mobile phase composition and flow rate for sample cleanup, transference and analytical separation of
the β-blockers were investigated and optimized. The developed method allowed for the simultaneous
pre-treatment and quantification of alprenolol (ALP) and propranolol (PRO) in human plasma in less
than 25 min. The method consisted in the injection of 100 μL of plasma samples on the RAM alkyl-
diol-silica column (Lichrospher® RP-18 ADS, 25 μm) with water/acetonitrile (98:2, v/v; at a flow rate
of 2.0 mL/min) and then transferred (via a six-port valve) to the analytical column (Luna PFP (2), 150
× 4.6 mm ID, 100 Å, 3 μm) with 0.1% (v/v) triethylamine in water acidified with trifluoroacetic acid
(pH = 3)/acetonitrile (74:26, v/v) at a flow rate of 0.6 mL/min in a back-flush mode. The column oven
temperature was optimized to 42 °C and the fluorescence detector set at 280 nm and 310 nm
(excitation and emission, respectively). The method was validated according to the European
Medicines Agency‘s guidelines and was linear (r2 > 0.999) over a dynamic range of 5.0 – 200 ng/mL.
Recoveries ranged from 90.2 and 107% and the lower limit of quantification was 5.0 ng/mL for both
compounds. Precision was expressed as a percentage of relative standard deviation and did not exceed
11%. Finally, the method was successfully applied to determine the plasma concentration of PRO in
four healthy volunteers.
An LC–MS/MS method for quantification of AC0010, a novel mutant-selective epidermal
growth factor receptor (EGFR) inhibitor, and its metabolites in human plasma and the
application to a pharmacokinetic study
Lu Wang, Xin Zheng, Weicong Wang, Pei Hu, Ji Jiang
ABSTRACT
AC0010 is an irreversible, mutant-selective EGFR inhibitor that effectively inhibits EGFR active and
T790 M resistance mutations in non-small cell lung cancer (NSCLC). A sensitive ultra-performance
liquid chromatography – tandem mass spectrometry (UPLC–MS/MS) method was developed and fully
validated for determining AC0010 and its metabolites in human plasma. The samples were purified by
solid-phase extraction (SPE) columns and separated on a BEH C18 column. Electrospray ionization
(ESI) in positive ion mode and multiple reaction monitoring (MRM) were used to monitor the ion
transitions of AC0010 (m/z 488 → 257) and its metabolites M1 (m/z 474 → 403), M2 (m/z 504 →
487), M4 (m/z 434 → 377), M7 (m/z 490 → 405), MII-1 (m/z 651 → 434) and MII-2 (m/z 609 →
434). The results revealed that the method had excellent selectivity and linearity. Intra-day and inter-
day precisions (in terms of relative standard deviation, RSD) were lower than 14.4% and the
accuracies (in terms of relative error, RE) were within the range of ±10.3% for all the analytes. The
lower limit of quantification (LLOQ), stability, matrix effect and extraction recovery were also
validated and satisfied the criteria of validation. Finally, the method was successfully applied to a
pharmacokinetic study of NSCLC patients after a single oral dose of 350 mg of AC0010.
155
Validation and application of an ultrahigh-performance liquid chromatographic-Orbitrap mass
spectrometric method for the simultaneous detection and quantification of volatile and non-
volatile organic acids in human faecal samples
Claudio Gardana, Cristian Del Bo‘, Paolo Simonetti
ABSTRACT
A simple and selective ultrahigh-performance liquid chromatographic-Orbitrap mass spectrometric
(UHPLC-HR-MS) method was developed and validated for the simultaneous detection and
quantification of short-chain fatty acids (SCFAs) such as acetic, propionic, butyric, isobutyric, valeric,
isovaleric, 2-methyl-butyric (IS) and lactic, pyruvic and succinic acid in human faecal samples. A
simple extraction procedure with 0.001% formic acid in water was performed on 40 samples. The
extracts were centrifuged and analyzed by UHPLC-HR-MS on a sub-2 μm column using gradient
elution; meanwhile, the same samples were analyzed by GC-FID and HPLC-UV as reference methods
The UHPLC-HR-MS method showed a recovery of 83–105%, a repeatability of 2.2–8.3% and an
intermediate precision of 2.9–9.4%. The LOD and LLOQ were in the range of 0.04–0.23 and 0.2–0.5
μg/ml, respectively. Regarding the SCFAs, statistical analysis showed a good correlation between the
data obtained by UHPLC-HR-MS and those provided by GC-FID (p > 0.05). On the contrary, the LC-
UV data were not in agreement with those obtained by UHPLC-HR-MS determination (p < 0.05). To
the best of our knowledge, this is the first method available for the simultaneous extraction and
quantification of SCFAs, lactic, pyruvic and succinic in faecal samples by UHPLC-HR-MS.
Validation of a SPE HPLC–UV method for the quantification of a new ER-specific
photosensitizer OR-141 in blood serum using total error concept
Emilie Bony, Yohan Mace, Adán Pinto, David Delvaux, Joëlle Quetin-Leclercq
ABSTRACT
The aim of this work is to develop the first validated HPLC–UV method quantification in blood serum
for a new endoplasmic reticulum (ER)-specific benzophenazine photosensitizer (OR-141). A fast solid
phase extraction (SPE) cleaning sample procedure was achieved on C18 encapped (ec) SPE cartridges
and the separation was performed on a RP-18e column (5 μM) using an isocratic elution with
methanol. The method has been fully validated according to accuracy profiles based on total error and
tolerance intervals. Calibration was performed in the matrix and trueness (<4.25% relative bias),
repeatability (<4.75% relative standard deviation (RSD)), intermediate precision (<5.37% RSD),
selectivity, response function, linearity, and dilution effect were evaluated for both OR-141 regio-
isomers. Afterwards the developed method was successfully applied to perform the quantitative
determination of OR-141 in mouse blood serum samples in a preliminary pharmacokinetic study.
156
Determination of a novel anticancer AMPK activator hernandezine in rat plasma and tissues
with a validated UHPLC–MS/MS method: Application to pharmacokinetics and tissue
distribution study
Yang Song, Zhibin Wang, Baozhen Zhang, Yujia Zhang, Xuesong Feng
ABSTRACT
Hernandezine, a novel anticancer AMPK activator, is a major active constituent of Thalictrum
Ranunculaceae. A simple, specific and sensitive liquid chromatography tandem mass spectrometry
(LC–MS/MS) method has been developed and validated for the quantification of hernandezine in rat
plasma and tissues after intravenous administration. Sample preparation was carried out through a
protein-precipitation extraction with acetonitrile using tetrandrine as internal standard (IS). The
chromatographic separation was achieved by using an Agilent ZORBAX Eclipse Plus C18 column
with a mobile phase of acetonitrile and water (containing 10 mM ammonium acetate) in an isocratic
elution way. The mass spectrometry (MS) analysis was conducted in positive ionization mode with
multiple reaction monitoring (MRM) transitions at m/z 653.4 → 411.2 for hernandezine and m/z 623.3
→ 381.3 for tetrandrine (IS). Calibration curves were linear over the ranges of 20.0–4000 ng/ml f or
both plasma samples and tissue samples (r > 0.991). The lower limit of quantification (LLOQ) was
20.0 ng/ml. The intra-day and inter-day precision (RSD%) were less than 14.0%, while the accuracy
was ranged from 85.2% to 114.9%. Finally, this developed method was successfully applied in the
pharmacokinetics and tissue distribution study of hernandezine after intravenous administration.
LC–MS/MS determination of tranexamic acid in human plasma after phospholipid clean-up
Nicolas Fabresse, Fanta Fall, Isabelle Etting, Philippe Devillier, Stanislas Grassin-Delyle
ABSTRACT
Tranexamic acid is a widely used antifibrinolytic drug but its pharmacology and pharmacokinetics
remains poorly understood. Owing to the recent knowledge on phospholipid-induced matrix effects
during human plasma analysis, our aim was to develop a liquid chromatography-mass spectrometry
method for the quantitation of tranexamic acid after efficient sample clean-up. Sample preparation
consisted in phospholipid removal and protein precipitation. Hydrophilic interaction liquid
chromatography was used and the detection was achieved with multiple reaction monitoring. The
method was validated according to the European Medicine Agency guideline in the range 1.0–1000.0
μg/mL. The performance of the method was excellent with a precision in the range 1.2-3.0%, an
accuracy between 88.4 and 96.6% and a coefficient of variation of the internal standard-normalized
matrix factor below 6.7%. This method is suitable for the quantification of tranexamic acid in the wide
range of concentrations observed during clinical studies, with all the advantages related to
phospholipid removal.
157
Metabolic profiles of neotuberostemonine and tuberostemonine in rats by high performance
liquid chromatography/quadrupole time-of-flight mass spectrometry
Yongbin Tong, Weitong Xu, Yan Wu, Liting Ou, Chaofeng Zhang
ABSTRACT
Neotuberostemonine (NS) and tuberostemonine (TS), a pair of stereoisomers, are the active
components contained in Stemona tuberosa, an antitussive herbal medicine in China. Two isomers
have different pharmacological efficacies, which will be related with their in vivo disposition.
However, the metabolic fates of NS and TS remain unknown. A method of high performance liquid
chromatography/quadrupole time-of-flight mass spectrometry coupled with mass detect filter
technique was established to investigate the metabolites in rat plasma, bile, urine, and feces after oral
administration of the equal doses of NS and TS. The results showed that NS produced 48 phase I
metabolites, including NS, 3 hydrolyzed, 14 hydroxylated, 20 monohydrolyzed + hydroxylated and 10
dihydrolyzed + hydroxylated metabolites. The number of detected NS metabolites was 11, 39, 22 and
30 in plasma, bile, urine and feces. TS yielded 23 phase I metabolites, including TS, 3 hydrolyzed, 7
hydroxylated, 9 monohydrolyzed + hydroxylated and 3 dihydrolyzed + hydroxylated metabolites.
Besides, TS yielded 9 phase II metabolites, including 1 glucuronic acid and 2 glutathione conjugates,
and the later further degraded and modified into cysteine-glycine, cysteine and N-acetylcysteine
conjugates. The number of detected TS metabolites was 9, 24, 24 and 15 in plasma, bile, urine and
feces. Different metabolic patterns may be one of the main reasons leading to different
pharmacological effects of NS and TS.
Comprehensive profiling and characterization of coumarins from roots, stems, leaves, branches,
and seeds of Chimonanthus nitens Oliv using ultra-performance liquid
chromatography/quadrupole-time-of-flight mass spectrometry combined with modified mass
defect filter
Ting Tan, Yun Luo, Chen-Cong Zhong, Xu Xu, Yulin Feng
ABSTRACT
Chimonanthus nitens Oliv (CNO), having been studied and developed as the tea beverages, tea raw
materials and preparations, an unique species in China, have been extensively used for treating colds
and influenza for centuries. In the present study, a method based on the modified mass defect filter
(MDF) was firstly developed and validated for comprehensive profiling of coumarins in the different
parts of CNO via ultra-performance liquid chromatography tandem quadrupole time-of-flight mass
spectrometry (UPLC-QTOF/MS). The five-point screening approach based on MDF and the visual
isotopic ion technique could rapidly screen the interested precursor ions. The fragmentation behavior
of coumarins was systematically investigated and a total of 42 coumarins including 27 potential new
ones were unambiguously or tentatively identified in the CNO. Collectively, the results obtained in the
present work may provide useful information for future utilization of CNO.
158
New analytical method for determination of epimer metabolites in rat plasma after oral
administration of Paeoniflorin by UPLC-TOF-MS following picolinoyl derivatization
Zhigang Wang, Shuhan Tang, Masao Hattori, Hailong Zhang, Ning Zhang
ABSTRACT
A highly sensitive analytical method was developed to study the in vivo metabolism of paeoniflorin,
one of the most principal components in traditional Chinese medicine. After hydrolyzation with
sulfatase, the epimer metabolites 7S-paeonimetabolin I and 7R-paeonimetabolin I of paeoniflorin in rat
plasma were successfully detected and well separated by LC–MS following picolinoyl derivatization
for the first time. Borneol was used as the internal standard to quantify 7S-paeonimetabolin I and 7R-
paeonimetabolin I in rat plasma. 7S-paeonimetabolin I and 7R-paeonimetabolin I show similar but
different pharmacokinetic behavior. 7S-paeonimetabolin I reached the maximum mean plasma
concentration of 45.7 ± 4.6 ng/mL at about 1.5 h after oral administration of paeoniflorin at a dose of 5
mg/kg, while 7R-paeonimetabolin I reached the maximum mean plasma concentration of 39.2 ± 3.5
ng/mL at about 1.5 h. The full metabolic pathway of paeoniflorin in rats was proposed. The
monoterpene compound paeoniflorin was found to be metabolized to the sulfate of 7S-
paeonimetabolin I and 7R-paeonimetabolin I in vivo which maybe responsible for the pharmacological
effect of paeoniflorin.
Application of 2D-NMR with room temperature NMR probes for the assessment of the higher
order structure of filgrastim
Robert G. Brinson, Houman Ghasriani, Derek J. Hodgson, Kristie M. Adams, John P. Marino
ABSTRACT
The higher order structure (HOS) of biotherapeutics is a critical quality attribute that can be evaluated
by nuclear magnetic resonance (NMR) spectroscopy at atomic resolution. NMR spectral mapping of
HOS can be used to establish HOS consistency of a biologic across manufacturing changes or to
compare a biosimilar to an innovator reference product. A previous inter-laboratory study performed
using filgrastim drug products demonstrated that two-dimensional (2D)-NMR 1HN-15NH
heteronuclear correlation spectroscopy is a highly robust and precise method for mapping the HOS of
biologic drugs at natural abundance using high sensitivity NMR ‗cold probes.‘ Here, the applicability
of the 2D-NMR method to fingerprint the HOS of filgrastim products is demonstrated using lower
sensitivity, room temperature NMR probes. Combined chemical shift deviation and principal
component analysis are used to illustrate the performance and inter-laboratory precision of the 2D-
NMR method when implemented on room temperature probes.
159
Influence of sulfur fumigation on the chemical profiles of Atractylodes macrocephala Koidz
evaluated by UFLC–QTOF–MS combined with multivariate statistical analysis
Xue Sun, Xiao-bing Cui, Hong-mei Wen, Chen-xiao Shan, Wei Li
ABSTRACT
In the present study, the chemical compositions of Atractylodes macrocephala Koidz. (AMK) were
analyzed systematically and influence of sulfur fumigation on the chemical profiles was evaluated by
ultrafast flow liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry
(UFLC–QTOF–MS) combined with multivariate statistical analysis. 52 components were detected
from non-fumigated AMK (NF-AMK) and 28 components were newly produced after sulfur
fumigation, out of which 59 major peaks were identified. The concentrations of 20 compounds
significantly decreased and 37 compounds obviously increased. The potential structural transformation
mechanism of terpenoids was explored to illustrate the correlation of the components contents before
and after sulfur fumigation. Eight sulfur-containing/dehydrated-integrated atractylenolides that
evolved from the NF-AMK were screened out as potential characteristic chemical markers to examine
the post-harvest handling procedures of commercial AMK with excessive sulfur fumigation and
maintain consistent quality.
A sandwich immunoassay for brucellosis diagnosis based on immune magnetic beads and
quantum dots
Li Li, Dehui Yin, Kun Xu, Yushen Liu, Juan Li
ABSTRACT
Brucellosis is a serious zoonosis with a rapid increase in incidence across epidemic regions. Currently,
there are numerous methods for diagnosing brucellosis. However, these studies often have a few
defects, such as low sensitivity, poor specificity, time consuming, laborious, and even potential
biological risk. To overcome these shortcomings, we have developed a rapid, sensitive and accurate
diagnosis procedure for brucellosis based on the immune magnetic beads (IMB) probe and quantum
dots (QDs) – staphylococcal protein A (SPA) probe. With the presence of Brucella antibody in the
tested serum, the QDs-SPA probe links to the IMB probe and an immune-sandwich complex is
formed. As a result, the fluorescence intensity from QDs increased significantly and was correlated
with the amount of Brucella antibody. Under the optimized conditions, 248 blood samples were
detected and the diagnosis effect was evaluated. The area under the receiver-operating characteristic
(ROC) curve was 0.970 (95% confidence interval (CI), 0.9479–0.9920). The diagnostic sensitivity was
96.15% (95% CI, 91.82–98.58%), the diagnostic specificity was 94.12% (95% CI, 87.64–97.81%)
with a fluorescence intensity cutoff value of 150.4 and the detection time was only 100 min. This
diagnostic procedure can be satisfactorily applied to the diagnosis of brucellosis.
160
Systematically characterize the absorbed effective substances of Wutou Decoction and their
metabolic pathways in rat plasma using UHPLC-Q-TOF-MS combined with a target network
pharmacological analysis
Tengfei Xu, Shizhe Li, Yufei Sun, Zifeng Pi, Zhiqiang Liu
ABSTRACT
Wu-tou Decoction (WTD) is a famous traditional Chinese medicine (TCM) formula which is applied
to treat arthritis and pain of joints. In this study, a sensitive and rapid method was developed for the
separation and identification of the absorbed constituents and metabolites of WTD in the rats plasma
by ultra high-performance liquid chromatography coupled with quadrupole time-of-flight mass
spectrometry (UHPLC-Q-TOF-MS). A total of 22 absorbed prototype constituents and 49 metabolites
were identified or tentatively characterized in dosed plasma. The possible metabolic pathways of these
constituents involved sulfation, glucuronidation, demethylation, hydroxylation and so on. What‘s
more, we optimized the conventional process ways of network pharmacology and proposed a new
concept called target-network pharmacology (T-NP). T-NP method used the absorbed constituents and
the corresponding target proteins to generate compound-target network, and compare to the
conventional method indifferent using the compounds collected from herbs, it could reduce the false
positive results. We found that the following proteins were related to the WTD therapeutic effects,
such as PTGS2, PTGS1, MAPK3, PPARG, TNF, IL4 and IL6. On the whole, the proposed method
clearly presented the metabolic processes of WTD and the results gave a comprehensive metabolic
profile of WTD in vivo for the first time.
Nontargeted metabolomics approach for the differentiation of cultivation ages of mountain
cultivated ginseng leaves using UHPLC/QTOF-MS
Xiangwei Chang, Juanjuan Zhang, Dekun Li, Dazheng Zhou, Zhengliang Ye
ABSTRACT
The adulteration or falsification of the cultivation age of mountain cultivated ginseng (MCG) has been
a serious problem in the commercial MCG market. To develop an efficient discrimination tool for the
cultivation age and to explore potential age-dependent markers, an optimized ultra high-performance
liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOF-MS)-based
metabolomics approach was applied in the global metabolite profiling of 156 MCG leaf (MGL)
samples aged from 6 to 18 years. Multivariate statistical methods such as principal component analysis
(PCA) and partial least squares discriminant analysis (PLS-DA) were used to compare the derived
patterns between MGL samples of different cultivation ages. The present study demonstrated that 6–
18-year-old MGL samples can be successfully discriminated using two simple successive steps,
together with four PLS-DA discrimination models. Furthermore, 39 robust age-dependent markers
enabling differentiation among the 6–18-year-old MGL samples were discovered. The results were
validated by a permutation test and an external test set to verify the predictability and reliability of the
established discrimination models.
161
Studies on the metabolites difference of psoralen/isopsoralen in human and six mammalian liver
microsomes in vitro by UHPLC–MS/MS
Aihong Yang, Junxiu Chen, Yetao Ma, Lili Wang, Xin He
ABSTRACT
Psoralen and isopsoralen are found in many fruits, vegetables and traditional Chinese medicines
(TCM), such as Ficus carica L., Celery, Fructus Psoraleae etc. Modern pharmacological studies found
that psoralen and isopsoralen can show estrogen-like activity, antitumor, and antibacterial activities
etc. However, some research results also show some liver damage associated with the use of
psoralen/isopsoralen or related medicines in human. Many studies focus on the pharmacological
activities of psoralen/isopsoralen, while it is important to choose the suitable pharmacological models
which are relevant to human in drug metabolism and pharmacokinetic process. The aim of this study is
to identify the metabolites of psoralen/isopsoralen by human and six mammalian liver microsomes,
and compare the metabolites difference of different species. Psoralen/isopsoralen are metabolized by
liver microsomes of different animals to form five and seven metabolites, respectively. The
metabolism of psoralen/isopsoralen undergoes hydroxylation, hydrogenation and hydrolysis, and
oxidation of the furan ring to generate a furanoepoxide or γ-ketoenal intermediate. Furanoepoxide then
forms a dihydrodiol, while γ-ketoenal forms 6-(7-hydroxycoumaryl)-acetic acid (in psoralen)/8-(7-
hydroxycoumaryl)-acetic acid (in isopsoralen). By comparing the types of metabolites in the seven
liver microsomes, it shows that the metabolic behaviors of psoralen by Beagle dog is most relevant to
human, while the metabolic behaviors of isopsoralen by Sprague-Dawley rat is most similar to human.
By comparing the relative amounts of the main metabolites, it shows that the metabolic capabilities of
Sprague-Dawley rat and Rhesus monkey for psoralen are most similar to human, while the metabolic
capabilities of Mouse, Dunkin-Hartley guinea pig, Sprague-Dawley rat, and human for isopsoralen are
similar. Furthermore, the results show that the metabolic capability of human for psoralen and
isopsoralen are weaker than other mammal species.
Al cation induces aggregation of serum proteins
P. Chanphai, L. Kreplak, H.A. Tajmir-Riahi
ABSTRACT
Al cation is known to induce protein fibrillation and causes several neurodegenerative disorders. We
report the spectroscopic, thermodynamic analysis and AFM imaging for the Al cation binding process
with human serum albumin (HSA), bovine serum albumin (BSA) and milk beta-lactoglobulin (b-LG)
in aqueous solution at physiological pH. Hydrophobicity played a major role in Al–protein interactions
with more hydrophobic b-LG forming stronger Al–protein complexes. Thermodynamic parameters
ΔS, ΔH and ΔG showed Al–protein bindings occur via hydrophobic and H-bonding contacts for b-LG,
while van der Waals and H-bonding interactions prevail in HSA and BSA adducts. AFM clearly
indicated that aluminum cations are able to force BSA and b-LG into larger or more robust aggregates
than HSA, with HSA 4 ± 0.2 (SE, n = 801) proteins per aggregate, for BSA 17 ± 2 (SE, n = 148), and
for b-LG 12 ± 3 (SE, n = 151). Thioflavin T test showed no major protein fibrillation in the presence of
Al cation. Al complexation induced major alterations of protein conformations with the order of
perturbations b-LG > BSA > HSA.
162
Volume 142 August 2017
Fighting falsified medicines: The analytical approach
Hervé Rebiere, Pauline Guinot, Denis Chauvey, Charlotte Brenier
ABSTRACT
Given the harm to human health, the fight against falsified medicines has become a priority issue that
involves numerous actors. Analytical laboratories contribute by performing analyses to chemically
characterise falsified samples and assess their hazards for patients. A wide range of techniques can be
used to obtain individual information on the organic and inorganic composition, the presence of an
active substance or impurities, or the crystalline arrangement of the formulation‘s compound. After a
presentation of these individual techniques, this review puts forward a methodology to combine them.
In order to illustrate this approach, examples from the scientific literature (products used for erectile
dysfunction treatment, weight loss and malaria) are placed in the centre of the proposed methodology.
Combining analytical techniques allows the analyst to conclude on the falsification of a sample, on its
compliance in terms of pharmaceutical quality and finally on the safety for patients.
Lipophilicity estimation of statins as a decisive physicochemical parameter for their hepato-
selectivity using reversed-phase thin layer chromatography
Azza H. Rageh, Noha N. Atia, Hamdy M. Abdel-Rahman
ABSTRACT
Lipophilicity plays a crucial role in determining the hepato-selectivity and hence, the biological
activity and the associated side effects of statins. Herein, the employment of RP-TLC for estimation of
lipophilicity of six statins namely; atorvastatin, simvastatin, pravastatin, lovastatin, rosuvastatin and
fluvastatin is examined. A very good correlation between the chromatographically-determined
retention parameters (relative lipophilicity (RM0) or lipophilic parameter (C0)) and both experimental
and computed log P values were obtained. However, the results indicate that the type of organic
modifier in the mobile phase system (methanol, acetonitrile and acetone) has a small influence on
RM0 or C0 values. Higher values of RM0 or C0 are ascribed to lipophilic statins and lower values of
RM0 or C0 are attributed to hydrophilic ones. Therefore, RM0 or C0 could be effectively used as
simple practical predictors of extra-hepatic distributions of statins and thus their expected side effects.
Furthermore, three QSPR (quantitative structure-property relationship) models were constructed to
describe the relationship between RM0 with log P and log D of the statins under investigation. These
models can be very useful to predict the lipophilicity of other members of statin drugs and might be
expanded to newly synthesized compounds with the same structural features.
163
Metabolite profiling of flavonols and in vitro antioxidant activity of young shoots of wild
Humulus lupulus L. (hop)
Annalisa Maietti, Virginia Brighenti, Gianpiero Bonetti, Paola Tedeschi, Federica Pellati
ABSTRACT
Humulus lupulus L., commonly named hop, is well-known for its sedative and estrogenic activity.
While hop cones are widely characterized, only few works have been carried out on the young shoots
of this plant. In the light of this, the aim of this study was to identify for the first time the flavonoids
present in young hop shoots and to compare the composition of samples harvested from different
locations in Northern Italy with their antioxidant activity. The samples were extracted by means of
dynamic maceration with methanol. The HPLC-UV/DAD, HPLC-ESI-MS and MS2 analysis were
carried out by using an Ascentis C18 column (250 × 4.6 mm I.D., 5 μm), with a mobile phase
composed of 0.1 M formic acid in both water and acetonitrile, under gradient elution. Quercetin and
kaempferol glycosides were the main compounds identified and quantified in hop shoot extracts. Total
flavonols ranged from 2698 ± 185 to 517 ± 48 μg/g (fresh weight). The antioxidant activity was
determined by means of the radical scavenging activity assay against diphenylpicrylhydrazyl (DPPH)
and by using a photochemiluscence assay with a Photochem® apparatus. The results showed that hop
shoots represent a new source of flavonols; therefore, they can be useful for a possible incorporation in
the diet as a functional food or applied in the nutraceutical ambit.
Development of assay for determination of eletriptan hydrobromide in loaded PLGA
nanoparticles
Ozgur Esim, Ayhan Savaser, Sevinc Kurbanoglu, Cansel K. Ozkan, Yalcin Ozkan
ABSTRACT
Eletriptan Hydrobromide is a serotonin 5-HT1 receptor agonist and it used for the treatment of
migraine headaches with or without aura. Even if the drug is well absorbed after oral administration, it
has some drawbacks like first pass metabolism and decrease in bioavailability after migraine attacks.
Encapsulation of drug into polymeric nanoparticles is one of the methods for protecting the drug
against degradation. The present work described a preparation of Eletriptan Hydrobromide loaded poly
(d,l-lactide-co-glycolide) nanoparticles prepared using o/w single emulsion solvent evaporation
method. In order to determine the factors affecting the physicochemical properties of the nanoparticles
on the particle size of poly (d,l-lactide-co-glycolide) nanoparticles, D-Optimal design is used.
Moreover, novel, simple, sensitive, selective, and fully validated chromatographic technique for the
quantification of Eletriptan Hydrobromide from Eletriptan Hydrobromide loaded poly(d,l-lactide-co-
glycolide) nanoparticles was developed. Poly(d,l-lactide-co-glycolide) concentration, sonication time
and sonication energy were found as significant factors (p < 0.05) on particle size of nanoparticles.
Limit of detection and limit of quantification values were calculated as 0.28 μg mL−1and 0.86 μg
mL−1, respectively.
164
Characterization of flavonol mono-, di-, tri- and tetra-O-glycosides by ultra-performance liquid
chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry and its
application for identification of flavonol glycosides in Viola tianschanica
Yan Qin, Boyan Gao, Haiming Shi, Jie Cao, Zhihong Cheng
ABSTRACT
In this study, 21 flavonol O-glycoside standards including flavonol mono-, di-, tri- and tetra-O-
glycosides have been systematically studied by ultra-high performance liquid chromatography-
electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS/MS) in
the negative ionization mode to analyze their fragmentation patterns. Here for the first time, the Z23−
fragment (corresponding to the loss of C-2‖ terminal sugar moiety) was observed in MS/MS spectra of
flavonol 3-O-triglycosides. The intensity ratio of [Y0-H]−/Y0− was proposed as a criterion to
distinguish the interglycosidic 1 → 2 and 1 → 6 linkages in flavonol 3-O-diglycosyl-7-O-
monoglycosides. The established fragmentation behaviors have been successfully applied to
characterization of flavonol glycosides in Viola tianschanica. A total of 30 flavonoid glycosides
including 3 flavonol mono-O-glycosides, 10 di-O-glycosides, 10 tri-O-glycosides, 4 tetra-O-glycosides
and 3 flavone di-C-glycosides were identified or tentatively identified on the base of their UV profiles,
MS/MS data and/or by comparing with reference substances. Among these 15 flavonoid glycosides
were reported from V. tianschanica for the first time.
Development and validation of a rapid reversed-phase HPLC method for the quantification of
monoclonal antibody bevacizumab from polyester-based nanoparticles
Flávia Sousa, Virgínia M.F. Gonçalves, Bruno Sarmento
ABSTRACT
Bevacizumab is a powerful human monoclonal antibody approved worldwide for treatment of several
types of cancer and ocular diseases due to its potential as antiangiogenic drug. Nowadays, in order to
improve the monoclonal antibody-based therapy, attempts have been focused in the formulation of
these biomacromolecules into nanoparticles. Thus, the aim of this work was to develop and validate a
reversed-phase high-performance liquid chromatography with fluorescence detection method for the
determination of bevacizumab from nanoparticulate systems, according to the International
Conference on Harmonization guidelines. Chromatographic analysis were performed on a RP-C8
column with a mobile phase composed by water-0.1% (v/v) TFA and acetonitrile-0.1% (v/v) TFA in
gradient mode at a flow rate of 1 mL min−1. Results showed that the proposed method is specific,
linear in the range of 10–100 μg mL−1 (r2 = 0.9997), accurate (recovery rate 100.50 ± 0.85%), precise
at the intraday and inter-day (relative standard deviation less than 1.79%) and robust. The detection
and quantification limits were calculated by specific linear calibration curve with less concentrated
standard (range of 1–20 μg mL−1). The LOD was 2.16 μg mL−1 and LOQ was 6.55 μg mL−1. This
method was also successfully used, for the first time, to quantify and compare the content of
bevacizumab encapsulated into poly(lactic-co-glycolic acid)-based nanoparticles before and after
lyophilization.
165
Quantification of active ingredients in semi-solid pharmaceutical formulations by near infrared
spectroscopy
Lisa B. Schlegel, Manfred Schubert-Zsilavecz, Mona Abdel-Tawab
ABSTRACT
Near infrared (NIR) spectroscopy is increasingly gaining significance in the pharmaceutical industry
for quality and in-process control. However, the potential of this method for quantitative quality
control in pharmacies has long been neglected and little data is available on its application in analysis
of creams and ointments. This study evaluated the applicability of NIR spectrometer with limited
wavelength range (1000–1900 nm) for quantitative quality control of six different dermatological
semi-solid pharmaceutical preparations. Each contained a frequently used active ingredient in a
common concentration either in a water-free lipid base or in an aqueous cream matrix. Based on direct
NIR transflectance measurements through standardized glass beakers and partial least squares (PLS)
multivariate calibration, quantitative models were generated comparing several data pre-processing
methods Whereas difficulties were observed for mixtures containing 2% (w/w) metronidazole or 4%
(w/w) erythromycin, content determination was possible with sufficient accuracy for salicylic acid (5
% (w/w)) and urea (10% (w/w)) in hydrophilic as well as in lipophilic formulations meeting the limit
of a maximum deviation of ± 5% (relative) from the reference values. Exemplarily, one of the methods
was successfully validated according to the EMA Guideline, determining several figures of merit such
as specificity, linearity, accuracy, precision and robustness.
Detection of regulated herbs and plants in plant food supplements and traditional medicines
using infrared spectroscopy
E. Deconinck, C.A. Sokeng Djiogo, J.L. Bothy, P. Courselle
ABSTRACT
The identification of a specific toxic or regulated plant in herbal preparations or plant food
supplements is a real challenge, since they are often powdered, mixed with other herbal or synthetic
powders and compressed into tablets or capsules. The classical identification approaches based on
micro- and macroscopy are therefore not possible anymore. In this paper infrared spectroscopy,
combined with attenuated total reflectance was evaluated for the screening of plant based preparations
for nine specific plants (five regulated and four common plants for herbal supplements). IR and NIR
spectra were recorded for a series of self-made triturations of the targeted plants. After pretreatment of
the spectral data chemometric classification techniques were applied to both data sets (IR and NIR)
separately and the combination of both. The results show that the screening of herbal preparations or
plant food supplements for specific plants, using infrared spectroscopy, is feasible. The best model was
obtained with the Mid-IR data, using SIMCA as modelling technique. During validation of the model,
using an external test set, 21 of 25 were correctly classified and six of the nine targeted plants showed
no misclassifications for the selected test set. For the other three a success rate of 50% was obtained.
Mid-IR combined with SIMCA can therefore be applied as a first step in the screening of unknown
samples, before applying more sophisticated fingerprint approaches or identification tests described in
several national and international pharmacopoeia. As a proof of concept five real suspicious samples
were successfully screened for the targeted regulated plants.
166
UV-induced electron transfer between triethylamine and 5-bromo-2′-deoxyuridine A puzzle
concerning the photochemical debromination of labeled DNA
Paweł Wityk, Magdalena Zdrowowicz, Justyna Wiczk, Janusz Rak
ABSTRACT
5-Bromo-2′-deoxyuridine (BrdU) photosensitizes DNA to strand break formation. However, this type
of photodamage is completely quenched by the presence of triethylamine (TEA) which originates from
RP-HPLC purification commonly employed by oligonucleotide providers. While the presence of TEA
in oligonucleotide samples does not interfere with PCR or other molecular biology applications, the
mechanism of photochemical reaction proceeding in the labeled DNA is dramatically changed due to
the photoinduced electron transfer (PET) between the photoexcited BrdU and the ground state TEA.
For the first time, we demonstrated that the latter process produces 2′-deoxyuridne2′-deoxyuridine
(debromination) in the labeled DNA instead of the expected strand break. PET between TEA and
BrdU was additionally confirmed by the UV irradiations of aqueous solutions containing both species.
Indeed, the efficient formation of 2′-deoxyuridine was observed in the studied photolytes. Moreover,
we showed the formation of an additional product in these binary mixtures, i.e. imidazole derivative,
that is not formed in DNA and was reported in the literature in the context of dark rather than
photochemical processes. Using mass spectrometry we demonstrated that the amount of TEA impurity
in the commercial samples of oligos exceeds up to 3 orders of magnitude that of the purchased DNA.
Development of an in vivo-relevant drug product performance method for an amorphous solid
dispersion
Brian W. Pack, Yelizaveta Babayan, Mark A. Schrad, Paul A. Stroud, Aktham Aburub
ABSTRACT
The purpose of this work was to develop a meaningful in vitro dissolution method for evacetrapib
spray-dried dispersion (SDD) tablets that is discriminating for crystalline drug substance (DS) content.
Justification of the method conditions included evaluation of dissolution media, rotation speed,
surfactant selection and level of surfactant to achieve sink conditions. Discrimination was illustrated
by testing SDD tablets spiked with 10%, 20%, and 30% crystalline DS. The results demonstrated a
13%, 22% and 32% drop in the dissolution end point, respectively, as compared to unspiked SDD
tablets. Additionally, tablets containing crystalline DS and tablets containing SDD were tested in a
relative bioavailability (RBA) study. Utilizing the proposed dissolution method, the dissolution end
point of SDD tablets was determined to be approximately 4 fold higher than that of the tablets
containing crystalline DS. These results compare favourably to the in vivo RBA study results where
SDD tablets had a 4.6 fold increase in exposure compared to tablets containing crystalline DS.
167
Ultrasound-assisted low-density solvent dispersive liquid–liquid microextraction for the
simultaneous determination of 12 new antidepressants and 2 antipsychotics in whole blood by
gas chromatography–mass spectrometry
Xiujuan Chen, Shuiqing Zheng, Jian Le, Zheyuan Qian, Yifeng Chai
ABSTRACT
Antidepressant drugs are widely used in the treatment of different psychiatric disorders, as well as in
conjunction with antipsychotics for the treatment of major depressive disorder. In this study, a simple
and rapid ultrasound-assisted low-density solvent dispersive liquid–liquid microextraction (UA-LDS-
DLLME) method was developed for the simultaneous determination of 12 new antidepressants
(norfluoxetine, fluoxetine, fluvoxamine, agomelatine, mirtazapine, moclobemide, melitracen, N-
desmethylmirtazapine, maprotiline, sertraline, citalopram, paroxetine) and 2 antipsychotics (clozapine
and haloperidol) in human whole blood by gas chromatography–mass spectrometry (GC–MS).
Different parameters affecting the UA-LDS-DLLME were optimized and the optimal conditions were
as follows: 100 μL of toluene as extraction solvent, extraction pH 12 and 3 min of ultrasound stirring.
Good linearity (R2 ≥ 0.991) was obtained at the concentration range of 15–1500 ng/mL for
norfluoxetine, fluoxetine, fluvoxamine, melitracen, maprotiline and citalopram, and 5–500 ng/mL for
agomelatine, mirtazapine, moclobemide, N-desmethylmirtazapine, sertraline, paroxetine, clozapine
and haloperidol. The intra-day and inter-day precision were all less than 10%, and accuracy of intra-
day and inter-day were in the range of −12.7% to 7.9% and −13.9 to 11.8%, respectively. The
extraction recoveries of most analytes were more than 60%. The UA-LDS-DLLME/GC–MS method
was demonstrated with acceptable precision, accuracy and good specificity for the simultaneous
determination of 12 antidepressants and 2 antipsychotics, and has been successfully applied in a real
case.
Application of protein A-modified capillary-channeled polymer polypropylene fibers to the
quantitation of IgG in complex matrices
Hung K. Trang, R. Kenneth Marcus
ABSTRACT
Polypropylene (PP) capillary-channeled polymer (C-CP) fibers loaded with recombinant
Staphyloccocus aureus protein A (rSPA) were used as an affinity chromatography stationary phase for
the quantitation of immunoglobulin G (IgG) in complex biological matrices. Optimization of the
chromatographic method regarding mobile phase components and load/elution conditions was
performed. The six-minute analysis, including a load step with 12 mM phosphate at pH 7.4, an elution
step with 0.025% phosphoric acid and a re-equilibration step, was employed for quantitation of IgG1
from 0.075 to 3.00 mg mL−1 in an IgG-free CHO cell supernatant matrix. Quantification of IgG1
content in a different CHO cell line was accomplished using the external calibration curve as well as
using a standard addition approach. The high level of agreement between the two approaches suggests
that the protein A-modified C-CP fiber phase is immune from matrix effects due to concomitant
species such as host cell proteins (HCPs), host cell DNA, media components and other leachables and
extractables. The inter-day and intra-day precision of the method were 3.1 and 3.5%RSD respectively
for a single column. Column-to-column variability was 1.31 and 6.62%RSD for elution time and peak
area, respectively, across columns prepared in different batches. The method reported here is well-
suited for IgG analysis in complex harvest cell culture media in both the development and production
environments.
168
Simple and rapid quantification of vancomycin in serum, urine and peritoneal/pleural effusion
via UHPLC–MS/MS applicable to personalized antibiotic dosing research
Lenka Javorska, Lenka Kujovska Krcmova, Petr Solich, Milan Kaska
ABSTRACT
Management of the therapy of life-threatening bacterial infection is extremely based on an optimal
antibiotic treatment. Achieving the correct vancomycin dosage in blood and target tissues can be
complicated in special situations, e.g., where large fluid sequestration and/or acute renal failure occur.
A UHPLC–MS/MS method operating in electrospray (ESI) positive ion mode was applied for the
determination of vancomycin in serum, urine and peritoneal/pleural effusion. Sample pretreatment was
composed of dilution and simple protein precipitation where only a small volume (50 μL) of serum,
urine or peritoneal/pleural effusion was required. The separation of vancomycin was performed on a
Meteoric Core C18 BIO column (100 × 4.6 mm, 2.7 μm) by gradient elution with 0.1% formic acid in
water and acetonitrile. The total time of analysis was 4.5 min. The method was found to be linear in
the range of 2–60 μM (or 0.5–10 μM) for serum, 0.27–10 μM (or 2–60 μM) for peritoneal/pleural
effusion and 25–300 μM for urine, which was adequate for the determination of vancomycin in patient
samples. The intra- and inter-day precision was below 8% RSD, and accuracy was from 89 to 104%.
The UHPLC/MS–MS method offers a fast and reliable approach to determine vancomycin
concentrations in three different human body fluid samples (serum, urine and peritoneal/pleural
effusion) with a simple sample pretreatment that was the same for all selected specimens.
Biphenyl based stationary phases for improved selectivity in complex steroid assays
Johanna M. Lindner, Michael Vogeser, Stefanie H. Grimm
ABSTRACT
The measurement of steroid hormones and their corticoid precursors is an important aspect in
endocrinology since these analytes are biomarkers for several endocrine disorders. Over the last few
years, HPLC–MS/MS has become the method of choice to analyze these compounds. There are
already several methods using stationary phases modified with C18 groups. However, since these
columns sometimes do not enable sufficient separation of some isobaric steroids, we investigated the
potential of a different RP modification using biphenyl groups for the separation of challenging isobars
such as corticosterone, 11- and 21-deoxycortisol. The aim of our work was the development of an
isotope dilution UHPLC–MS/MS assay for clinical research that combines simple and effective sample
preparation with a powerful MS method quantifying a broad steroid panel (aldosterone, corticosterone,
cortisol, cortisone, 11‐deoxycorticosterone, 11-deoxycortisol, 21-deoxycortisol,
dehydroepiandrosterone, dehydroepiandrosterone sulfate, 17-OH-progesterone, progesterone, and
testosterone) in human serum. After a manual protein precipitation step using zinc trifluoroacetate
(ZnTFA) in methanol, the supernatants were directly injected into the UHPLC–MS system.
Chromatographic baseline separation of all isobaric compounds (corticosterone ↔ 11-deoxycortisol ↔
21-deoxycortisol, 17-OH-progesterone ↔ 11‐deoxycorticosterone, and aldosterone ↔ cortisone) was
achieved using a Kinetex Biphenyl column (150 × 2.1 mm, 1.7 μm) with a mobile phase consisting of
0.2 mM ammonium fluoride in water and methanol. The total run time was 10 min. For detection we
used a Xevo TQ-S mass spectrometer operating in the ESI positive and negative modes. The method
was validated according to the EMA guideline for bioanalytical method validation. The results for
accuracy (within-run: 92.3%–115%, between-run: 92.4 %–113%) and imprecision (within-run:
0.80%–9.05%, between-run: 1.98 %–15.2%) were satisfying.
169
Ultra-sensitive and selective quantification of endothelin-1 in human plasma using ultra-
performance liquid chromatography coupled to tandem mass spectrometry
Yosuke Suzuki, Lukas Witt, Walter Mier, Gerd Mikus, Jürgen Burhenne
ABSTRACT
Endothelin-1 (ET-1) is a potent endogenous vasoconstrictor peptide and the plasma concentrations are
commonly quantified by immunoassays such as enzyme-linked immuno-sorbent assays (ELISA) with
the disadvantage of possible cross-reactivity with closely related endothelin derivatives. The aim of
this study was to develop and validate an ultra-sensitive and selective assay for the quantification of
ET-1 in human plasma, using ultra-performance liquid chromatography with tandem mass
spectrometry (UPLC-MS/MS) after solid phase extraction. The assay fulfilled the requirements of the
US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) guidelines for
assay validation, with a lower limit of quantification of 1.5 pg/mL for ET-1. Recovery rates from
plasma ranged between 80.8% and 93.6%, and matrix effect varied between 121% and 135%. The
assay was successfully applied to assess the time course of plasma ET-1 concentrations in two human
volunteers after co-administration of bosentan and clarithromycin. In this trial, the concentrations
measured by UPLC-MS/MS were slightly lower than those measured by ELISA, with a strong positive
correlation between the two methods. Our novel UPLC-MS/MS method is applicable to the clinical
setting and may have better selectivity for ET-1 than ELISA.
Serum metabolomics reveals the mechanistic role of functional foods and exercise for obesity
management in rats
Naglaa M. Ammar, Mohamed A. Farag, Tahani E. Kholeif, Nadia S. Metwally, Abdel- Hamid Z.
Abdel- Hamid
ABSTRACT
Obesity is one of the independent risk factors for several health problems, leading to metabolic
perturbations and for which analytical approaches i.e., ―metabolomics‖ is needed to monitor the
underlying metabolic changes. In this study, obesity associated changes were assessed via serum
metabolites analysis of obese rats fed on high fat diet. Obese rats were subsequently treated with
different functional foods used for obesity management including pomegranate, grapefruit, and red
cabbage in parallel to swimming exercise. Serum samples were analyzed using gas chromatography-
mass spectrometry (GC–MS) followed by multivariate data analysis to classify samples and determine
if such treatments can help revert obesity related metabolic changes back to normal status. Results led
to the identification of several novel metabolites biomarkers for obesity related to lipids, amino acids
and central tricarboxylic acid (TCA) pathways. Distinct variations in metabolite levels were recorded
in obese rats compared to normal ones including l-aspartic, l-alanine, l-glutamine, l-glycine,
phenylethanolamine, α-aminobutyric acid and β-hydroxybutyric acid. Metabolomics approach
developed herein provides novel insight onto the metabolic disturbances associated with obesity,
which will assist in future drug design that can help mitigate against such changes.
170
Systematic screening and characterization of prototype constituents and metabolites of total
astragalosides using HPLC-ESI-IT-TOF-MSn after oral administration to rats
Hong-Fu Li, Feng Xu, Ping Yang, Guang-Xue Liu, Shao-Qing Cai
ABSTRACT
Astragalosides (AGs) are the main bioactive constituents in Astragali Radix (AR), and have a wide
range of pharmacological properties, including immunoregulatory, cardioprotective, neuroprotective,
antioxidative, antidiabetic, and antinociceptive effects. However, the metabolism of total AGs remains
unclear. To clarify the metabolic fate of AGs after oral administration to rats, total AGs were isolated
from AR extracts using AB-8 macroporous resin chromatography and preparative HPLC, and then
analyzed using HPLC-DAD-ELSD and LC–MS. HPLC-ESI-IT-TOF-MSn was used to systematically
screen and characterize prototype constituents and metabolites of total AGs in rat feces, urine, and
plasma samples. As a result, 123 AG-related compounds from feces were detected and structurally
characterized. Among the 123 compounds, 107 were phase I metabolites, of which 91 were new
metabolites, and 73 were new compounds. In addition, six prototype constituents in urine, and one in
plasma were detected. The main metabolic sites in the structure of cycloastragenol (CAG), the
aglycone of AGs, were found to be the 9, 19-cyclopropane ring (E ring) and the 20, 24-furan ring (F
ring). The cleavage mode of CAG derivatives in negative ion mode was identified, and was found to
be highly dependent on the integrity of the E ring. Mono- to tetra-hydroxylated and carboxyl
substituted metabolites were tentatively identified. Deglycosylation, hydroxylation, dehydrogenation,
isomerization, ring cleavage, and carboxyl substitution were considered to be the major metabolic
reactions involved in the formation of the metabolites, among which carboxyl substitution was a novel
metabolic reaction.
Development, validation and application of a novel liquid chromatography tandem mass
spectrometry assay measuring uracil, 5,6-dihydrouracil, 5-fluorouracil, 5,6-dihydro-5-
fluorouracil, α-fluoro-β-ureidopropionic acid and α-fluoro-β-alanine in human plasma
Ottiniel Chavani, Berit Packert Jensen, R. Matthew Strother, Christopher M. Florkowski, Peter M.
George
ABSTRACT
The plasma 5,6-dihydrouracil/uracil (UH2/U) ratio is a possible phenotypic marker of
dihydropyrimidine dehydrogenase (DPD) activity, hence an index of 5-fluorouracil (5-FU) response
and toxicity. Studies have re-affirmed the value of 5-FU and 5,6-dihydro-5-fluorouracil (FUH2) for
therapeutic drug monitoring (TDM). However, FUH2 has limited stability in plasma, necessitating
expedited plasma separation and freezing, where routine compliance may not be easy. The metabolites
α-fluoro-β-ureidopropionic acid (FUPA) and α-fluoro-β-alanine (FβAL) are stable in plasma and are
probable candidates for TDM. This paper describes development, validation and application of an LC–
MS/MS assay quantifying U, UH2, 5-FU, FUH2, FUPA and FβAL in human plasma. Extraction was
by salt-assisted liquid–liquid extraction (LLE) in two-stages with pH adjustment. The supernatants
were mixed, dried and reconstituted. Analytes were resolved on the Luna PFP (2) (150 × 2.00 mm, 3
μ) column by gradient elution and analyzed by tandem mass spectrometry via electrospray ionisation
in positive polarity. The analytical response was linear (r2 ≥ 0.99) in the concentration (ng/mL) ranges:
50–10 000 for FβAL and FUH2, 50–5 000 for FUPA, 50–100 000 for 5–FU, 5–200 for U and 10–400
for UH2. Within- and between-run accuracy and precision were ≤ 10.2% and ≤ 9.8% respectively
across the QC range and inclusive of LLOQ.
171
Multi-platform metabolomics and a genetic approach support the authentication of agarwood
produced by Aquilaria crassna and Aquilaria malaccensis
Huy Truong Nguyen, Jung-Eun Min, Nguyen Phuoc Long, Ma Chi Thanh, Sung Won Kwon
ABSTRACT
Agarwood, the resinous heartwood produced by some Aquilaria species such as Aquilaria crassna,
Aquilaria malaccensis and Aquilaria sinensis, has been traditionally and widely used in medicine,
incenses and especially perfumes. However, up to now, the authentication of agarwood has been
largely based on morphological characteristics, a method which is prone to errors and lacks
reproducibility. Hence, in this study, we applied metabolomics and a genetic approach to the
authentication of two common agarwood chips, those produced by Aquilaria crassna and Aquilaria
malaccensis. Primary metabolites, secondary metabolites and DNA markers of agarwood were
authenticated by 1H NMR metabolomics, GC–MS metabolomics and DNA-based techniques,
respectively. The results indicated that agarwood chips could be classified accurately by all the
methods illustrated in this study. Additionally, the pros and cons of each method are also discussed. To
the best of our knowledge, our research is the first study detailing all the differences in the primary and
secondary metabolites, as well as the DNA markers between the agarwood produced by these two
species.
Apoptosis induction activity and molecular docking studies of survivin siRNA carried by Fe3O4-
PEG-LAC-chitosan-PEI nanoparticles in MCF-7 human breast cancer cells
Sanam Arami, Majid Mahdavi, Mohammad-Reza Rashidi, Reza Yekta, Nasser Samadi
ABSTRACT
Delivery of small interfering RNAs (siRNAs) into cells still remains a challenge in gene delivery
studies. Here, we investigated the ability of synthesized Fe3O4-PEG-LAC-chitosan-PEI nanoparticles
for siRNA delivery of survivin as the model gene into cells. The cellular uptake of survivin siRNA
carried by synthesized nanoparticles into MCF-7 breast cancer cell line was evaluated by florescent
microscopy and flowcytometry, both proving the efficacy of nanoparticles in delivery of up to 64.7%
in comparison with lipofectamine 2000. Furthermore, the delivery of survivin siRNA by the
nanoparticles (nanoplex) induced apoptosis that was assessed through DAPI staining and Annexin
V/PI assays. In addition, we evaluated the efficacy of treatment with nanoplexes in the presence of
mitoxantrone, as a chemotherapeutic agent. Our data indicated that inhibition of survivin expression
increased the cell sensitivity to mitoxantrone. Real-time PCR and western blotting analysis revealed a
significant reduction in mRNA and protein levels of survivin upon delivery of siRNA. Molecular
docking studies showed that nanoparticles can bind to centeral BIR domain of survivin, exactly above
zinc ion location with high affinity (ΔG: −10.3 Kcal/mol). Also, thermodynamic studies proved the
experimental results theoretically, revealing that the siRNA-loaded nanoparticles have a suppressing
effect on survivin mRNA. Therefore, delivery of survivin siRNA into MCF-7 cells using Fe3O4-PEG-
LAC-chitosan-PEI nanoparticles as a carrier enhances the cell death.
172
Exploring the neuroprotective effects of ginkgolides injection in a rodent model of cerebral
ischemia–reperfusion injury by GC–MS based metabolomic profiling
Jian-liang Geng, Ji-ye Aa, Si-qi Feng, Shu-yao Wang, Guang-ji Wang
ABSTRACT
Cerebral ischemia–reperfusion (I/R) injury usually contributes to mortality and disability after
ischemic stroke. Ginkgolides injection (GIn), a standard preparation composed of ginkgo diterpene
lactones extract, is clinically used for neuroprotective treatment on reconvalescents of cerebral
infarction. However, the understanding about its therapeutic mechanism is still lacking. In this study, a
gas chromatography–mass spectrometry (GC–MS) based metabolomic approach coupled with
multivariate data analysis (MVDA) was applied to explore the neuroprotective effects of GIn in a
rodent model of focal ischemic stroke induced by transient middle cerebral artery occlusion (tMCAO).
Metabolomic profiling revealed a series of metabolic perturbations that underlie the cerebral I/R
pathological events. GIn can reverse the I/R induced brain metabolic deviations by modulating
multiple metabolic pathways, such as glycolysis, Krebs cycle, pentose phosphate pathway (PPP), γ-
aminobutyrate (GABA) shunt and lipid metabolism. Moreover, the main bioactive components of GIn
were distributed to brain tissue much more easily in tMCAO rats than in normal rats after an
intravenous administration, suggesting that the increased cerebral exposure to ginkgolides in I/R
pathological condition potentially facilitated the neuroprotective effects of GIn by directly targeting at
brain. The present study provided valuable information for our understanding about metabolic changes
of cerebral I/R injury and clinical application of GIn.
Quantitative LC–HRMS determination of selected cardiovascular drugs, in dried blood spots, as
an indicator of adherence to medication
Dennis Bernieh, Graham Lawson, Sangeeta Tanna
ABSTRACT
Dried blood spot (DBS) sampling was investigated as a means of obtaining micro-volume blood
samples for the quantitative analyses of ten commonly UK prescribed cardiovascular drugs as an
indicator of medication adherence. An 8 mm disc was punched out from each DBS from calibration,
quality control and volunteer samples and extracted using methanol containing the internal standard.
Each extract was evaporated to dryness, the residue reconstituted in methanol:water (40:60 v/v)
containing 0.1% formic acid and analysed by LC–HRMS. Chromatography was performed using
gradient elution on a Zorbax Eclipse C18 HD 100 mm × 2.1 mm, 1.8 μm pore size column with the
column oven temperature at 40 °C. Flow rate of the mobile phase was 0.6 ml/min with a run time of
2.5 min. Electrospray positive ionization was used for MS detection. Drug recoveries from spiked
blood spots were 68% for simvastatin and ≥87% for all other target drugs. Compound specificity was
obtained operating the MS with a 5 ppm mass window. The LC–HRMS method was validated, with
results for accuracy and precision within acceptable limits; analytes were stable at room temperature
for at least 10 weeks and different blood spot volumes and haematocrit values had no significant
effect. The LC–HRMS assay was used to analyse DBS samples from volunteers, some of whom were
prescribed one or more of the target drugs. In results from 37 volunteers the assay successfully
identified volunteers who were known to be either adherent or nonadherent; confirmed the correct
drug/drugs for multiple prescriptions; demonstrated no false positives from other cardiovascular drugs;
revealed several examples of unsuspected non-adherence. These results indicated that the developed
assay was suitable for trials with patients.
173
Determination of thiopurine S-methyltransferase activity by hydrophilic interaction liquid
chromatography hyphenated with mass spectrometry
Daniel Pecher, Svetlana Dokupilová, Zuzana Zelinková, Maikel Peppelenbosch, Peter Mikuń
ABSTRACT
Thiopurine S-methyltransferase (TPMT) plays an important role in the metabolism of thiopurines used
in the therapy of inflammatory bowel diseases (IBD). In this work a new progressive method for the
determination of TPMT activity in red blood cells lysates was developed. Analysis was carried out by
means of hydrophilic interaction liquid chromatography (HILIC) hyphenated with mass spectrometry
(MS). In comparison with reversed-phase high-performance liquid chromatography (RP-HPLC), that
has been typically applied in determination of TPMT activity, the HILIC significantly improved the
analytical signal provided by MS, shortened analysis time, and improved chromatographic resolution.
The HILIC-HPLC-MS method was optimized and validated, providing favorable parameters of
detection and quantitation limits (5.5 and 16.5 pmol/mL, respectively), linearity (coefficient of
determination 0.9999 in the range of 0.01–1.0 nmol/mL), recovery and precision (93.25–100.37% with
RSD 1.06-1.32% in the whole concentration range of QC samples). Moreover, in contrast to the
conventional RP-HPLC-UV approach, the complex phenotype TPMT profiles can be reliably and
without interferences monitored using the HILIC-HPLC-MS method. Such advanced monitoring can
provide valuable detail information on the thiopurines (e.g. evaluating ratio of methylated and non-
methylated 6-mercaptopurine) and, by that, TPMT action in biological systems before and during the
therapy of IBD.
Workflow methodology for rat brain metabolome exploration using NMR, LC–MS and GC–MS
analytical platforms
Binta Diémé, Antoine Lefèvre, Lydie Nadal-Desbarats, Laurent Galineau, Sylvie Mavel
ABSTRACT
We developed a multi-platform approach for the metabolome exploration of rat brain tissue, using
liquid chromatography coupled with mass spectrometry (LC–MS), nuclear magnetic resonance
spectroscopy (NMR) and gas-chromatography coupled with mass spectrometry (GC–MS). The critical
steps for metabolite exploration of cerebral tissues are tissue lysis and metabolites extraction. We first
evaluated the impact of freeze-drying compared to wet tissue metabolites extraction using NMR and
LC–MS with a reversed phase liquid chromatography. Then, we compared four metabolite extraction
methods Based on the number of metabolites extracted, their intensity and their coefficient of variation
(%CV), the most reproducible protocol (one-step extraction with acetonitrile on lyophilized material)
was chosen to further evaluate the impact of sample mass on method performance (3, 6, and 9 mg were
essayed). GC–MS analysis was also investigated by analyzing four different methoximation/silylation
derivatization combinations. The optimal analytical protocols were proposed to establish the reliability
required to realize untargeted brain tissue metabolomics exploration. The most reliable workflow was
then exemplified by analyzing three rat brain regions (cerebellum, frontal and parietal cortices, n = 12)
by 1H NMR, LC–MS and GC–MS, allowing their clustering based on their metabolic profiles. We
present here an example of development of methodology that should be done before running analysis
campaigns.
174
Bioelectrical impedimetric sensor for single cell analysis based on nanoroughened quartz
substrate; suitable for cancer therapeutic purposes
Milad Gharooni, Mohammad Abdolahad
ABSTRACT
Single cells analysis has been interested in recent decade. Apart from scientific benefits to achieve new
biological phenomena in cell study, many diagnostic and therapeutic protocols in non-communicable
diseases were introduced by single cell analysis. Moreover, non-invasive methods to maintain the
investigated cell for time dependent monitoring has been widely studied because of its importance in
some crucial cases such as drug resistance in cancer. Bioelectrical monitoring is one of such methods
Although the procedures reported based on electrical probing might not induce cell disruption, indirect
connection between recording electrodes and cell membrane (mostly in microfluidic approaches)
reduced the quality of response and limited the precision of the results. Here, a bioelectronic sensor for
monitoring the effect of anticancer drugs on single breast cancer cells was fabricated based on nano-
roughened gold electrodes on a quartz substrate applied direct contacts to cell membrane. Whole of the
surface except a microcircle surrounded the sensing region was passivated by overbaked photoresist
layer. Cells were dropped on the sensor without the assistance of any micropipette or microfluidic
systems and just individual regions for attachment of one cell has been opened on the sensing region
arrays. MCF-7 cancer cells were time tracked under the effect of Paclitaxel and Mebendazole anti-
tubulin drugs in low and high doses. Inducing non regulated depolymerization and polymerization in
tubulin structures of the single cancer cells were monitored by the electrical signals recorded before
and after drug treatment.
Highly sensitive UHPLC–MS/MS method for the simultaneous estimation of propafenone and its
metabolites 5-hydroxypropafenone and N-depropylpropafenone on human dried blood spots
technique and application to a pharmacokinetic study
Adinarayana Andy, Raja Reddy Kallem, Ramesh Mullangi, Divya Andy, J.V.L.N. Seshagiri Rao
ABSTRACT
A highly sensitive, rapid and selective UHPLC–MS/MS method has been developed and validated for
quantification of the propafenone (PF), 5-hydroxypropafenone (5-OHPF) and N-depropylpropafenone
(N-DPF) on human dried blood spot (DBS). The assay procedure involves a solid–liquid extraction of
PF, 5-OHPF and N-DPF and amlodipine (internal standard, I.S.) from dried human DBS cards using
water and acetonitrile. The chromatographic resolution was achieved on a BEH C18 column using a
gradient mobile phase consisting of 0.1% formic acid in water and acetonitrile with 0.1% formic acid
at flow rate of 0.6 mL/min. The UHPLC–MS/MS was operated under the multiple-reaction monitoring
mode using electrospray ionization. Total run time of analysis was 1.1 min and elution of PF, 5-OHPF,
N-DPF and I.S. occurred at 0.69, 0.6, 0.68 and 0.73 min, respectively. A detailed method validation
was performed as per the regulatory guidelines and the standard curves found to be linear in the range
of 5.11–1000 ng/mL for PF and 5-OHPF and 0.51–100 ng/mL for N-DPF with a correlation
coefficient of ≥0.99 for all the drugs. The intra- and inter-day accuracies were in the range of 95.6–107
and 93.5–103; 93.4–106 and 96.3–107 and 87.9–103 and 96.5–102%, for PF, 5-OHPF and N-DPF,
respectively. The intra- and inter-day precisions were in the range of 2.50–5.52 and 3.38–5.18; 2.16–
6.34 and 3.23–4.94 and 2.63–7.55 and 1.56–10.2%, for PF, 5-OHPF and N-DPF, respectively. The
validated assay method was successfully applied to a pharmacokinetic study in humans.
175
Simple determination of L-hydroxyproline in idiopathic pulmonary fibrosis lung tissues of rats
using non-extractive high-performance liquid chromatography coupled with fluorescence
detection after pre-column derivatization with novel synthetic 9-acetylimidazol-carbazole
Yan Ren, Juanjuan Zhao, Yanan Shi, Caiyun Chen, Changjun Lv
ABSTRACT
L-Hydroxyproline (L-Hyp) is an important biomarker for idiopathic pulmonary fibrosis (IPF). The
quantitative methods based on high-performance liquid chromatography coupled with fluorescence
detection after pre-column derivatization typically requires complicated derivatization conditions and
obtains unstable derivatives. Here, a novel derivatization reagent, 9-acetylimidazol-carbazole, was
synthesized for the first time to efficiently and rapidly label the amino groups of L-Hyp. The high-
performance liquid chromatography method with pre-column derivatization was performed on an
Agilent ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm). The product was measured using
fluorescence detection at excitation and emission wavelengths of 232 and 370 nm, respectively. The
method was validated in specificity, linearity, limit of detection (66.7 fmol), limit of quantification
(333.3 fmol), intra-day precision (0.75%), inter-day precision (3.82%), stability (3.15%), and recovery
(90.7–109.4%). The validated method was successfully applied to the determination of L-Hyp in the
lung tissues of healthy and IPF rats. The results showed that the concentration of L-Hyp (3.64 mg/g) in
the IPF model was significantly higher than the concentration (2.33 mg/g) in the healthy control group
with P < 0.01. This is a new method for the determination of L-Hyp and can be used for other amino
acid-related studies in the future.
A simple method for assaying colistimethate sodium in pharmaceutical aerosol samples using
high performance liquid chromatography
Adam P. Metcalf, Lucy E.A. Hardaker, Ross H.M. Hatley
ABSTRACT
A rapid and simple reversed-phase high performance liquid chromatography (HPLC) method for the
quantitation of colistimethate sodium in pharmaceutical formulations has been developed. The
chromatographic separation was performed using a Phenomenex Kinetex XB-C18 column with
gradient elution using a mobile phase containing acetonitrile and 32 mM sodium sulphate. Quantitation
is based on the sum of the areas of two prominent peaks in the chromatogram, which produces a total
peak area that is stable for 120 sample injections. The HPLC method was validated over the range
0.05–7 mg/mL, and was shown to be suitable for the analysis of aerosolised pharmaceuticals in terms
of aerosol output onto filter and for the analysis of samples from a cascade impactor, which is used for
the determination of aerosol particle size.
176
Quantification of apigenin trimethyl ether in rat plasma by liquid chromatography–tandem
mass spectrometry: Application to a pre-clinical pharmacokinetic study
Mai Gamal Elhennawy, Hai-Shu Lin
ABSTRACT
Apigenin trimethyl ether (5,7,4′-trimethoxyflavone, ATE) is a naturally occurring polymethoxyflavone
with a wide range of health-promoting activities. In this study, a sensitive liquid chromatography–
tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of
ATE in rat plasma. Protein precipitation was applied as plasma clean-up procedure; the electrospray
ionization was operated in its positive ion mode while ATE and formononetin (internal standard) were
measured by multiple reactions monitoring (ATE: m/z 313.1 → 298.1; formononetin: 269.2 → 213.3).
This LC–MS/MS method displayed good selectivity, sensitivity (lower limit of quantification = 2.5
ng/ml), accuracy (both intra- and inter-day analytical recovery within 100 ± 10%) and precision (both
intra- and inter-day RSD <10%). The matrix effect was found to be insignificant. The pharmacokinetic
profiles of ATE were subsequently examined in Sprague-Dawley rats after single oral administration
(10 mg/kg). When given in an aqueous suspension, ATE was slowly absorbed with quite low plasma
exposure (AUC). Fasting further attenuated its oral absorption and led to ∼70% drops in average
maximal plasma concentration (Cmax) and AUC. When dosed in a solution formulated with 2-
hydroxypropyl-β-cyclodextrin, the oral absorption of ATE was substantially improved with ∼500%
increases in average Cmax and AUC. Clearly, aqueous solubility has been identified as a barrier to the
oral absorption of ATE. The information obtained from this study will facilitate further medicinal
exploration on ATE.
Simultaneous analysis of regorafenib and sorafenib and three of their metabolites in human
plasma using LC–MS/MS
Marie Allard, Nihel Khoudour, Benoît Rousseau, Charlotte Joly, Anne Hulin
ABSTRACT
A new liquid chromatography-tandem mass spectrometry (LC–MS/MS) method, performed by
electrospray ionization in positive mode using a triple quadrupole mass spectrometry, has been
developed and validated for the simultaneous determination of regorafenib (REGO), its two
metabolites regorafenib-M2 and regorafenib-M5, sorafenib (SORA), and its N-oxide metabolite in
human plasma. Separation is achieved on an Hypersil Gold® column using a gradient elution of 10
mM ammonium formate containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid
(B) at a flow rate of 0.3 mL/min. After addition of two internal standards and a protein precipitation,
the supernatant is diluted two-fold in a 0.1% (v/v) formic acid solution. Two selected reaction
monitoring transitions are used, for each analyte, one for quantitation and the second one for
confirmation. The standard curves are ranged from 50 to 5 000 ng/mL for REGO and its metabolites
and 80 to 5 000 ng/mL for SORA and its metabolite and were fitted to a 1/x weighted linear regression
model. The method also showed satisfactory results in terms of sensitivity, specificity, precision (intra-
and inter-day CV from 2.4 to 10.2%), accuracy (from 91.0 to 111.7%), recovery as well as stability of
the analytes under various conditions. The method is usually used in clinical practice in order to
improve the SORA treatment for renal carcinoma, REGO treatment for colorectal cancer and both for
hepatocellular carcinoma.
177
Stability behavior of antiretroviral drugs and their combinations 7: Comparative degradation
pathways of lamivudine and emtricitabine and explanation to their differential degradation
behavior by density functional theory
Moolchand Kurmi, Saranjit Singh
ABSTRACT
The interest in this study was to establish comparative degradation behavior of lamivudine (3TC) and
emtricitabine (FTC) under solution and solid state stress conditions. Structurally, the two drugs differ
only in terms of additional fluorine at 5 position in FTC. Along with the known degradation products
of both the drugs, one additional degradation product was observed in each case, which was
characterized by mass spectrometry. Both the drugs degraded via the same route, but at a differential
rate in acid, base and oxidative stress conditions. The variable rate of degradation in acid and base
conditions was justified by the application of density functional theory (DFT).
An atmospheric pressure ionization source using a high voltage target compared to electrospray
ionization for the LC/MS analysis of pharmaceutical compounds
Arnaud Lubin, Ronald De Vries, Deirdre Cabooter, Patrick Augustijns, Filip Cuyckens
ABSTRACT
The type and design of an ionization source can have a significant influence on the performances of a
bioanalytical method. It is, therefore, of high interest to evaluate the performances of newly introduced
sources to highlight their benefits and limitations in comparison to other well established sources. In
this paper, liquid chromatography – mass spectrometry (LC/MS) performances of a new atmospheric
pressure ionization (API) source, commercialized as UniSpray, is evaluated. The dynamic range of 24
pharmaceutical and biological compounds is compared between the new API source and electrospray
ionization (ESI) for 3 different mobile phase conditions. Matrix effects are also compared with ESI on
a refined selection of 19 pharmaceutical and biological compounds in 4 matrices commonly
encountered in bioanalysis. A slightly better dynamic range towards lower concentrations was often
observed with the new API source. Matrix effects were quite similar between the two sources with a
small, but statistically significant, lower percentage of matrix effects observed for the new API source
in plasma and bile in the positive ion mode, and bile in negative ion mode for ESI. Finally, the
sensitivity of late eluting compounds could be improved on the new API source by post-column
addition of water.
178
UHPLC–MS/MS method with sample dilution to test therapeutic adherence through
quantification of ten antihypertensive drugs in urine samples
Amedeo De Nicolò, Valeria Avataneo, Franco Rabbia, Mauro Sciandra, Antonio D‘Avolio
ABSTRACT
Nowadays, hypertension represents an important health problem, particularly in developed countries.
In some cases the standard therapeutic approaches are not able to reestablish the normal blood pressure
values: this condition is called ―resistant hypertension‖. However, a fraction of cases of resistant
hypertension are actually due to poor adherence to the prescribed therapy. Therapeutic Drug
Monitoring could represent a direct and useful tool to correctly identify non-compliant patients.
Nevertheless, high throughput methods for the simultaneous monitoring of a wide panel of drugs in the
same analysis are lacking and, furthermore, there is not a generally acknowledged ―standard‖ matrix
for this test (plasma or urine). In this work, we validated a UHPLC–MS/MS assay to quantify ten
among the most used antihypertensive agents in urine samples, covering all the current classes:
amlodipine, atenolol, clonidine, chlortalidone, doxazosin, hydrochlorothiazide, nifedipine, olmesartan,
ramipril and telmisartan. Both standards and quality controls were prepared in urine matrix. Only 100
μL of each sample were added with 40 μL of internal standard and 860 μL of water:acetonitrile 90:10,
acidified with 0.05% formic acid. Chromatographic separation was performed on an Acquity® UPLC
HSS T3 1.8 μm 2.1 × 150 mm column, with a gradient of water and acetonitrile, both added with
0.05% formic acid. Accuracy, intra-day and inter-day precision fitted FDA guidelines for all analytes,
while matrix effects resulted reproducible among different urine lots. Method performances were
tested on urine samples from hypertensive patients with good results. This simple analytical method
could represent a useful tool for the management of antihypertensive therapy.
A novel carbohydrate labeling method utilizing transfer hydrogenation-mediated reductive
amination
Zsuzsanna Kovács, Gábor Papp, Henrietta Horváth, Ferenc Joó, András Guttman
ABSTRACT
One of the most frequently used high-resolution glycan analysis methods in the biopharmaceutical and
biomedical fields are capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection.
Glycans are usually labeled by reductive amination with a charged fluorophore containing a primary
amine, which reacts with the aldehyde group at the reducing end of the glycan structures. In this
reaction, first a Schiff base is formed that is reduced to form a stable conjugate by a hydrogenation
reagent, such as sodium cyanoborohydride. In large scale biopharmaceutical applications, such as
clone selection for glycoprotein therapeutics, hundreds of reactions are accomplished simultaneously,
so the HCN generated in the process poses a safety concern. To alleviate this issue, here we propose
catalytic hydrogen transfer from formic acid catalyzed by water-soluble iridium(III)- and
ruthenium(II)-phosphine complexes as a novel alternative to hydrogenation. The easily synthesized
water-soluble iridium(III) and the ruthenium(II) hydrido complexes showed high catalytic activity in
carbohydrate labeling. This procedure is environmentally friendly and reduces the health risks for the
industry. Using carbohydrate standards, oligosaccharides released from glycoproteins with highly
sialylated (fetuin), high mannose (ribonuclease B) and mixed sialo and neutral (human plasma) N-
glycans, we demonstrated similar labeling efficiencies for iridium(III) dihydride to that of the
conventionally used sodium cyanoborohydride based reaction. The derivatization reaction time was
less than 20 min with no bias towards the above mentioned specific glycan structures.
179
Non-volatile extractable analysis of prefilled syringes for parenteral administration of drug
products
Noemí Dorival-García, Iben Larsson, Jonathan Bones
ABSTRACT
The determination of extractable profiles for single-use technologies represents an important aspect of
pharmaceutical production to minimize any possible compromise in drug product quality or potential
risk to patients by identifying substances that may potentially leach from such devices. An approach
for the extractable assessment of prefilled syringes, a promising alternative for parenteral
administration of pharmaceutical products, is described herein. Four extraction solvents were selected:
a mixture 2-propanol:water (1:1), was intended to represent aggressive conditions to extract a broad
spectrum of extractables, including organic additives and substances which are poorly water-soluble.
Extractions with buffers at three different working pH values spanning a range standardly used in
pharmaceutical formulations were also evaluated to identify substances that require specific conditions
for their extraction due to their individual chemical properties. Syringes from two different brands
were analysed along with their corresponding plunger stoppers. Syringes were extracted at 40 °C for 4
days, the plunger stoppers were extracted with 2-propanol at 70 °C for 24 h according to ISO 10993-
12:2012. Extractables were identified by UHPLC–MS on a quadrupole time of flight instrument using
a non-targeted discovery strategy. A total of 25 compounds were identified, mostly polymer additives
and their degradation products. The presented methodology represents a reference point for further
studies focused on the characterisation of extractables and leachables from prefilled syringes.
A rapid microextraction by packed sorbent − liquid chromatography tandem mass spectrometry
method for the determination of dexamethasone disodium phosphate and dexamethasone in
aqueous humor of patients with uveitis
Federica Bianchi, Monica Mattarozzi, Nicolò Riboni, Paolo Mora, Maria Careri
ABSTRACT
A new method based on microextraction by packed sorbent (MEPS) coupled with liquid
chromatography-tandem mass spectrometry (LC–MS/MS) was developed and validated for the
determination of dexamethasone and dexamethasone disodium phosphate in human aqueous humor. A
central composite design was applied to investigate the effects of both loading and eluting cycles in the
MEPS procedure; subsequently the multicriteria method of the desirability functions allowed to find
the best conditions for the simultaneous extraction of both the analytes. Detection was performed on a
LTQ XL linear ion trap mass spectrometer operating in the positive electrospray ionization mode
applying multiple reaction monitoring mode. The assay was validated in accordance with the
guidelines bioanalytical method validation obtaining quantitation limits in the low μg/L range, a
precision characterized by RSD ≤ 16% and recovery rates in the 91–119% range.
180
Isotope-coded derivatization based LC/ESI-MS/MS methods using a pair of novel reagents for
quantification of hydroxycinnamic acids and hydroxybenzoic acids in fermented brown rice
product
Shoujiro Ogawa, Kiriko Takafuji, Sumi Tsubuku, Yukiko Horie, Tatsuya Higashi
ABSTRACT
Hydroxycinnamic acids (HCAs) and hydroxybenzoic acids (HBAs) are antioxidant phytochemicals
found in rice and effective for the prevention of human diseases including cancer. FBRA, which is a
functional food manufactured by fermenting brown rice and rice bran with Aspergillus oryzae, has
been demonstrated to have chemopreventive effects against carcinogenesis in various organs. In this
study, we developed methods for the relative and absolute quantification of ferulic acid, sinapic acid,
caffeic acid, protocatechuic acid and syringic acid in the FBRA and raw material (RM; unfermented
brown rice and rice bran) samples by LC/ESI-MS/MS combined with derivatization using a newly
developed reagent, N-(2-aminoethyl)-4-(diethylamino)benzamide (ADB) and its deuterium-coded
analog, d-ADB. For the relative quantification, the FBRA and RM samples were derivatized with
ADB and d-ADB, respectively, then the resulting derivatives were mixed and subjected to LC/ESI-
MS/MS; by this method, we found that the fermentation process significantly increased the free HCA
and HBA contents. The HCA and HBA contents in the FBRA were also determined, in which the d-
ADB-derivatized standards of known amounts were used as the internal standards. The ADB-
derivatization enabled the sensitive and specific detection, and the use of d-ADB significantly
improved the assay precision.
Rapid discovery of cyclopamine analogs from Fritillaria and Veratrum plants using LC-Q-TOF-
MS and LC-QqQ-MS
Yuan Du, Zu-Guo Zheng, Yue Yu, Zi-Tian Wu, Hui-Jun Li
ABSTRACT
Cyclopamine, an inhibitor of the Hedgehog (Hh) signaling pathway, has been paid much attention in
treating a wide variety of tumors. However, isolation and purification of cyclopamine analogs from
medicinal plants remain challengeable. We herein proposed an efficient strategy using liquid
chromatography quadrupole-time-of-flight mass spectrometry (LC-Q-TOF-MS) and liquid
chromatography triple-quadrupole mass spectrometry (LC-QqQ-MS) for rapid screening and targeted
isolation of cyclopamine analogs in Fritillaria and Veratrum plants. Firstly, fifteen reference
compounds were characterized by LC-Q-TOF-MS and their characteristic fragment ions were
summarized. Secondly, according to the characteristic fragment ions at m/z 67.1, 84.1, 109.1 and
114.1, rapid chemical screening of plant extracts was carried out by LC-QqQ-MS using precursor ion
scan mode and 69 pre-target compounds were screened out. Thirdly, 24 real target compounds were
verified by LC-Q-TOF-MS based on relative abundances (over 20%) of characteristic fragment ions.
Fourthly, the targeted isolation of Fritillaria ussuriensis bulb and Veratrum dahuricum rhizome
afforded a novel cyclopamine analog namely 15β-hydroxy-23-isopengbeisine B as well as four known
ones, whose structures were determined by nuclear magnetic resonance (NMR) analysis. Additionally,
these five analogs were evaluated for the inhibitory activity of Hh signaling pathway in NIH/3T3 cell
and cytotoxicity in PANC-1 and HepG2 cells. These results indicated that the proposed strategy was
reliable for rapid discovery and targeted isolation of important natural products from chemically
complex plant matrices.
181
Development and validation of a HS/GC–MS method for the simultaneous analysis of diacetyl
and acetylpropionyl in electronic cigarette refills
Sophia Barhdadi, Michaël Canfyn, Patricia Courselle, Vera Rogiers, Eric Deconinck
ABSTRACT
The use of e-cigarettes as alternative for tobacco cigarettes has become increasingly popular, even
though their safety has not yet been scientifically established. One of the frequently raised concerns is
the potential toxicity of certain flavours present in the e-liquids, such as diacetyl and acetylpropionyl.
It is therefore important to be able to identify and quantify both compounds. Numerous analytical
methods have been published for determining e-liquid compositions, but concerns exist with respect to
the lack of analytical evaluation. Hence in this study, a new HS/GC–MS-based method was developed
for the screening and quantification of diacetyl and acetylpropionyl in e-liquids. This method was fully
validated using the ‗total error‘ approach. The LOQ of the analytical method was 5 ppm for diacetyl
and acetylpropionyl. The obtained accuracy profiles show that the β-expectation tolerance intervals did
not exceed the acceptance limits of ± 10%, meaning that 95% of future measurements will be included
in the [-10%, 10%] bias limits. As proof of applicability, the validated method was successfully
applied on a small set of e-liquid samples, indicating that this methodology could be used for routine
quality control analyses of e-liquids.
Structural characterization and discrimination of the Paris polyphylla var. yunnanensis and
Paris vietnamensis based on metabolite profiling analysis
Li-ping Kang, Yuan-yuan Huang, Zhi-lai Zhan, Da-hui Liu, Lu-qi Huang
ABSTRACT
This study aimed to distinguish the rhizomes of Paris polyphylla var. yunnanensis (Franch) Hand
Mazz (PPY) and Paris veitnamensis (Takht.) H. Li (PV) using metabolomics-based ultra high-
performance liquid chromatography coupled with quadrupole time-of-fligh mass spectrometry
(UHPLC/Q-TOF MS). First, the UHPLC/Q-TOF MS approach was optimized for metabolite profiling.
Then, the MS data were processed using UNIFI™ combined with an in-house library to automatically
characterize the metabolites. Based on the exact mass information, the fragmentation characteristics,
and the retention time of compounds, and the fragmentation mechanism and retention behavior of
steroidal glycosides in the references, the structures identified by UNIFI were further verified. Overall,
146 metabolites, including 42 potential new compounds, were identified or tentatively identified.
Pattern recognition analysis of the PPY and PV MS data revealed that they were clearly separated, and
15 potential biomarkers for differentiating between them were selected. These biomarkers were
subsequently used to successfully predict the genus of PPY and PV samples. These results indicated
that metabolite profiling by UHPLC/Q-TOF MS is an effective, robust approach for determining the
characteristic biomarkers that differentiate between TCM species with multiple botanical origins.
182
Volume 143 September 2017
Reliability of point-of-collection testing devices for drugs of abuse in oral fluid: A systematic
review and meta-analysis
Juliana Nichterwitz Scherer, Taís Regina Fiorentin, Bruna Tassi Borille, Graciela Pasa, Flavio
Pechansky
ABSTRACT
Point-of-collection testing (POCT) devices for drugs of abuse are used to screen for the presence of
psychoactive substances (PAS) in different types of settings and environments. However, these quick
and advantageous tools also present disadvantages, including low-reliability measures in comparison
to chromatographic assays. Therefore, this article presents a systematic review and meta-analysis of
studies evaluating the reliability of measurements of PAS detection in oral fluid using POCT devices.
The reliability measures for detection of the five most important drug classes – cocaine,
amphetamines, benzodiazepines, cannabinoids and opioids, are reported. The article also presents a
subgroup analysis considering the reliability estimates for the different POCT devices that were
evaluated by the studies contemplated in the review. A discussion considering the strengths and
limitations of POCT techniques was performed in order to guide policymakers, traffic agents and other
professionals who also conduct such tests. The use of POCT devices often involves legal and moral
aspects of the subjects tested, which demands critical evaluation of these devices before they are
implemented in different settings.
High-throughput thermofluor-based assays for inhibitor screening of STAT SH2 domains
Elvin D. de Araujo, Pimyupa Manaswiyoungkul, Johan Israelian, Jisung Park, Patrick T. Gunning
ABSTRACT
The development of STAT protein-specific inhibitors has been the focus of a number of drug
discovery programs. STAT activation occurs through phosphorylation at the STAT SH2 domain,
resulting in dimerization, translocation to the nucleus, and transcription of proliferative genes. Due to
the functional significance of the SH2 domain in mediating multiple components of the STAT
signalling cascade, many libraries of inhibitors have been designed to target the SH2 domain. This has
triggered the requirement for effective high-throughput screening platforms for analyzing binding by
larger chemical libraries to STAT proteins. Herein, we present strategies for the development of a
high-throughput thermal denaturation-based assay for identifying STAT inhibitors as well as high-
yielding recombinant expression and purification of untagged STAT1, STAT3, and STAT5 proteins.
This assay reports changes in the fluorescence of a labelled peptide bound to the STAT protein as a
function of increasing temperature. STAT inhibitors which displace the labelled peptide elicit a change
in the melt profile, which is quantitatively determined as a change in the area under the curve. This
assay offers an alternative, but complimentary, high-throughput screening strategy for identifying new
inhibitors of STAT proteins as well as characterizing further, the mode of inhibition by existing
libraries of compounds.
183
Trace level determination of 5-hydroxytryptamine and its related indoles in amniotic fluid by
gas chromatography–mass spectrometry
Hongmei Shi, Bo Wang, Lingmei Niu, Mengsi Cao, Pingping Zhang
ABSTRACT
5-hydroxytryptamine (5-HT) and its derivatives are endogenously active substances involved in
multiple physiological and pathological processes. A novel method of detetermining 5-hydroxyindole
ethanol (5-HTOL), 5-hydroxyindole acetic acid (5-HIAA), 5-hydroxytryptophan (5-HTP) and 5-HT in
amniotic fluid by gas chromatography-mass spectrometry (GC–MS) was established based on a
modified method of derivatization by silanization, in combination with solid-phase extraction
pretreatment. Good linearity was achieved in the tested calibration range. The limits of detection
(LOD) were 0.05, 0.08, 0.56, 0.43 μg/L for 5-HTOL, 5-HIAA, 5-HTP and 5-HT, respectively.
Accuracy (92.4-103.3) and precision (RSD < 5.4%) for all analytes was also determined. Then the
method was used to analyze samples of amniotic fluid from 12 patients carrying foetuses with trisomy
21 and 12 healthy controls. Compared with normal fetuses, the levels of 5-HTOL, 5-HTP and 5-HT in
the amniotic fluid were significantly altered in the fetuses with trisomy 21 (P < 0.01); the level of 5-
HIAA showed no significant difference between the two groups (P>0.05). This is a rapid, sensitive and
reliable method for the determination of 5-HTOL, 5-HIAA, 5-HTP and 5-HT, and the study provide
both potential trisomy 21 markers and elucidation of the physiological and pathological roles of 5-HT.
Identification of related substances in tofacitinib citrate by LC-MS techniques for synthetic
process optimization
Xiao Wu, Xuefang Zeng, Lei Wang, Taijun Hang, Min Song
ABSTRACT
A specific LC-MS method was developed for separation, identification and characterization of the
process-related substances and degradation products in tofacitinib citrate. The separation was achieved
on a LiChrospher C18 column (250 mm × 4.6 mm, 5 μm) by linear gradient elution of 0.1%
ammonium acetate solution (pH adjusted to 4.0 by formic acid) and acetonitrile at a flow rate of 1.0
mL/min. Forced degradation studies were conducted under hydrolytic (acidic, basic), oxidative,
photolytic and thermal stress conditions as described in ICH. It was found that tofacitinib was stable
under photolytic condition, but degraded obviously in acidic, basic, thermal and oxidative conditions.
The high resolution TOF-MS and MS/MS were used for determination and structural identification of
the related substances. Eleven major related substances were detected and identified as five process-
related substances and six degradation products, and three of them were further synthesized and
characterized by NMR spectroscopy. The most plausible mechanisms involved in the formation of the
related substances were also proposed. Since related substances have a significant impact on drug
safety, quality and efficacy, the data obtained are valuable for process monitoring and quality
assurance of tofacitinib citrate.
184
Macro- and microstructural tracking of ageing-related changes of papaverine hydrochloride-
loaded electrospun nanofibrous buccal sheets
Adrienn Kazsoki, Péter Szabó, Károly Süvegh, Tamás Vörös, Romána Zelkó
ABSTRACT
Electrospun papaverine hydrochloride-loaded nanofibrous sheets consist of hydroxypropyl
cellulose/poly(vinyl alcohol) composite were prepared for buccal administration for cerebral ischemia.
The nanofibrous drug delivery system was subjected to accelerated stability test for four weeks in
order to scrutinize the solid state changes relating to the stress induced (40 ± 2 °C/75 ± 5% relative
humidity) physical ageing. Micro- and macrostructural alterations were detected using scanning
electron microscopy (SEM), Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR) and
positron annihilation lifetime spectroscopy (PALS). Significant changes were revealed at both
supramolecular and macroscopic levels. Microscopic morphology uncovered major morphological
transitions. Subtle variations of Raman and FTIR spectra indicated that the local chemical environment
of papaverine was altered suggesting a partial phase transition of the active. Discrete o-Ps lifetimes and
lifetime-distributions unveiled a two-step ageing process of the drug carrier. In addition to the tracking
of the glassy-to-rubbery transition of the fiber forming polymers, the Raman spectroscopy enabled
monitoring the kinetics of the phase transition observed.
A simplified guide for charged aerosol detection of non-chromophoric compounds—Analytical
method development and validation for the HPLC assay of aerosol particle size distribution for
amikacin
Arianne Soliven, Imad A. Haidar Ahmad, James Tam, Nani Kadrichu, Andrei Blasko
ABSTRACT
Amikacin, an aminoglycoside antibiotic lacking a UV chromophore, was developed into a drug
product for delivery by inhalation. A robust method for amikacin assay analysis and aerosol particle
size distribution (aPSD) determination, with comparable performance to the conventional UV detector
was developed using a charged aerosol detector (CAD). The CAD approach involved more parameters
for optimization than UV detection due to its sensitivity to trace impurities, non-linear response and
narrow dynamic range of signal versus concentration. Through careful selection of the power
transformation function value and evaporation temperature, a wider linear dynamic range, improved
signal-to-noise ratio and high repeatability were obtained. The influences of mobile phase grade and
glassware binding of amikacin during sample preparation were addressed. A weighed (1/X2) least
square regression was used for the calibration curve. The limit of quantitation (LOQ) and limit of
detection (LOD) for this method were determined to be 5 μg/mL and 2 μg/mL, respectively. The
method was validated over a concentration range of 0.05–2 mg/mL. The correlation coefficient for the
peak area versus concentration was 1.00 and the y-intercept was 0.2%. The recovery accuracies of
triplicate preparations at 0.05, 1.0, and 2.0 mg/mL were in the range of 100–101%. The relative
standard deviation (Srel) of six replicates at 1.0 mg/mL was 1%, and Srel of five injections at the limit
of quantitation was 4%. A robust HPLC-CAD method was developed and validated for the
determination of the aPSD for amikacin. The CAD method development produced a simplified
procedure with minimal variability in results during: routine operation, transfer from one instrument to
another, and between different analysts.
185
Proton dissociation properties of arylphosphonates: Determination of accurate Hammett
equation parameters
Gergő Dargó, Adrienn Bölcskei, Alajos Grün, Szabolcs Béni, György T. Balogh
ABSTRACT
Determination of the proton dissociation constants of several arylphosphonic acid derivatives was
carried out to investigate the accuracy of the Hammett equations available for this family of
compounds. For the measurement of the pKa values modern, accurate methods, such as the differential
potentiometric titration and NMR-pH titration were used. We found our results significantly different
from the pKa values reported before (pKa1: MAE = 0.16 pKa2: MAE = 0.59). Based on our recently
measured pKa values, refined Hammett equations were determined that might be used for predicting
highly accurate ionization constants of newly synthesized compounds (pKa1 = 1.70–0.894σ, pKa2 =
6.92–0.934σ).
Sub–1 min separation in sequential injection chromatography for determination of synthetic
water-soluble dyes in pharmaceutical formulation
Polina Davletbaeva, Petr Chocholouń, Andrey Bulatov, Dalibor Ńatínský, Petr Solich
ABSTRACT
Sequential Injection Chromatography (SIC) evolved from fast and automated non-separation
Sequential Injection Analysis (SIA) into chromatographic separation method for multi-element
analysis. However, the speed of the measurement (sample throughput) is due to chromatography
significantly reduced. In this paper, a sub–1 min separation using medium polar cyano monolithic
column (5 mm × 4.6 mm) resulted in fast and green separation with sample throughput comparable
with non-separation flow methods The separation of three synthetic water-soluble dyes (sunset yellow
FCF, carmoisine and green S) was in a gradient elution mode (0.02% ammonium acetate, pH 6.7 –
water) with flow rate of 3.0 mL min−1 corresponding with sample throughput of 30 h−1.
Spectrophotometric detection wavelengths were set to 480, 516 and 630 nm and 10 Hz data collection
rate. The performance of the separation was described and discussed (peak capacities 3.48–7.67, peak
symmetries 1.72–1.84 and resolutions 1.42–1.88). The method was represented by validation
parameters: LODs of 0.15-0.35 mg L−1, LOQs of 0.50–1.25 mg L−1, calibration ranges 0.50-150.00
mg L−1 (r > 0.998) and repeatability at 10.0 mg L−1 of RSD ≤ 0.98% (n = 6). The method was used
for determination of the dyes in ―forest berries‖ colored pharmaceutical cough-cold formulation. The
sample matrix – pharmaceuticals and excipients were not interfering with vis determination because of
no retention in the separation column and colorless nature. The results proved the concept of fast and
green chromatography approach using very short medium polar monolithic column in SIC.
186
Impurity profiling of liothyronine sodium by means of reversed phase HPLC, high resolution
mass spectrometry, on-line H/D exchange and UV/Vis absorption
M. Ruggenthaler, J. Grass, W. Schuh, C.G. Huber, R.J. Reischl
ABSTRACT
For the first time, a comprehensive investigation of the impurity profile of the synthetic thyroid API
(active pharmaceutical ingredient) liothyronine sodium (LT3Na) was performed by using reversed
phase HPLC and advanced structural elucidation techniques including high resolution tandem mass
spectrometry (HRMS/MS) and on-line hydrogen-deuterium (H/D) exchange. Overall, 39 compounds
were characterized and 25 of these related substances were previously unknown to literature. The
impurity classification system recently developed for the closely related API levothyroxine sodium
(LT4Na) could be applied to the newly characterized liothyronine sodium impurities resulting in a
wholistic thyroid API impurity classification system. Furthermore, the mass-spectrometric CID-
fragmentation of specific related substances was discussed and rationalized by detailed fragmentation
pathways. Moreover, the UV/Vis absorption characteristics of the API and selected impurities were
investigated to corroborate chemical structure assignments derived from MS data.
A UHPLC method for the rapid separation and quantification of anthocyanins in acai berry and
dry blueberry extracts
Jakub Fibigr, Dalibor Ńatínský, Petr Solich
ABSTRACT
The presented work describes the development and validation of a rapid UHPLC-UV method using a
core-shell particle column with a pentafluorophenyl stationary phase for the separation and
quantitative analysis of the six anthocyanins in acai berry and dry blueberry extracts. The anthocyanins
(cyanidin-3-glucoside, cyanidin-3-rutenoside, delphinidin-3-galactoside, delphinidin-3-glucoside,
delphinidin-3-rutenoside, and peonidin-3-glucoside) were separated and analyzed in 5 min. The
chromatographic separation was performed on a Kinetex PFP (150 × 2.1 mm) core-shell column with a
particle size of 1.7 μm at a temperature of 50 °C. Acetonitrile was used as mobile phase B and 5%
formic acid, filtrated through a 0.22 μm filter, as mobile phase A. They were delivered at a flow rate of
0.55 mL min−1 according to the elution gradient program. The detection wavelength was set at 520
nm. A solid-liquid extraction with a solution of methanol and a 5% water solution of formic acid (25 +
75 v/v) using an ultrasonic bath was chosen for the preparation of the available commercial samples of
food supplements with a content of acai berry extract and blueberry extract. Under optimal
chromatographic conditions, the method was validated. Recoveries for all analyzed anthocyanins were
97.8–102.6% and the relative standard deviation ranged from 0.4% to 3.0% for within-day and from
0.6% to 3.1% for between-day repeatability. The limits of detection were in the range of 0.11–0.14 μg
mL−1.
187
Combined computational-experimental approach to predict blood–brain barrier (BBB)
permeation based on ―green‖ salting-out thin layer chromatography supported by simple
molecular descriptors
Krzesimir Ciura, Mariusz Belka, Piotr Kawczak, Tomasz Bączek, Joanna Nowakowska
ABSTRACT
The objective of this paper is to build QSRR/QSAR model for predicting the blood–brain barrier
(BBB) permeability. The obtained models are based on salting-out thin layer chromatography
(SOTLC) constants and calculated molecular descriptors. Among chromatographic methods SOTLC
was chosen, since the mobile phases are free of organic solvent. As consequences, there are less toxic,
and have lower environmental impact compared to classical reserved phases liquid chromatography
(RPLC). During the study three stationary phase silica gel, cellulose plates and neutral aluminum oxide
were examined. The model set of solutes presents a wide range of log BB values, containing
compounds which cross the BBB readily and molecules poorly distributed to the brain including drugs
acting on the nervous system as well as peripheral acting drugs. Additionally, the comparison of three
regression models: multiple linear regression (MLR), partial least-squares (PLS) and orthogonal partial
least squares (OPLS) were performed. The designed QSRR/QSAR models could be useful to predict
BBB of systematically synthesized newly compounds in the drug development pipeline and are
attractive alternatives of time-consuming and demanding directed methods for log BB measurement.
The study also shown that among several regression techniques, significant differences can be obtained
in models performance, measured by R2 and Q2, hence it is strongly suggested to evaluate all
available options as MLR, PLS and OPLS.
Development of a new extraction technique and HPLC method for the analysis of non-
psychoactive cannabinoids in fibre-type Cannabis sativa L. (hemp)
Virginia Brighenti, Federica Pellati, Marleen Steinbach, Davide Maran, Stefania Benvenuti
ABSTRACT
The present work was aimed at the development and validation of a new, efficient and reliable
technique for the analysis of the main non-psychoactive cannabinoids in fibre-type Cannabis sativa L.
(hemp) inflorescences belonging to different varieties. This study was designed to identify samples
with a high content of bioactive compounds, with a view to underscoring the importance of quality
control in derived products as well. Different extraction methods, including dynamic maceration
(DM), ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE) and supercritical-
fluid extraction (SFE) were applied and compared in order to obtain a high yield of the target analytes
from hemp. Dynamic maceration for 45 min with ethanol (EtOH) at room temperature proved to be the
most suitable technique for the extraction of cannabinoids in hemp samples. The analysis of the target
analytes in hemp extracts was carried out by developing a new reversed-phase high-performance liquid
chromatography (HPLC) method coupled with diode array (UV/DAD) and electrospray ionization-
mass spectrometry (ESI–MS) detection, by using an ion trap mass analyser. An Ascentis Express C18
column (150 mm × 3.0 mm I.D., 2.7 μm) was selected for the HPLC analysis, with a mobile phase
composed of 0.1% formic acid in both water and acetonitrile, under gradient elution. The application
of the fused-core technology allowed us to obtain a significant improvement of the HPLC performance
compared with that of conventional particulate stationary phases, with a shorter analysis time and a
remarkable reduction of solvent usage.
188
Analytical profiling of selected antioxidants and total antioxidant capacity of goji (Lycium spp.)
berries
Michele Protti, Isacco Gualandi, Roberto Mandrioli, Sergio Zappoli, Laura Mercolini
ABSTRACT
Goji berries and derived products represent a relevant source of micronutrients, most of which are
natural antioxidants and contribute to the high nutritional quality of these fruits. Three brands of dried
goji berries have been analysed by a multidisciplinary approach to get an insight into both their content
of selected antioxidants and their antioxidant capacity (AC). The former goal has been achieved by
developing a liquid chromatographic method coupled to mass spectrometry and combined to a fast
solid phase extraction. Several significant representative antioxidant compounds belonging to the
following classes: flavonoids, flavan-3-ols, phenolic acids, amino acids and derivatives, and
carotenoids have been taken into account. Quercetin and rutin were found to be the predominant
flavonoids, chlorogenic acid was the most abundant phenolic acid and zeaxanthin was the major
carotenoid. The AC of the goji berries has been evaluated by four analytical methods in order to
estimate the contributions of different reactions involved in radicals scavenging. In particular, AC has
been determined using 3 standardised methods (DPPH, ABTS, ORAC) and a recently proposed
electrochemical method, which measures the scavenging activity of antioxidants towards OH radicals
generated both by hydrogen peroxide photolysis and the Fenton reaction. The results obtained from
chemical composition and antioxidant capacity assays confirm the high nutritional and commercial
value of goji berries and highlight that the three brands do not exhibit significant differences.
Characterization and inhibition studies of tissue nonspecific alkaline phosphatase by
aminoalkanol derivatives of 1,7-dimethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5,10-
trione, new competitive and non-competitive inhibitors, by capillary electrophoresis
Błażej Grodner, Mariola Napiórkowska
ABSTRACT
The article describes the inhibitory effect of two new aminoalkanol derivatives on the enzymatic
kinetic of tissue non-specific alkaline phosphatase with use of capillary zone electrophoresis to
evaluate the inhibitory effect. This technique allows to investigate of the enzymatic kinetic by the
measure of the amounts of the substrate and product in the presence of compound (I) or (II) in the
reaction mixture. The separation process was conducted using an eCAP fused-silica capillary. The
detector was set at 200 nm. The best parameters for the analysis were: 25 mM sodium dihydrogen
phosphate adjusted to pH = 2.5, temperature 25 °C, and voltage −15 kV. Lineweaver-Burk plots were
constructed and determined by comparison of the Km, of alkaline phosphatase in the presence of
inhibitor (I) or (II) with the Km in a solution without inhibitor. The influence of replacement the
propylamine group by the dimethylamine group on tissue non-specific alkaline phosphatase inhibition
activity of new derivatives (I) and (II) was investigated. The tested compounds (I) and (II) were found
to be tissue non-specific alkaline phosphatase inhibitors. Detailed kinetic studies indicated a
competitive mode of inhibition against tissue non-specific alkaline phosphatase for compound (I) and
non-competitive mode of inhibition for compound (II).
189
A validated UHPLC-QTOF-MS method for quantification of metformin and teneligliptin in rat
plasma: Application to pharmacokinetic interaction study
David Paul, Lingesh Allakonda, Nanjappan Satheeshkumar
ABSTRACT
In this study a sensitive UHPLC-QTOF-MS method was developed and validated for the quantitation
of the metformin (MET) and teneligliptin (TEN) in rat plasma using dapagliflozin as an internal
standard (IS). Chromatographic separation were carried out on a Acquity UPLC HSS Cyano column
(100 mm x 2.1 mm, 1.8 μm) using gradient mobile phase system consisting of 0.1% formic acid and
acetonitrile at a flow rate of 0.4 mL/min, within a run time of 6 min. Protein precipitation method was
used as sample preparation approach. Detection of target ions [M+H]+ at m/z 130.1085 for MET, m/z
427.2277 for TEN and m/z 409.1623 for IS was performed at positive ion electrospray ionization
mode. Linearity was assessed in the range of 0.98–1000 ng/mL for both MET and TEN. The
developed assay was validated as per US-FDA and EMA bioanalytical guidelines and successfully
applied to pharmacokinetic interaction study in SD rats. A 1.1 fold increment in the AUC levels of
MET and TEN was observed when co-administered together in rats.
Overcoming interference with the detection of a stable isotopically labeled microtracer in the
evaluation of beclabuvir absolute bioavailability using a concomitant microtracer approach
Hao Jiang, Craig Titsch, Jianing Zeng, Barry Jones, Mark E. Arnold
ABSTRACT
The oral absolute bioavailability of beclabuvir in healthy subjects was determined using a microdose
(100 μg) of the stable isotopically labeled tracer via intravenous (IV) infusion started after oral dosing
of beclabuvir (150 mg). To simultaneously analyze the concentrations of the IV microtracer
([13C6]beclabuvir) and beclabuvir in plasma samples, a liquid chromatography-triple quadrupole mass
spectrometry (LC–MS/MS) method was initially developed. Surprisingly beclabuvir significantly
interfered with the IV microtracer detection when using the selected reaction monitoring (SRM) in the
assay. An interfering component from the drug substance was observed using a high resolution mass
spectrometer (HRMS). The mass-to-charge (m/z) of the interfering component was −32 ppm different
from the nominal value for the IV microtracer and thus could not be differentiated in the SRM assay
by the unit mass resolution. To overcome this interference, we evaluated two approaches by either
monitoring an alternative product ion using the SRM assay or isolating the interfering component
using the parallel reaction monitoring (PRM) assay on the HRMS. This case study has demonstrated
two practical approaches for overcoming interferences with the detection of stable isotopically labeled
IV microtracers in the evaluation of absolute bioavailability, which provides users the flexibility in
using either LC–MS/MS or HRMS to mitigate unpredicted interferences in the assay to support
microtracer absolute bioavailability studies.
190
Ex-vivo measurement of scalp follicular infundibulum delivery of zinc pyrithione and climbazole
from an anti-dandruff shampoo
Guoqiang Chen, Chengdong Ji, Miao Miao, Kang Yang, Hans-Gerd Janssen
ABSTRACT
Efficient delivery of anti-dandruff (AD) actives into the scalp follicular infundibulum as well as onto
the scalp surface is critical for the efficacy of AD shampoos. A method involving scalp cyanoacrylate
(CA) biopsy sampling, a tailor made cutting device, ultra-high-performance liquid chromatography–
tandem mass spectrometry (UHPLC–MS/MS) analysis, scanning electron microscopy (SEM)
measurement and Raman imaging has been developed for the measurement of delivery of zinc
pyrithione (ZPT) and climbazole (CBZ) from an AD shampoo into the scalp follicular infundibulum.
Scalp CA biopsy enables the sampling of ZPT and CBZ delivered into the scalp follicular infundibula
as well as onto the scalp surface. Raman imaging of scalp CA biopsy samples allows the visualization
of the spatial distribution of ZPT and CBZ deposited on the scalp. A tailor made cutting device enables
the separation of the scalp follicular infundibulum sample (20 μm below the scalp surface) from the
scalp surface samples (including top 20 μm of infundibula). UHPLC–MS/MS was used as a sensitive
and specific methodology enabling the quantification of ZPT and CBZ without interference. Using this
method, both ZPT and CBZ were successfully quantified and spacially visualized within the scalp
follicular infundibulum, after scalp was washed with an AD shampoo.
Pooled human liver preparations, HepaRG, or HepG2 cell lines for metabolism studies of new
psychoactive substances? A study using MDMA, MDBD, butylone, MDPPP, MDPV, MDPB, 5-
MAPB, and 5-API as examples
Lilian H.J. Richter, Veit Flockerzi, Hans H. Maurer, Markus R. Meyer
ABSTRACT
Metabolism studies play an important role in clinical and forensic toxicology. Because of potential
species differences in metabolism, human samples are best suitable for elucidating metabolism.
However, in the case of new psychoactive substances (NPS), human samples of controlled studies are
not available. Primary human hepatocytes have been described as gold standard for in vitro
metabolism studies, but there are some disadvantages such as high costs, limited availability, and
variability of metabolic enzymes. Therefore, the aim of our study was to investigate and compare the
metabolism of six methylenedioxy derivatives (MDMA, MDBD, butylone, MDPPP, MDPV, MDPB)
and two bioisosteric analogues (5-MAPB, 5-API) using pooled human liver microsomes (pHLM)
combined with cytosol (pHLC) or pooled human liver S9 fraction (pS9) all after addition of co-
substrates for six phase I and II reactions. In addition, HepaRG and HepG2 cell lines were used.
Results of the different in vitro tools were compared to each other, to corresponding published data,
and to metabolites identified in human urine after consumption of MDMA, MDPV, or 5-MAPB.
Incubations with pHLM plus pHLC showed similar results as pS9. A more cost efficient model for
prediction of targets for toxicological screening procedures in human urine should be identified. As
expected, the incubations with HepaRG provided better results than those with HepG2 concerning
number and signal abundance of the metabolites. Due to easy handling without special equipment,
incubations with pooled liver preparations should be the most suitable alternative to find targets for
toxicological screening procedures for methylenedioxy derivatives and bioisosteric analogues.
191
Evaluation of pharmacokinetics and blood-brain barrier permeability of mitragynine using in
vivo microdialysis technique
Wai Mun Kong, Zahurin Mohamed, Mohammed A. Alshawsh, Zamri Chik
ABSTRACT
A microdialysis system coupled with a sensitive ultra-fast liquid chromatography–mass spectrometry
(UFLC-MS) method was developed for the pharmacokinetic analysis of mitragynine in rat blood and
striatum. Mitragynine is an active alkaloid of Mitragyna speciosa and has been proposed to be used for
opioid withdrawal therapy. In this study, chromatographic separation was performed in a gradient
elution mode with 0.1% formic acid and acetonitrile on a Zorbax Eclipse C18 column. The mass
spectrometric (MS) analysis was carried out in a positive electrospray mode and mitragynine ion (m/z
399.2) was monitored in extracted ion chromatography. A good linearity range was obtained from 10-
1000 ng/mL with acceptable accuracy and precision parameters. The microdialysate was collected
simultaneously from the striatum and the right jugular vein using microdialysis probes. After a single
intravenous administration of 10 mg/kg mitragynine, mitragynine showed a two-compartmental drug
elimination pattern with half-life (T1/2) of approximately 13 h. The percent of AUCbrain/AUCplasma
of mitragynine was calculated and shown to be 65.8 ± 4.5%. The results indicated that mitragynine
could be a suitable molecule to develop into an opioid replacement drug based on its ideal
pharmacokinetic properties, namely, small molecular size, lipophilic in nature and with excellent
blood–brain barrier (BBB) permeability.
1H NMR spectral identification of medication in cerebrospinal fluid of pediatric meningitis
Shayne Mason, Carolus J. Reinecke, Regan Solomons, Ron A. Wevers, Udo F.H. Engelke
ABSTRACT
Exploratory metabolomics studies of cerebrospinal fluid (CSF), using proton nuclear magnetic
resonance (1H NMR) spectroscopy, hold major potential application in neurodiagnostics. Such studies,
however, rely upon established databases of known metabolites. Here we address the ‗unknowns‘ in
the 1H NMR spectra of CSF from treated pediatric meningitis cases. Through knowledge of the
clinical information given by the pediatrician and analytical application of 1H NMR spectroscopy on
pure reference compounds of the medication used, we identified four of the previously unknown
compounds in the 1H NMR CSF spectra — the drugs pyrazinamide, isoniazid, acyclovir, and
sulfamethoxazole. We report on the one- and two-dimensional 1H NMR spectral data and chemical
information of these four compounds. By expanding our knowledge of 1H NMR CSF spectra from
treated meningitis cases, we are able to bring 1H NMR closer to the forefront of neurodiagnostics.
192
Therapeutic drug monitoring of beta-lactam antibiotics – Influence of sample stability on the
analysis of piperacillin, meropenem, ceftazidime and flucloxacillin by HPLC-UV
Nadine Pinder, Thorsten Brenner, Stefanie Swoboda, Markus A. Weigand, Torsten Hoppe-Tichy
ABSTRACT
Introduction: Therapeutic drug monitoring (TDM) is a useful tool to optimize antibiotic therapy.
Increasing interest in alternative dosing strategies of beta-lactam antibiotics, e.g. continuous or
prolonged infusion, require a feasible analytical method for quantification of these antimicrobial
agents. However, pre-analytical issues including sample handling and stability are to be considered to
provide valuable analytical results.
Methods: For the simultaneous determination of piperacillin, meropenem, ceftazidime and
flucloxacillin, a high performance liquid chromatography (HPLC) method including protein
precipitation was established utilizing ertapenem as internal standard. Long-term stability of stock
solutions and plasma samples were monitored. Furthermore, whole blood stability of the analytes in
heparinized blood tubes was investigated comparing storage under ambient conditions and 2–8 °C.
Results: A calibration range of 5–200 μg/ml (piperacillin, ceftazidime, flucloxacillin) and 2–200
μg/ml (meropenem) was linear with r2 > 0.999, precision and inaccuracy were <9% and <11%,
respectively. The successfully validated HPLC assay was applied to clinical samples and stability
investigations. At −80 °C, plasma samples were stable for 9 months (piperacillin, meropenem) or 13
months (ceftazidime, flucloxacillin). Concentrations of the four beta-lactam antibiotics in whole blood
tubes were found to remain within specifications for 8 h when stored at 2–8 °C but not at room
temperature.
Conclusions: The presented method is a rapid and simple option for routine TDM of piperacillin,
meropenem, ceftazidime and flucloxacillin. Whereas long-term storage of beta-lactam samples at −80
°C is possible for at least 9 months, whole blood tubes are recommended to be kept refrigerated until
analysis.
193
Development and validation of an ultra-performance liquid chromatography–tandem mass
spectrometry method for quantification of SR1001, an inverse agonist of retinoid-related orphan
receptors, and its application to pharmacokinetic studies in streptozotocin-induced diabetic mice
Cuipei Lin, Hanqing Wang, Hua Sun, Chengju Xiao, Zhijie Li
ABSTRACT
Retinoic acid receptor-related orphan receptors (RORs) play critical roles in the onset and progression
of type I diabetes, an autoimmune disease characterized by the destruction of pancreatic β-cells.
SR1001, an ROR inverse agonist, has been proven to be an effective diabetes treatment in the non-
obese diabetic (NOD) mouse model. However, optimization of this treatment is challenging because
knowledge of SR1001 pharmacokinetic (PK) behaviors in type I diabetic animals is limited. The aim
of our study was to develop and validate a specific and sensitive ultra-performance liquid
chromatography-tandem mass spectrometric (UPLC–MS/MS) method to measure the concentrations
of SR1001 in plasma and biological samples. Using the developed UPLC–MS/MS method, SR1001
linearity ranges in biological matrices were determined to be 5–1000 ng/mL, with correlation
coefficients of >0.99. The limit of detection (LOD) and limit of quantification (LOQ) values of
SR1001 were 1 and 5 ng/mL, respectively. And the intra-day and inter-day variances were less than
10%, and accuracy was within 90%–110%. The extraction recoveries of SR1001 were ≥80%, and no
significant matrix effect was observed. Using the validated UPLC–MS/MS method, levels of SR1001
in plasma and six major organs (heart, liver, spleen, lung, kidney, and brain) were determined in
streptozotocin (STZ) −induced diabetic mice. The PK parameters of SR1001 were also calculated. The
SR1001 drug concentration–time curves for organs and plasma showed similar trends, and the
elimination half-lives of SR1001 in diabetic mice were about 12 h. SR1001 was highly bound to
plasma protein, resulting in a much higher maximum concentration (Cmax = 144394 ng/mL) and area
under the concentration–time curve (AUC0-t = 2728258 ng/mL*h), but a low tissue/plasma partition
coefficient (Kp) value of <0.3.
Quantum dot-linked immunosorbent assay (QLISA) using orientation-directed antibodies
Miho Suzuki, Hikari Udaka, Takeshi Fukuda
ABSTRACT
An approach similar to the enzyme-linked immunosorbent assay (ELISA), with the advantage of
saving time and effort but exhibiting high performance, was developed using orientation-directed half-
part antibodies immobilized on CdSe/ZnS quantum dots. ELISA is a widely accepted assay used to
detect the presence of a target substance. However, it takes time to quantify the target with specificity
and sensitivity owing to signal amplification. In this study, CdSe/ZnS quantum dots are introduced as
bright and photobleaching-tolerant fluorescent materials. Since hydrophilic surface coating of quantum
dots rendered biocompatibility and functional groups for chemical reactions, the quantum dots were
modified with half-sized antibodies after partial reduction. The half-sized antibody could be bound to a
quantum dot through a unique thiol site to properly display the recognition domain for the core process
of ELISA, which is an antigen-antibody interaction. The reducing conditions were investigated to
generate efficient conjugates of quantum dots and half-sized antibodies. This was applied to IL-6
detection, as the quantification of IL-6 is significant owing to its close relationships with various
biomedical phenomena that cause different diseases. An ELISA-like assay with CdSe/ZnS quantum
dot institution (QLISA; Quantum dot-linked immunosorbent assay) was developed to detect 0.05
ng/mL IL-6, which makes it sufficiently sensitive as an immunosorbent assay.
194
Validation of liquid and gaseous calibration techniques for quantification of propofol in breath
with sorbent tube Thermal Desorption System GC–MS
Felix Maurer, Martin Geiger, Thomas Volk, Daniel I. Sessler, Sascha Kreuer
ABSTRACT
Plasma concentrations of intravenous drugs cannot currently be evaluated in real time to guide clinical
dosing. However, a system for estimating plasma concentration of the anesthetic propofol from
exhaled breath may soon be available. Developing reliable calibration and analytical validation
techniques is thus necessary. We therefore compared the established sorbent tube liquid injection
technique with a gas injection procedure using a reference gas generator. We then quantified propofol
with Tenax sorbent tubes in combination with gas-chromatography coupled mass spectrometry in the
breath of 15 patients (101 measurements). Over the clinically relevant concentration range from 10 to
50 ppbv, coefficient of determination was 0.995 for gas calibration; and over the range from 10 to 100
ng, coefficient of determination was 0.996 for liquid calibration. A regression comparing gas to liquid
calibration had a coefficient of determination of 0.89; slope 1.05 ± 0.01 (standard deviation). The limit
of detection was 0.74 ng and the lower limit of quantification was 1.12 ng for liquid; the limit of
detection was 0.90 ppbv and the lower limit of quantification was 1.36 ppbv for gas. Loaded sorbent
tubes were stable for at least 14 days without significant propofol loss as determined with either
method. Measurements from liquid or gas samples were comparably suitable for evaluation of patient
breath samples.
Development and validation of a bioanalytical method based on LC–MS/MS analysis for the
quantitation of CIGB-814 peptide in plasma from Rheumatoid Arthritis patients
Ania Cabrales-Rico, Yassel Ramos, Vladimir Besada, María del Carmen Domínguez, Luis Javier
González
ABSTRACT
CIGB-814, originally named as E18-3 APL1 or APL1 in preclinical experiments, is a novel therapeutic
peptide candidate for Rheumatoid Arthritis (RA). It is an altered peptide ligand containing a novel
CD4+ T-cell epitope of human heat shock protein 60 (83–109, MW 2988.38 g/mol) with a mutation
(D100 → L) that increases its affinity for HLA-II type molecules associated to RA. A bioanalytical
method, based on LC–MS/MS analysis, in the SRM mode was developed and fully validated to
quantify this peptide in human plasma. An internal standard with the same amino acid sequence but
labeled with three (13C615N2)-Lys residues was used for quantitation. The method provides a linear
range from 1.5 to 48 ng/mL (without matrix effect and carry over) and an accuracy and precision good
enough for monitoring more than 80% of the AUC of the PK profile in a phase I clinical trial. The
peptide was administered subcutaneously in three dose levels (1, 2.5 and 5 mg) not normalized to the
body weight of patients with RA. The low doses imposed an analytical challenge; however, a LLOQ of
1.5 ng/mL enabled the PK analysis. The Cmax, reached at 0.5 h, showed a great variability, that was
most likely due to the non-normalized doses; the proposed mechanism for this peptide; and the
variability between patients. A rapid clearance of this peptide (4–6 h) is advantageous for an
immunomodulatory drug, because the therapeutic schedule requires repeated dosages to restore
peripheral tolerance.
195
Application of volumetric absorptive microsampling device for quantification of tacrolimus in
human blood as a model drug of high blood cell partition
Kenji Kita, Yuji Mano
ABSTRACT
Volumetric absorptive microsampling device (VAMS) was evaluated for bioanalysis of tacrolimus,
which was used as a model drug with high blood cell partition. Aliquots of blood (ca. 10 μL) with
different hematocrits and fortified with tacrolimus were wicked up by VAMS and tacrolimus was
extracted with a methanol-water mixture (1:1, v/v) via sonication. After chromatography on an
AQUITY UPLC HSS T3 column (100 × 2.1 i.d., mm, 1.8 μm), tacrolimus and the internal standard
ascomycin, were detected in the positive ion mode with electrospray ionization by monitoring of
transitions m/z 826.6 → 616.4 and m/z 814.6 → 604.0, respectively. An assay method to quantify
tacrolimus from 1 to 250 ng/mL in whole blood was qualified by ensuring that linearity, selectivity,
intra- and inter-batch reproducibility, and stability were within the acceptance criteria. Consistent and
high extraction recovery of tacrolimus was ensured from blood with low- (20%), mid- (45%), and
high-hematocrit (65%) levels with minimal matrix effects. Apparent instability at ambient temperature
or 4 °C possibly due to reduced recovery suggests that tacrolimus in VAMS should be stored at −25 °C
until assay. Potential reduced recovery over time from VAMS should be taken into consideration in
method optimization.
Fast and efficient zirconia-based reversed phase chromatography for selective determination of
triptans in rat plasma
Sameh Ahmed, Noha N. Atia
ABSTRACT
Selective and fast chromatographic method was essential for the determination of triptans, selective
serotonin receptor (5-HT1) agonists, in biological specimens. However, selective chromatographic
separation of these highly basic drugs is a challenging problem on silica-based stationary phases.
Zirconia-based stationary phases have been introduced as an efficient alternative for silica-based
columns offering unique stability, selectivity, and retention ability for various classes of drugs. Herein,
a new selective, fast and reproducible high performance liquid chromatographic method (HPLC) was
developed and validated for the determination of four triptans in plasma samples namely; Sumatriptan
(SMT), Zolmitriptan (ZLT), Eletriptan (ELT) and Rizatriptan (RZT). Zirconia-based polybutadiene
(PBD) column was used for the separation and quantitation of the studied triptans in rat plasma based
on mixed mode ion exchange and reversed phase chromatography. Zirconia-PBD column (ZirChrom-
PBD) has enhanced chemical and thermal stability, selectivity and provides high resolution for the
investigated triptans in short analysis time compared with the commonly used C18 columns. A simple
isocratic separation mode was used with a mobile phase consisted of acetonitrile and 10 mM
phosphate buffer adjusted to pH 3.0 (20:80; v/v) at flow rate 1.0 mL min−1. The column was
maintained at 50 °C and effluent was monitored by photodiode array detector (PDA). The developed
method was validated in agreement with US-FDA guidelines and was appropriate for analysis of
triptans in plasma samples. The linearity range obtained for the developed HPLC method was 15–2000
ng mL−1 with detection limits of 4.8- 6.2 ng mL−1 for all the studied triptans. The developed
zirchonia-PBD-HPLC method was applied successfully to study the pharmacokinetics of ZLT in rats
after a single oral dose. The method was proved to be valuable for therapeutic drug monitoring and
bioavailability studies of the studied triptans.
196
Monitoring breast cancer treatment using a Fourier transform infrared spectroscopy-based
computational model
J. Depciuch, E. Kaznowska, S. Golowski, A. Koziorowska, J. Cebulski
ABSTRACT
Breast cancer affects one in four women, therefore, the search for new diagnostic technologies and
therapeutic approaches are of critical importance. This involves the development of diagnostic tools to
facilitate the detection of cancer cells, which is useful for assessing the efficacy of cancer therapies.
One of the major challenges for chemotherapy is the lack of tools to monitor efficacy during the course
of treatment. Vibrational spectroscopy appears to be a promising tool for such a purpose, as it yields
Fourier transformation infrared (FTIR) spectra which can be used to provide information on the
chemical composition of the tissue. Previous research by our group has demonstrated significant
differences between the infrared spectra of healthy, cancerous and post-chemotherapy breast tissue.
Furthermore, the results obtained for three extreme patient cases revealed that the infrared spectra of
post-chemotherapy breast tissue closely resembles that of healthy breast tissue when chemotherapy is
effective (i.e., a good therapeutic response is achieved), or that of cancerous breast tissue when
chemotherapy is ineffective. In the current study, we compared the infrared spectra of healthy,
cancerous and post-chemotherapy breast tissue. Characteristic parameters were designated for the
obtained spectra, spreading the function of absorbance using the Kramers–Kronig transformation and
the best fit procedure to obtain Lorentz functions, which represent components of the bands. The
Lorentz function parameters were used to develop a physics-based computational model to verify the
efficacy of a given chemotherapy protocol in a given case. The results obtained using this model
reflected the actual patient data retrieved from medical records (health improvement or no
improvement). Therefore, we propose this model as a useful tool for monitoring the efficacy of
chemotherapy in patients with breast cancer.
Enhancement in recovery of drugs with high protein binding efficiency from human plasma
using magnetic nanoparticles
Aniruddha Bhati, Rucha P. Desai, C.N. Ramchand
ABSTRACT
In this paper, we propose an alternate method for bioanalytical extraction of drugs from human plasma
samples using bare magnetic nanoparticles. The magnetic nanoparticles (MNPs) were used for
deproteination of biological samples that further assist in extraction of plasma bound drugs for
bioanalytical studies. The method uses basic solvents (ethanol, methanol, etc.) rather than the
expensive and toxic solvents. The MNPs provide several advantages like avoiding the use of
centrifuge machine, and making extraction time effective. The average time involved for the sample
preparation is around 30–40 min. The developed method was examined for seven different drugs
having moderate (40–70%) to high (>80%) plasma protein binding efficiency. The present study
focuses on the principle of magnetic nanoparticle based extraction of drug that binds with the plasma
protein. In calcitriol (protein binding efficiency >99%), it was observed that the drug extraction
efficiency could be enhanced by 16% using the present method. However, we assume that still there is
a scope for improving the extraction efficiency by optimizing proper solvent for the specific drug. The
use of magnetic nanoparticles makes the extraction cost effective and quick with improved efficiency.
197
Simultaneous quantification of endothelin receptor antagonists and phosphodiesterase 5
inhibitors currently used in pulmonary arterial hypertension
Yeliz Enderle, Lukas Witt, Heinrike Wilkens, Ekkehard Grünig, Jürgen Burhenne
ABSTRACT
Combination treatment with endothelin receptor antagonists (ERA) and phosphodiesterase 5 inhibitors
(PDE5I) improved efficacy of pulmonary arterial hypertension (PAH) therapy. However, drug–drug
interactions, variable exposure, non-adherence can influence plasma levels. For these reasons, drug
quantification may be advantageous particularly in patients with poor treatment responses. We
developed, validated, and applied an assay for the simultaneous quantification of ambrisentan,
bosentan, macitentan, sildenafil, and tadalafil as well as their main (and partly active) metabolites in
human plasma. This method is based on LC–MS/MS separation for a rapid and sensitive quantification
with stable isotopically labelled analogues as internal standards for each drug and metabolite. Sample
preparation was carried out using a solid phase extraction protocol based on Oasis HLB material. The
separation was achieved on a Kinetex C18 column and multiple reaction monitoring in negative
ionization mode was used for sensitive detection. The calibrations were linear for all analytes with
correlation coefficients >0.99 within the concentration range observed under a therapeutic PAH dosing
scheme with lower limits of quantification between 0.34 ng/mL (OH-ambrisentan) and 10 ng/mL
(despropyl-macitentan). Intra- and inter-day precision at LLOQ and QC levels ranged between 2.03%
and 19.8%, and 0.65% and 14.0%, respectively. The sample turnover time was 12 min. The
applicability of this versatile LC/MS/MS assay was verified by the successful analysis of clinical
routine samples of patients on PAH medication.
Application of 1H NMR spectroscopy to the metabolic phenotyping of rodent brain extracts: A
metabonomic study of gut microbial influence on host brain metabolism
J.R. Swann, I. Garcia-Perez, V. Braniste, I.D. Wilson, E. Holmes
ABSTRACT
1H NMR Spectroscopy has been applied to determine the neurochemical profiles of brain extracts
from the frontal cortex and hippocampal regions of germ free and normal mice and rats. The results
revealed a number of differences between germ free (GF) and conventional (CV) rats or specific
pathogen-free (SPF) mice with microbiome-associated metabolic variation found to be both species-
and region-dependent. In the mouse, the GF frontal cortex contained lower amounts of creatine, N-
acetyl-aspartate (NAA), glycerophosphocholine and lactate, but greater amounts of choline compared
to that of specific pathogen free (SPF) mice. In the hippocampus, the GF mice had greater creatine,
NAA, lactate and taurine content compared to those of the SPF animals, but lower relative quantities
of succinate and an unidentified lipid-related component. The GF rat frontal cortex contained higher
relative quantities of lactate, creatine and NAA compared to the CV animals whilst the GF
hippocampus was characterized by higher taurine and phosphocholine concentrations and lower
quantities of NAA, N-acetylaspartylglutamate and choline compared to the CV animals. Of note is
that, in both rat and mouse brain extracts, concentrations of hippocampal taurine were found to be
greater in the absence of an established microbiome. The results provide further evidence that brain
biochemistry can be influenced by gut microbial status, specifically metabolites involved in energy
metabolism demonstrating biochemical dialogue between the microbiome and brain.
198
A re-investigation of the phytochemical composition of the edible herb Amaranthus retroflexus
L
Serena Fiorito, Francesco Epifano, Roberta Palmisano, Salvatore Genovese, Vito Alessandro Taddeo
ABSTRACT
In this paper the presence of selected prenylated and unprenylated phenylpropanoids of nutraceutical
value, namely umbelliferone, apigenin, 4′-geranyloxyferulic acid, 7-isopentenyloxycoumarin,
auraptene, and umbelliprenin have been determined in all parts of the edible herb Amaranthus
retroflexus extracted with different methodologies. Roots were seen to contain the widest variety of
unprenylated and prenylated phenylpropanoids both in terms of number of secondary metabolites and
their quantitites. Findings described in the present study underline how A. retroflexus can be
considered as a potential nutraceutical for human welfare.
A vote for robustness: Monitoring serum enzyme activity by thin-layer chromatography of
dabsylated bradykinin products
Malte Bayer, Simone König
ABSTRACT
High-end analytical methods provide excellent data but may lack the robustness required in large
analytical studies. In particular complex chemical matrices may cause difficulties and increase the
need for extensive sample preparation. For screening of patients we thus developed a low-tech assay to
monitor bradykinin degradation by serum proteases. The bradykinin concentration mirrors the activity
of angiotensin-converting enzyme (ACE). Dabsylated bradykinin (DBK) and its labeled fragments
DBK1-8 and DBK1-5 were visualized by thin-layer chromatography using only 3 μL of serum. Lower
DBK1-5 levels indicated reduced ACE activity due to medication (ACE-inhibitors) or disease.
Provided that purified DBK is available, the assay protocol itself is very simple and does not require
any expensive high-end equipment.
199
Salting-out assisted liquid–liquid extraction combined with gas chromatography-mass
spectrometry for the determination of pyrethroid insecticides in high salinity and biological
samples
Zongliang Niu, Chunwei Yu, Xiaowen He, Jun Zhang, Yingying Wen
ABSTRACT
A salting-out assisted liquid–liquid extraction (SALLE) combined with gas chromatography-mass
spectrometry (GC–MS) method was developed for the determination of four pyrethroid insecticides
(PYRs) in high salinity and biological samples. Several parameters including sample pH, salting-out
solution volume and salting-out solution pH influencing the extraction efficiency were systematically
investigated with the aid of orthogonal design. The optimal extraction conditions of SALLE were: 4
mL of salting-out solution with pH = 4 and the sample pH = 3. Under the optimum extraction and
determination conditions, good responses for four PYRs were obtained in a range of 5–5000 ng/mL,
with linear coefficients greater than 0.998. The recoveries of the four PYRs ranged from 74% to 110%,
with standard deviations ranging from 1.8% to 9.8%. The limits of detection based on a signal-to-noise
ratio of 3 were between 1.5–60.6 ng/mL. The method was applied to the determination of PYRs in
urine, seawater and wastewater samples with a satisfactory result. The results demonstrated that this
SALLE-GC–MS method was successfully applied to determine PYRs in high salinity and biological
samples. SALLE avoided the need for the elimination of salinity and protein in the sample matrix, as
well as clean-up of the extractant. Most of all, no centrifugation or any special apparatus are required,
make this a promising method for rapid sample preparation procedure.
HPLC–MS/MS method for troventol determination in human plasma and its application to
biological samples
Aleksandra Nikitina, Alexander Grigoriev, Alla Sidorova
ABSTRACT
For the first time, an HPLC–MS/MS method for the determination pmol/l levels of troventol (TRV)
and clenbuterol as an internal standard (IS) in human plasma was developed, validated and tested on
biological samples. The method included solid phase extraction by Waters Oasis WCX cartridges and
chromatographic separation on a YMC-Pack SIL (100 mm × 2.1 mm, 5 μm, 12 nm) analytical column
with acetonitrile–water–formic acid (50:50:0.1, v/v/v) as the mobile phase; the selected ion transitions
were m/z 332.2→138.2 and m/z 277.0→203.1 for TRV and IS, respectively, in positive ionization
mode. The calibration curve for TRV showed good linearity in the concentration range of 35–500
pg/ml. The method was applied to real samples taken from healthy subjects after inhalation of an
aerosol containing 640 μg of TRV.
200
1,4-Anthraquinone: A new useful pre-column reagent for the determination of N-acetylcysteine
and captopril in pharmaceuticals by high performance liquid chromatography
Rita Gatti, Rita Morigi
ABSTRACT
1,4-Anthraquinone (ANQ) is proposed as a novel pre-column reagent for high performance liquid
chromatography (HPLC) determination of N-acetylcysteine (NAC) and captopril (CAP) in
pharmaceutical formulations. The derivatization reactions were carried out at room temperature: NAC
at pH 8 for 1 min, while CAP at pH 7.5 for 20 min. Both reactions reached completeness at a reagent
to thiol molar ratio of about 2.5. The synthesised derivatives were characterized by 1H NMR and IR.
The chromatographic separations were performed on a C18 Phenomenex Synergi Fusion 4 μm (250
mm × 4.6 mm I.D.) stainless steel column with detection at λ = 300 nm. The mobile phase consisted of
methanol/triethylammonium (TEA) phosphate buffer (pH 3; 0.05 mol/L) 75:25 (v/v) at a flow-rate of
0.4 mL/min for NAC and 88:12 (v/v), at a flow-rate of 0.6 mL/min for CAP. The validation
parameters (linearity, sensitivity, accuracy, precision, specificity and stability) were highly
satisfactory. Linear response was observed (determination coefficient ≥0.9996). Detection limits were
about 8 and 18 ng/mL for NAC and CAP, respectively. Intra-day precision (relative standard
deviation, R.S.D.) was ≤1.58%, for thiol to internal standard (IS) peak area ratio and ≤0.33%, for thiol
and IS retention times (tR), without significant differences between intra- and inter-day data. Thiol
recovery studies were satisfactory (99.50%) with R.S.D. ≤0.56%. The results highlight the high
sensitivity of the method and the remarkable reactivity and selectivity of the reagent towards the thiol
function.
Development a validated highly sensitive LC–MS/MS method for simultaneous quantification of
Ledipasvir, sofosbuvir and its major metabolite GS-331007 in human plasma: Application to a
human pharmacokinetic study
Ola M. Abdallah, Ahmed M. Abdel-Megied, Amira S. Gouda
ABSTRACT
A highly sensitive and rapid LC–MS/MS method was developed, fully optimized and validated for the
simultaneous determination of Ledipasvir (LED) and Sofosbuvir (SOF) in the presence of its major
metabolite GS-331007 in human plasma using Daclatasvir as internal standard (IS). The extraction of
analytes and IS from plasma was performed using liquid-liquid extraction with ethyl acetate. The
chromatographic separation of these prepared samples was achieved on Xterra MS C8 column (4.6 ×
50 mm,5 μm) using gradient elution with a mobile phase of ammonium formate buffer (pH 3.5; 10
mM), acetonitrile and methanol pumped at a flow rate 0.7 mL min−1.The detection was performed on
API4000 triple quadrupole tandem mass spectrometer using multiple reaction monitoring (MRM)
positive electrospray ionization interface. The method was validated according to FDA guidelines for
bio-analytical methods with respect to linearity, accuracy, precision, selectivity, carry-over, stability
and dilution integrity. Linearity was obtained over a concentration range of 0.1–1000, 0.3–3000 and
3.0–3000 ng mL−1 for LED, SOF and GS-331007; respectively by applying weighted least-squares
linear regression method (1/x2). The wider range of quantification in a shorter period of separation
time less than 5.0 min allowed monitoring the serum concentration of analytes up to 144 h. The
proposed method can be successfully applied for pharmacokinetic and bioequivalence studies in
healthy human volunteers.
201
Chemometrics and chromatographic fingerprints to classify plant food supplements according to
the content of regulated plants
E. Deconinck, C.A. Sokeng Djiogo, P. Courselle
ABSTRACT
Plant food supplements are gaining popularity, resulting in a broader spectrum of available products
and an increased consumption. Next to the problem of adulteration of these products with synthetic
drugs the presence of regulated or toxic plants is an important issue, especially when the products are
purchased from irregular sources. This paper focusses on this problem by using specific
chromatographic fingerprints for five targeted plants and chemometric classification techniques in
order to extract the important information from the fingerprints and determine the presence of the
targeted plants in plant food supplements in an objective way. Two approaches were followed: (1) a
multiclass model, (2) 2-class model for each of the targeted plants separately. For both approaches
good classification models were obtained, especially when using SIMCA and PLS-DA. For each
model, misclassification rates for the external test set of maximum one sample could be obtained. The
models were applied to five real samples resulting in the identification of the correct plants, confirmed
by mass spectrometry. Therefore chromatographic fingerprinting combined with chemometric
modelling can be considered interesting to make a more objective decision on whether a regulated
plant is present in a plant food supplement or not, especially when no mass spectrometry equipment is
available.
Surrogate CD16-expressing effector cell lines for determining the bioactivity of therapeutic
monoclonal antibodies
Shalom A. Gurjar, Jeremy P. Derrick, Rebecca J. Dearman, Robin Thorpe, Meenu Wadhwa
ABSTRACT
Traditional antibody dependent cellular cytotoxicity (ADCC) assays use donor derived natural killer
(NK) or peripheral blood mononuclear cells, but donor genetic variability and the technically
challenging nature of the assay means that alternative in vitro assay formats are required. We explored
the utility of two reporter gene cell lines, the J2 and J9, as surrogate effector cells for ADCC assays.
Both express the ADCC relevant Fcγ receptor CD16, crosslinking of which leads to firefly luciferase
expression. For anti-CD20 rituximab and anti-HER2 trastuzumab (both IgG1 monoclonal antibodies,
mAbs) a dose dependent firefly luciferase response was observed exclusively in the presence of their
respective targets, representing the molecular interaction which potentiates ADCC activity.
Importantly, both surrogate effector and NK cell based assays gave statistically similar values for
rituximab ADCC activity. Increased engagement with target cell bound mAbs was determined to be
cytotoxic for the J2 and J9 cell lines at the assay end point (at which luciferase expression is
measured). However, use of the J9 cells containing the constitutively expressed renilla luciferase gene
enabled data normalisation and corrected for fluctuations in both cell number and viability providing
an advantage over currently available surrogate effector cell-lines. Abrogated ADCC activity with
IgG4 mAbs, but enhanced activity with an IgG1 non-fucosylated mAb, was seen with the J9 cell line,
as expected. Additionally, two rituximab products (biosimilars in development) with similar binding
by flow cytometry, N-glycan profiles using HPLC and CD16 binding by surface plasmon resonance
showed comparable ADCC activity to Mabthera. The ADCC activity of another anti-CD20 mAb,
ofatumumab, reported only with primary cell based assays to date was also measured.
202
Dual-target screening of bioactive components from traditional Chinese medicines by hollow
fiber-based ligand fishing combined with liquid chromatography–mass spectrometry
Liang Chen, Xin Wang, Youping Liu, Xin Di
ABSTRACT
A novel strategy was developed for dual-target screening of bioactive components from traditional
Chinese medicines (TCMs). This strategy was based on the use of low-cost microporous hollow fibers
filled with target enzymes as baits to ―fish out‖ the ligands in TCM extracts, followed by identification
of the ligands dissociated from the target-ligand complexes by liquid chromatography–mass
spectrometry. Ganjiang Huangqin Huanglian Renshen Decoction (GHHRD), a classical TCM
prescription for diabetes treatment, was chosen as a model sample to evaluate the feasibility of the
proposed strategy. Three bioactive components were successfully screened out from GHHRD.
Coptisine was identified as the ligand of α-glucosidase and baicalin as the ligand of angiotensin-
converting enzyme (ACE). Berberine was found to be a dual inhibitor of α-glucosidase and ACE. The
results were further verified by enzyme inhibitory assay and molecular docking simulation. The study
suggested that our developed strategy would be a powerful tool for screening bioactive components
from multi-component and multi-target TCMs.
Volume 144 September 2017
Circular dichroism analysis of the calicheamicin-DNA interaction revisited
Gloria Proni, Kristi Tami, Nina Berova, George A. Ellestad
ABSTRACT
Calicheamicin, γ1I, is a remarkable DNA binding-cleaving, enediyne-containing, natural product that
exhibits potent antitumor activity. In this study, we used electronic circular dichroism spectroscopy to
monitor potential drug-induced DNA conformational changes and DNA induced conformational
changes in the calicheamicin aglycone. Three DNA dodecamer sequences were examined: one
containing a primary TCCT binding/cleavage site and two dodecamers containing less prominent
CTCT and TCTC sites. The binding was monitored by taking advantage of the drug‘s unique negative
exciton couplet (−313 nm/+275 nm) in phosphate buffer/ethanol 10%. Specifically the CD analysis
focused at the longest wavelength region around 313 nm where there is no interference by the positive
CD contributions of the DNA. Upon binding at a DNA/drug ratio of 1/1.2 and 1/2.7 a slight red shift
from 313 nm to 319 nm was observed. At a ratio of 1/1.2, the CE intensity remained practically
unchanged from that of free drug, which indicates no conformational changes in the bound aglycone
itself. A larger amount of drug, at a molar ratio of DNA/drug of 1/2.7 but especially at 1/6 and up to
1/10, however, caused a surprisingly distinct decrease in the intensity at this negative CD band and a
further small red-shift to 322 nm, evidence for non-specific binding.
203
Induced circularly polarized luminescence for revealing DNA binding with fluorescent dyes
Marcin Górecki, Francesco Zinna, Tarita Biver, Lorenzo Di Bari
ABSTRACT
To the best of our knowledge this is the first report on the application of induced circularly polarized
luminescence (CPL) for sensing the binding of fluorescent dyes to double stranded DNA. Using
Thiazole Orange (TO) and 4′,6-diamidino-2-phenylindole (DAPI) as models, we show utility and
limitations of CPL in DNA binding studies. The results obtained indicate that CPL can be used as a
new chiroptical tool for this purpose, however, special attention while recording CPL data must be
used, in order to exclude measurement artefacts caused by linear polarization components.
Analysis of stereoselective drug interactions with serum proteins by high-performance affinity
chromatography: A historical perspective
Zhao Li, David S. Hage
ABSTRACT
The interactions of drugs with serum proteins are often stereoselective and can affect the distribution,
activity, toxicity and rate of excretion of these drugs in the body. A number of approaches based on
affinity chromatography, and particularly high-performance affinity chromatography (HPAC), have
been used as tools to study these interactions. This review describes the general principles of affinity
chromatography and HPAC as related to their use in drug binding studies. The types of serum agents
that have been examined with these methods are also discussed, including human serum albumin, α1-
acid glycoprotein, and lipoproteins. This is followed by a description of the various formats based on
affinity chromatography and HPAC that have been used to investigate drug interactions with serum
proteins and the historical development for each of these formats. Specific techniques that are
discussed include zonal elution, frontal analysis, and kinetic methods such as those that make use of
band-broadening measurements, peak decay analysis, or ultrafast affinity extraction.
204
An indirect stereoselective analysis of nebivolol glucuronides in plasma by LC–MS/MS:
Application to clinical pharmacokinetics
Carolina Pinto Vieira, Daniel Valente Neves, Evandro José Cesarino, Adriana Rocha, Vera Lucia
Lanchote
ABSTRACT
Nebivolol is a racemate of the d-isomer responsible for β1 adrenergic receptor antagonism and the l-
isomer responsible for the release of nitric oxide from endothelial cells. Nebivolol is mainly
metabolized to nebivolol glucuronide, which also contribute to the nebivolol β1 adrenoreceptor
antagonism. This study reports the development and validation of an indirect stereoselective method of
analysis of nebivolol glucuronides in plasma by LC–MS/MS. The method was applied to the
investigation of stereoselectivity in the glucuronidation of nebivolol in elderly hypertensive patients (n
= 11) CYP2D6 phenotyped as EM and treated with a single oral dose of the racemate. One-milliliter
plasma aliquots spiked with internal standard (S)-(−)-metoprolol were incubated with 25 μL of β-
glucuronidase (final concentration 2500 unit/mL) at pH 5.0 for 16 h at 37 °C. Linearity for total
nebivolol was 0.2–125 ng of each isomer per mL plasma and permitted analysis of nebivolol
glucuronide isomers up to 48 h after administration of a single oral dose of 10 mg racemate. Regarding
to the nebivolol glucuronide isomers, higher plasma concentrations of the d-isomer were observed
compared to the l-isomer (d/l AUC = 5.4), explaining at least in part the plasma accumulation of
unchanged l-nebivolol (l/d AUC = 1.8). This study also showed metabolic glucuronide
nebivolol/unchanged nebivolol ratios of approximately 6.5 for the l-isomer (AUC 65.3 vs 10.1 ng
h/mL) and approximately 62.1 (335.2 vs 5.4 ng h/mL) for the d-isomer. Considering that d-nebivolol
glucuronide also contributes for β1 adrenergic receptor antagonism, future studies regarding PK-PD of
nebivolol should evaluate not only plasma concentrations of unchanged nebivolol isomers but also
glucuronide nebivolol isomers.
N-Decyl-S-trityl-(R)-cysteine, a new chiral selector for ―green‖ ligand-exchange chromatography
applications
Andrea Carotti, Federica Ianni, Emidio Camaioni, Lucia Pucciarini, Benedetto Natalini
ABSTRACT
In search for new enantioselectivity profiles, the N-decyl-S-trityl-(R)-cysteine [C10-(R)-STC] was
synthesized through a one-step procedure and then hydrophobically adsorbed onto an octadecylsilica
surface to generate a stable chiral stationary phase for ligand-exchange chromatography (CLEC-CSP)
applications. The CLEC analysis was carried out on underivatized amino acids, by using a Cu(II)
sulphate (1.0 mM) containing aqueous eluent system. Most of the analysed compounds (34 out of 45)
were enantiodiscriminated by the C10-(R)-STC-based CSP, with resolution factor (RS) values up to
8.86. Conformationally rigid and hydrophobic ligands often experienced the largest enantioselectivity
effects. A high loadability emerged from the analysis of rac-NorVal (selected as prototype test
compound): up to 20 mg/mL were efficiently enantioseparated with the CLEC-CSP. Two in-line hand-
made cartridges filled with a strong cation-exchange resin allowed the effective catching of Cu(II) ions
after the semi-preparative enantioseparation. The quantitative recovery of the rac-NorVal enantiomers
was made possible by flowing through the cartridge a 5% (v) ammonia solution. The CLEC phase
proved successful in the enantioselective analysis of a commercially available (S)-Leu containing
tablet. Furthermore, in order to understand the molecular basis for a successful use of the C10-(R)-
STC-based CLEC system, a descriptive structure-separation relationship study was performed.
205
The role of chirality in a set of key intermediates of pharmaceutical interest, 3-aryl-substituted-
γ-butyrolactones, evidenced by chiral HPLC separation and by chiroptical spectroscopies
Daniela Rossi, Rita Nasti, Simona Collina, Giuseppe Mazzeo, Sergio Abbate
ABSTRACT
The enantiomers of four chiral 3-aryl-substituted-γ-butyrolactones, key intermediates for the
preparation of compounds of pharmaceutical interest, were successfully isolated by enantioselective
chromatography, employing the Chiralpak AD-H chiral stationary phase. For all compounds the same
elution order was observed, as monitored by a full set of chiroptical methods that we employed,
namely ORD (optical rotatory dispersion), ECD (electronic circular dichroism, or CD in the UV
range), and VCD (vibrational circular dichroism, or CD in the IR range). By density functional theory
(DFT) calculations we were able to determine that the first eluted enantiomer has (S) absolute
configuration in all four cases. We were able to justify the elution order by molecular docking
calculations for all four enantiomeric pairs and suitable modeling of the stationary and mobile phases
of the employed columns. The optimal performance of the chiroptical spectroscopies and of the DFT
calculations allows us to formulate a lactone chirality rule out of the CO stretching region of the VCD
spectra.
Determination of the absolute configuration of a novel tetrasubstituted isoindolinone by
vibrational circular dichroism
Antonio Massa, Paola Rizzo, Francesco Scorzelli, Guglielmo Monaco, Riccardo Zanasi
ABSTRACT
The absolute configuration of a recently prepared asymmetric 3,3-disubstituted isoindolinone (ethyl 2-
benzyl-3-oxo-1-(3-oxobutyl)isoindoline-1-carboxylate), possessing highly promising pharmaceutical
activity, has been determined by means of VCD spectroscopy and DFT calculations. The great
flexibility of the molecule reduces to a few relevant conformers, all contributing in the same way to the
shape of the VCD spectrum for the carbonyl stretching region. Two of the three CO groups of the
molecule interact with each other during the stretching vibration, thus providing a non-conservative
VCD couplet whose signature, together with the VCD sign of the third CO stretching mode,
unequivocally determines the absolute configuration of the molecule, which is found to be (S) for the
(–) optical isomer.
206
GC–MS based Gestational Diabetes Mellitus longitudinal study: Identification of 2-and 3-
hydroxybutyrate as potential prognostic biomarkers
Danuta Dudzik, Marcin Zorawski, Mariusz Skotnicki, Wieslaw Zarzycki, M. Pilar Ramos
ABSTRACT
Gestational Diabetes Mellitus (GDM) causes severe short- and long-term complications for the
mother, fetus and neonate, including type 2-diabetes (T2DM) later in life. In this pilot study, GC–
Q/MS analysis was applied for plasma metabolomics fingerprinting of 24 healthy and 24 women with
GDM at different stages of gestation (second and third trimester) and postpartum (one and three
months). Multivariate (unsupervised and supervised) statistical analysis was performed to investigate
variance in the data, identify outliers and for unbiased assessment of data quality. Plasma fingerprints
allowed for the discrimination of GDM pregnant women from controls both in the 2nd and 3rd
trimesters of gestation. However, metabolic profiles tended to be similar after delivery. Follow up of
these women revealed that 4 of them developed T2DM within 2 years postpartum. Multivariate PLS-
DA models limited to women with GDM showed clear separation 3 months postpartum. In the 2nd
trimester of gestation there was also a clear separation between GDM women that were
normoglycemic after pregnancy and those with recognized postpartum T2DM. Metabolites that had
the strongest discriminative power between these groups in the 2nd trimester of gestation were 2-
hydroxybutyrate, 3-hydroxybutyrate, and stearic acid. We have described, that early GDM comprises
metabotypes that are associated with the risk of future complications, including postpartum T2DM. In
this pilot study, we provide evidence that 2-hydroxybutyrate and 3-hydroxybutyrate may be considered
as future prognostic biomarkers to predict the onset of diabetic complications in women with
gestational diabetes after delivery.
Development and validation of a quantification method for cucurbitacins E and I in rat plasma:
Application to population pharmacokinetic studies
Giovana Maria Lanchoti Fiori, Salvatore D‘Agate, Adriana Rocha, Ana Maria Soares Pereira,
Norberto Peporine Lopes
ABSTRACT
Cucurbitacin E is a potential drug candidate due to its anticancer activity, recognition of its molecular
targets, and synergism with other drugs used for cancer treatment. However, the use of cucurbitacin E
in clinical practice is not possible because of important knowledge gaps in its preclinical and clinical
pharmacokinetic characteristics. Cucurbitacin E is hydrolyzed to cucurbitacin I in plasma and in
human liver microsomes. The aim of this study was to evaluate the population pharmacokinetics of
cucurbitacin E and of its metabolite cucurbitacin I in rats. The method for the sequential analysis of
cucurbitacins E and I in rat plasma was developed using LC–MS/MS. Plasma aliquots of 50 μL were
deproteinized with acetonitrile and clobazam was added as internal standard. The extracts were
injected into an RP-18 column and eluted with a mobile phase consisting of a mixture of
acetonitrile:water:methanol (32:35:33, v/v/v). The method was precise and accurate, showing linearity
in the range of 1–100 ng cucurbitacin E/mL plasma and of 0.4–200 ng cucurbitacin I/mL plasma. The
method was applied to the pharmacokinetic evaluation of cucurbitacin E administered intravenously to
male Wistar rats (1 mg/kg). Serial blood samples were collected up to 24 h after administration. The
plasma concentrations of cucurbitacin E were quantified up to 16 h, while the plasma concentrations of
cucurbitacin I remained below the limit of quantification. A population pharmacokinetic model was
developed for cucurbitacin E using the NONMEM program, with adequate goodness of fit and
predictive performance.
207
Ultra-fast quantitation of voriconazole in human plasma by coated blade spray mass
spectrometry
Marcos Tascon, Germán Augusto Gómez-Ríos, Nathaly Reyes-Garcés, Justen Poole, Janusz Pawliszyn
ABSTRACT
Voriconazole is a triazole broad-spectrum antifungal medication often used to treat fungal infections
caused by Aspergillus and Fusarium species. One of the main challenges associated with the
implementation of this medication is its narrow therapeutic concentration range, demonstrating toxicity
at concentrations above 6 μg/mL and limited efficacy at concentrations below 2 μg/mL. As a result,
methodologies which permit the rapid and accurate quantitation of voriconazole in patients are highly
desirable. In this work two different approaches based on coated blade spray directly coupled to mass
spectrometry (CBS-MS) are introduced; each enabling the quantitation of voriconazole in plasma
samples with a simple and fast sample preparation and no chromatographic step. The first approach
involves a rapid extraction (1 min) of the target analyte from 300 μL of human plasma using
conventional laboratory vessels (e.g. vial, 96-well plate). Alternatively, the second strategy consists of
a 2 min extraction from a plasma droplet (10 μL) placed on the coated area of the blade. Both
procedures were successfully validated and good linearity (R2 ≥ 0.998), accuracy (91–122%) and
precision (<8%) were attained in the concentration range evaluated (0.1–50 μg/mL). Moreover, very
good results in terms of relative matrix effects were obtained given that the slopes of the calibration
curves constructed in five different plasma lots exhibited relative standard deviation (RSD) values
below 7%. Herein we demonstrated that CBS-MS is a technology suitable for the ultra-fast
determination of voriconazole in human plasma samples. Indeed, the proposed methodology can be
easily used either for routine drug monitoring or for in vitro pharmacokinetic studies in applications
where very small sample volumes are available and great temporal resolution is needed.
Pharmacokinetic profile of bilberry anthocyanins in rats and the role of glucose transporters:
LC–MS/MS and computational studies
G. Baron, A. Altomare, L. Regazzoni, V. Redaelli, G. Aldini
ABSTRACT
The aim of the present investigation was to better understand the pharmacokinetic profile of bilberry
(Vaccinium Myrtillus) anthocyanins and the role of glucose transporters (sGLT1 and GLUT2) on their
absorption. In particular, the absorption of 15 different anthocyanins contained in a standardized
bilberry extract (Mirtoselect®) was measured in rats by a validated LC-ESI–MS/MS approach. The
plasma concentration peak (Cmax) of 11.1 ng/mL was reached after 30 min and fasting condition
significantly increased the bioavailability of anthocyanins by more than 7 fold in respect to fed rats.
Glucose co-administration did not interfere with the overall anthocyanin uptake. Bioavailability of
each anthocyanin was then estimated by comparing the relative content in plasma vs extract. The 15
anthocyanins behaved differently in term of bioavailability and both the aglycone and the sugar moiety
were found to affect the absorption. For instance, arabinoside moiety was detrimental while cyanidin
enhanced bioavailability. Computational studies permitted to rationalize such results, highlighting the
role of glucose transporters (sGLT1 and GLUT2) in anthocyanins absorption. In particular a
significant correlation was found for the 15 anthocyanins between sGLT1 and GLUT2 recognition and
absorption.
208
Comparative pharmacodynamic analysis of imidazoline compounds using rat model of ocular
mydriasis with a test of quantitative structure–activity relationships
Joanna Raczak-Gutknecht, Antoni Nasal, Teresa Frąckowiak, Anita Kornicka, Roman Kaliszan
ABSTRACT
Imidazol(in)e derivatives, having the chemical structure similar to clonidine, exert diverse
pharmacological activities connected with their interactions with alpha2-adrenergic receptors, e.g.
hypotension, bradycardia, sedation as well as antinociceptive, anxiolytic, antiarrhythmic, muscle
relaxant and mydriatic effects. The mechanism of pupillary dilation observed after systemic
administration of imidazol(in)es to rats, mice and cats depends on the stimulation of postsynaptic
alpha2-adrenoceptors within the brain. It was proved that the central nervous system (CNS)-localized
I1-imidazoline receptors are not engaged in those effects. It appeared interesting to analyze the CNS-
mediated pharmacodynamics of imidazole(in)e agents in terms of their chromatographic and
calculation chemistry-derived parameters. In the present study a systematic determination and
comparative pharmacometric analysis of mydriatic effects in rats were performed on a series of 20
imidazol(in)e agents, composed of the well-known drugs and of the substances used in experimental
pharmacology. The eye pupil dilatory activities of the compounds were assessed in anesthetized Wistar
rats according to the established Koss method. Among twenty imidazol(in)e derivatives studied, 18
produced diverse dose-dependent mydriatic effects. In the quantitative structure–activity relationships
(QSAR) analysis, the pharmacological data (half maximum mydriatic effect – ED50 in μmol/kg) were
considered along with the structural parameters of the agents from molecular modeling. The
theoretically calculated lipophilicity parameters, CLOGP, of imidazol(in)es, as well as their
lipophilicity parameters from HPLC, log kw, were also considered. The attempts to derive statistically
significant QSAR equations for a full series of the agents under study were unsuccessful. However, for
a subgroup of eight apparently structurally related imidazol(in)es a significant relationship between
log(1/ED50) and log kw values was obtained.
Characterization of oxycodone in vitro metabolism by human cytochromes P450 and UDP-
glucuronosyltransferases
Stéphanie Romand, Dany Spaggiari, Niloufar Marsousi, Caroline Samer, Serge Rudaz
ABSTRACT
The hepatic metabolism of oxycodone by cytochromes P450 (CYP) and the UDP-
glucuronosyltransferases (UGT), the main metabolic enzymes of phase I and phase II, respectively,
was assessed in vitro. The N-demethylation by CYP3A4/5 and the O-demethylation by CYP2D6 in
human liver microsomes (HLM) followed Michaelis-Menten kinetics, with intrinsic clearances of 1.46
μL/min/mg and 0.35 μL/min/mg, respectively. Although noroxycodone and oxymorphone mainly
contribute to the elimination of oxycodone, the simulated total in vivo clearance using in vitro phase I
metabolism was underestimated. For the first time, metabolism of oxycodone by UGT was deeply
investigated using HLM, recombinant enzymes and selective inhibitors. Oxycodone-glucuronide was
mainly produced by UGT2B7 (Km = 762 ± 153 μM, Vmax = 344 ± 20 peak area/min/mg) and to a
lesser extent by UGT2B4 (Km = 2454 ± 497 μM, Vmax = 201 ± 19 peak area/min/mg). Finally, the
kinetics of the drug–drug interactions were assessed using two CYP and UGT cocktail approaches.
Incubations of HLM with phase I and phase II drug probes showed that oxycodone mainly decreased
the in vitro activities of CYP2D6, CYP3A4/5, UGT1A3, UGT1A6 and UGT2B subfamily with an
important impact on UGT2B7.
209
Structural and functional integrity of human serum albumin: Analytical approaches and clinical
relevance in patients with liver cirrhosis
Marina Naldi, Maurizio Baldassarre, Marco Domenicali, Manuela Bartolini, Paolo Caraceni
ABSTRACT
Human serum albumin (HSA) is the most abundant circulating plasma protein. Besides a significant
contribution to the osmotic pressure, it is also involved in the fine regulation of many other
physiological processes, including the balance of the redox state, the inflammatory and/or
immunological responses, and the pharmacokinetic and pharmacodynamics of many drugs. Growing
evidence suggests that HSA undergoes structural and functional damage in diseases characterized by
an enhanced systemic inflammatory response and oxidative stress, as it occurs in chronic liver disease.
Based on their clinical relevance, this review provides a summary of the most common post-
translational modifications affecting HSA structural integrity and functions and their clinical relevance
in the field of liver disease. The review also provides a critical description of the analytical approaches
employed for the investigation of conformational alterations and the identification/quantitation of
specific post-translational modifications affecting HSA. Finally, the analytical methods available for
the assessment of two of the most clinically relevant non-oncotic properties of HSA, namely the
binding capacity and the antioxidant activity, are critically reviewed. Among the available techniques
particular attention is given to those proposed for the in vitro and in vivo investigation of structurally
modified albumin.
Targeted proteomics of cannabinoid receptor CB1 and the CB1b isoform
Soumita Ghosh, Isabel González-Mariscal, Josephine M. Egan, Ruin Moaddel
ABSTRACT
Cannabinoid receptors (CBR), including CB1 and CB2 have been therapeutic targets for a number of
conditions. Recently, splice variants of the CB1R have been identified in humans. The isoforms differ
in their N-terminus sequence and pharmacological activity relative to the CB1R, as a result, the
differentiation between the CB1 receptor and its isoform is required. As a result, a selected reaction
monitoring mass spectrometry (SRM-MS) method was developed for the quantitation of CB1 and the
CB1b isoform in CHO cells transduced with CB1 and CB1b. The SRM-MS protocol was assessed
with isotopically labeled peptide standards and had high reproducibility of intra-day assay (CVs from
1.9 to 4.3% for CB1 and 0.5 to 5.9% for CB1b) and inter-day assay (CVs from 1.2 to 5.2% for CB1
and 1.2 to 6.1% for CB1b).
210
Application of an ESI-QTOF method for the detailed characterization of GSK-3β inhibitors
Angela De Simone, Jessica Fiori, Marina Naldi, Annalisa D‘Urzo, Vincenza Andrisano
ABSTRACT
The crucial role of Glycogen Synthase Kinase 3 (GSK-3β) as a pivotal player in Alzheimer's Disease
(AD) has recently inspired significant attempts to design and synthesize potent kinase inhibitors. In
fact GSK-3β is considered the main kinase which catalyzes the microtubule-associated protein tau
hyper-phosphorylation and the neurofibrillary tangles (NFT) in vitro and in vivo, The first classes of
GSK-3β inhibitors were classified as ATP-competitive and, therefore, they lack of an efficient degree
of selectivity over other kinases. In light of this consideration, many efforts are devoted to characterize
new non ATP-competitive GSK-3β inhibitors, endowed with high selectivity. In parallel, there is an
urgent need to develop new analytical methodologies for the hit selection (highthroughput screening)
and ligand binding characterization in terms of potency, affinity and mechanism of action. The new
methodology for GSK-3β enzymatic activity determination can be adopted as a realistic alternative to
the currently used radioactive, luminescence and fluorescence detection methods, each showing
limitations in terms of safety and interferences. Herein, we propose an alternative and selective
electrospray ionization quadrupole time-of-flight (ESI-QTOF) method, based on the direct
quantification of phosphorylated substrate muscle glycogen synthase GSM, a peptide resembling the
high affinity sequence of natural substrate muscle glycogen synthase 1, for the detailed
characterization of GSK-3β inhibitors. The method was validated in terms of accuracy and
reproducibility of GSM signal intensity with a relative standard deviation RSD% value of 3.55%;
Limit of Detection (LOD): 0.006 μM; Lower Limit of Quantification (LLOQ): 0.02 μM; linearity r2
0.9951. The kinetic constants (KM and vmax) of the GSK-3β catalyzed kinase reaction and the
inhibitory potency of known ligands (IC50), were determined. All the obtained results were in
agreement with those reported in literature or obtained in house by the standard reference
luminometric approach. The proposed method was applied to the elucidation of well known inhibitors
mechanism of action by the construction of a Lineweaver–Burk plot and the Ki determination.
Furthermore, the potency, affinity and mechanism of action of a new non ATP-competitive compound
were established.
Quantitative estimation of cholinesterase-specific drug metabolism of carbamate inhibitors
provided by the analysis of the area under the inhibition-time curve
Huimin Zhou, Qiaoling Xiao, Wen Tan, Yiyi Zhan, Marco Pistolozzi
ABSTRACT
Several molecules containing carbamate groups are metabolized by cholinesterases. This metabolism
includes a time-dependent catalytic step which temporary inhibits the enzymes. In this paper we
demonstrate that the analysis of the area under the inhibition versus time curve (AUIC) can be used to
obtain a quantitative estimation of the amount of carbamate metabolized by the enzyme. (R)-
bambuterol monocarbamate and plasma butyrylcholinesterase were used as model carbamate-
cholinesterase system. The inhibition of different concentrations of the enzyme was monitored for 5 h
upon incubation with different concentrations of carbamate and the resulting AUICs were analyzed.
The amount of carbamate metabolized could be estimated with <15% accuracy (RE%) and ≤23%
precision (RSD%). Since the knowledge of the inhibition kinetics is not required for the analysis, this
approach could be used to determine the amount of drug metabolized by cholinesterases in a selected
compartment in which the cholinesterase is confined (e.g. in vitro solutions, tissues or body fluids),
either in vitro or in vivo.
211
A new method to characterize the kinetics of cholinesterases inhibited by carbamates
Qiaoling Xiao, Huimin Zhou, Hong Wei, Huaqiao Du, Marco Pistolozzi
ABSTRACT
The inhibition of cholinesterases (ChEs) by carbamates includes a carbamylation (inhibition) step, in
which the drug transfers its carbamate moiety to the active site of the enzyme and a decarbamylation
(activity recovery) step, in which the carbamyl group is hydrolyzed from the enzyme. The
carbamylation and decarbamylation kinetics decide the extent and the duration of the inhibition, thus
the full characterization of candidate carbamate inhibitors requires the measurement of the kinetic
constants describing both steps. Carbamylation and decarbamylation rate constants are traditionally
measured by two separate set of experiments, thus making the full characterization of candidate
inhibitors time-consuming. In this communication we show that by the analysis of the area under the
inhibition-time curve of cholinesterases inhibited by carbamates it is possible to calculate the
decarbamylation rate constant from the same data traditionally used to characterize only the
carbamylation kinetics, therefore it is possible to obtain a full characterization of the inhibition with a
single set of experiments. The characterization of the inhibition kinetics of human and dog plasma
butyrylcholinesterase and of human acetylcholinesterase by bambuterol and bambuterol
monocarbamate enantiomers was used to demonstrate the validity of the approach. The results showed
that the proposed method provides reliable estimations of carbamylation and decarbamylation rate
constants thus representing a simple and useful approach to reduce the time required for the
characterization of carbamate inhibitors.
Cyclodextrins as inhibitors of the precipitation of riboflavin-5’-phosphate due to presence of zinc
chloride: A NMR investigation
Federica Aiello, Gloria Uccello-Barretta, Niccolò Falugiani, Francesca Nardelli, Federica Balzano
ABSTRACT
Several cyclodextrins (CDs) were probed in order to counteract the precipitation of riboflavin-5‘-
phosphate (or flavin mononucleotide, FMN-P) due to the presence of divalent cations, by exploiting
Nuclear Magnetic Resonance (NMR) spectroscopy both for quantitative analyses and stereochemical
characterizations. Among CDs, β-cyclodextrin (β-CD) showed the best solubilizing power in virtue of
the formation of a 1–2 FMN-P/β-CD complex, the stereochemistry of which was ascertained by
ROESY (Rotating-frame Overhauser Enhanced SpectroscopY) measurements.
212
Monitoring drug–serum protein interactions for early ADME prediction through Surface
Plasmon Resonance technology
Edoardo Fabini, U. Helena Danielson
ABSTRACT
Many molecules fail to reach the market due to poor pharmacokinetic (PK) properties, rendering the
potential drug virtually unavailable for the primary target despite efficient administration to the body.
PK properties of endogenous and exogenous compounds in mammals are dependent, among other
factors, on their ability to interact with serum proteins. The extent of binding can greatly influence
their ADME (adsorption, distribution, metabolism and execration) profile. Reliable and cost-effective
bioavailability studies, early in the drug discovery process, can lead to an improvement of the success
rate for compounds entering clinical trials. Optical biosensors based on surface plasmon resonance
(SPR) detection emerged as an efficient approach to obtain large amounts of information about the
binding of small molecules to serum proteins. Simple, automated and fast assays provide a good
throughput, versatility and highly informative data output, rendering the methodology particularly
suited for early screening. The ability to provide basic information on PK can be easily coupled to
structure–activity relationship analysis. In this review, features of the technology and its employment
for the study of serum protein–small molecule interactions are presented and discussed.
Capillary electrophoresis in the context of drug discovery
Elena Farcaş, Lionel Pochet, Jacques Crommen, Anne-Catherine Servais, Marianne Fillet
ABSTRACT
Capillary Electrophoresis is a very efficient and resolutive separation technique used for many years in
the analytical field. Despite all its assets, CE remains poorly used in drug discovery. This can be
explained by the relatively low number of experienced CE practitioners, the maturity of HPLC in the
pharmaceutical industry and some intrinsic limitations of the technique. The objective of this review is
to focus our attention on recent developments of this technique in three different drug discovery areas:
bioassays, drug-plasma interactions and drug metabolism studies. These developments were based on
two important abilities of CE: the capacity to measure non-covalent interactions in solution and the
ability to use a portion of the capillary as a reactor while the rest of the capillary is used for the
separation of the product of the reaction.
213
Simultaneous analysis of nucleobases, nucleosides and ginsenosides in ginseng extracts using
supercritical fluid chromatography coupled with single quadrupole mass spectrometry
Yang Huang, Tingting Zhang, Yumei Zhao, Haibo Zhou, Zhengjin Jiang
ABSTRACT
Nucleobases, nucleosides and ginsenosides, which have a significant impact on the physiological
activity of organisms, are reported to be the active components of ginseng, while they are less present
in ginseng extracts. Few analytical methods have been developed so far to simultaneously analyze
these three classes of compounds with different polarities present in ginseng extracts. In the present
study, a simple and efficient analytical method was successfully developed for the simultaneous
separation of 17 nucleobases, nucleosides and ginsenosides in ginseng extracts using supercritical fluid
chromatography coupled with single quadrupole mass spectrometry (SFC-MS). The effect of various
experimental factors on the separation performance, such as the column type, temperature and
backpressure, the type of modifier and additive, and the concentration of make-up solvent were
systematically investigated. Under the selected conditions, the developed method was successfully
applied to the quality evaluation of 14 batches of ginseng extracts from different origins. The results
obtained for the different batches indicate that this method could be employed for the quality
assessment of ginseng extracts.
Combined approach using capillary electrophoresis, NMR and molecular modeling for
ambrisentan related substances analysis: Investigation of intermolecular affinities, complexation
and separation mechanism
Benedetta Pasquini, Fabrizio Melani, Claudia Caprini, Massimo Del Bubba, Sandra Furlanetto
ABSTRACT
A comprehensive investigation on the CE separation mechanisms and on the inclusion complexation
with CyDs of the chiral drug S-ambrisentan (S-AMB), its R-enantiomer and other impurities was
performed by different techniques. A CE method was previously set up allowing the simultaneous
determination of the enantiomeric purity and of impurities of S-AMB, based on the addition of SDS
micelles and γ-cyclodextrin (γCyD) to borate buffer. In this study, the electrophoretic behavior of the
analytes in terms of selectivity and mobility with respect to the addition of different CyDs was first
investigated, evidencing the presence of interactions for all the CyDs, but the unique ability of γCyD
for obtaining the separation of all the compounds. By molecular modeling, aggregates between SDS
micelles and analytes, and inclusion complexes between CyDs, SDS and/or analytes of different
stoichiometries were simulated. The potential and the gain energy of complexes were calculated on the
minimized conformations, showing the great tendency of γCyD of forming mixed complexes with one
or two SDS molecules and with the analyte, even if with different affinities among the analytes. For
1:1:1 mixed complexes with different CyDs, the highest difference of potential energy between the
enantiomers‘ complexes was observed for γCyD. Two-dimensional NOE spectroscopy experiments
were performed for S-AMB and I1 and pointed out the interactions of the aromatic moiety of the
analytes and of SDS aliphatic chain with γCyD protons, confirming the existence of γCyD mixed
complexes. The high affinity of SDS for the γCyD cavity was suggested to justify the fundamental role
of SDS in modulating and achieving the CE separation, due to its influence both on the stability and on
the type of complexes between γCyD and the analytes.
214
Molecularly imprinted polymer for glutathione by modified precipitation polymerization and its
application to determination of glutathione in supplements
Yukari Nakamura, Shizuka Masumoto, Hisami Matsunaga, Jun Haginaka
ABSTRACT
Molecularly imprinted polymers (MIP) particles for glutathione (GSH) with a narrow particle size
distribution were prepared by modified precipitation polymerization using methacrylic acid as a
functional monomer, divinylbenzene as a crosslinker and water as a co-solvent. The particle diameters
of the MIP and non-imprinted polymer (NIP) prepared under the optimum conditions were 3.81 ± 0.95
(average ± standard deviation) and 3.39 ± 1.22 μm, respectively. The retention and molecular-
recognition properties of the prepared MIP were evaluated using a mixture of acetonitrile and water as
a mobile phase in hydrophilic interaction chromatography. With an increase of acetonitrile content, the
retention factor of GSH was increased on the MIP. In addition to shape recognition, hydrophilic
interactions seem to work for the recognition of GSH on the MIP. The MIP had a specific molecular-
recognition ability for GSH, while glutathione disulfide, l-Glu, l-Cys, Gly-Gly and l-Cys-Gly could not
be retained or recognized on the MIP. The effect of column temperature revealed that the separation of
GSH on the MIP was entropically driven. Binding experiments and Scatchard analyses revealed that
one binding sites were formed on both the MIP and NIP, while the MIP gave higher affinity and
capacity for GSH than the NIP. Furthermore, the MIP was successfully applied for determination of
GSH in the supplements.
Efficacy of a titanium dioxide nanoparticles − based indoor anti-odor product as assessed by
electronic nose and gaschromatography–mass spectrometry
Mara Mirasoli, Roberto Gotti, Massimo Di Fusco, Giulia Basaglia, Aldo Roda
ABSTRACT
Indoor air pollutants and odorants may have psychological and physical impact on exposed individuals
and the unpleasant room air is considered as one of the factors associated with sick building syndrome
comprising general symptoms such as headache and lethargy. Approaches for improving the quality of
indoor air are thus important as support for human health and well-being. Photo-oxidation catalyzed by
titanium dioxide (TiO2), is one of the methods used for elimination of volatile organic compounds,
which are the cause of odor nuisance in indoor and outdoor air. In the present investigation, the
efficacy of an experimental anti-odor air freshener based on TiO2 nanoparticles was estimated by
testing its ability in removing from a small air chamber (200 mL) the odor of triethylamine solutions
(50 μL at concentrations between 0.700 to 700 mM), used as a model volatile molecule for simulating
fish-like unpleasant indoor environment. The evaluation was performed by electronic nose which
provided a holistic and objective data on the efficacy of the product, demonstrating that the effects of
triethylamine even at the highest tested concentrations can be completely removed by application of
3.0 g of the product at 25% TiO2 nanoparticles concentration. The obtained results were confirmed by
gaschromatography-mass spectrometry (GC–MS) analysis addressed to the quantitative determination
of residual triethylamine in the environment after treatment by the anti-odor product.
215
Comprehensive study on the effects of sodium and potassium additives in size exclusion
chromatographic separations of protein biopharmaceuticals
Alexandre Goyon, Alain Beck, Jean-Luc Veuthey, Davy Guillarme, Szabolcs Fekete
ABSTRACT
To separate proteins solely based on their difference in hydrodynamic volume in size exclusion
chromatography (SEC), the ionic strength of the mobile phase has to be increased in order to avoid
secondary ionic interactions between proteins and the stationary phase. However, adding salts to the
mobile phase can have a serious effect on protein aggregation and can lead to artifacts. In the present
study, several monoclonal antibodies (mAbs) and the antibody-drug conjugate (ADC), trastuzumab
emtansine were selected to study the effect of mobile phase salt additive on aggregation
measurements. In a first instance, the same aggregation ratios between the dimeric and monomeric
forms of ten mAbs approved by the Food and Drug Administration (FDA) and the European Medicine
Agency (EMA) were obtained with three UHP-SEC columns. However, SEC analysis using various
amounts of NaCl provided surprising results for rituximab, e.g. presence of 0.8% aggregates with a
mobile phase containing 0.2 M NaCl, while no aggregates were observed without NaCl in the mobile
phase. Despite the absence of monomeric protein adsorption at the surface of the SEC resin, the
comparison of sodium- and potassium-based salts demonstrated the superiority of potassium-based
salts to reduce possible secondary electrostatic interactions, mainly between protein dimers and the
SEC support as well as to lower protein-salts interaction. To investigate the effect of mobile phase salt
additives on SEC measurements, fluorescence spectroscopy provided insights related to the possible
contribution of protein tertiary structure. Indeed, biopharmaceuticals could be classified depending on
the exposure of their tryptophan residues to the solvent in order to understand their propensity to
interact with the stationary phase or/and to undergo self-association.
Application of a rapid HILIC-UV method for synthesis optimization and stability studies of
immunogenic neo-glycoconjugates
F. Rinaldi, S. Tengattini, E. Calleri, T. Bavaro, C. Temporini
ABSTRACT
Proteins and glycoproteins with therapeutic activity are susceptible to environmental factors, which
can cause their degradation and the loss of their activity. Thus, the maintenance of their stability during
the production process is a critical factor. In this work, a simple and rapid hydrophilic interaction
liquid chromatography HILIC-UV method was validated in terms of accuracy, precision, linearity,
LOD, LOQ and specificity and applied to the investigation of the stability of intact proteins and their
neo-glycoconjugates with antigenic activity against tuberculosis. The method proved to be suitable for
the estimation of the degradation of the proteins under critical conditions (i.e. freeze-thaw cycles) and
for the monitoring of their coupling reaction with saccharidic moieties, without the need of sample
preparation. In addition, the chromatographic analysis allowed calculating the yields of the protein
glycosylation reaction.
216
Simple and rapid LC–MS method for the determination of circulating albumin
microheterogeneity in veal calves exposed to heat stress
Maurizio Baldassarre, Marina Naldi, Marco Domenicali, Sabrina Volo, Angelo Peli
ABSTRACT
Heat stress has a major impact on veal calves welfare and productivity. Prolonged exposure to warm
temperature is associated with several alterations of physiologic processes and increased systemic
inflammation and oxidative stress. Bovine serum albumin (BSA) is the most abundant plasma protein
and, besides the regulation of osmotic pressure, carries several additional functions, including
antioxidant, immunomodulatory, binding and transport activities. Such non-oncotic properties are
closely related to structural integrity of the circulating molecule and may be compromised in stressful
microenvironments as it occurs in heat stressed animals. Thus, in the present study we developed and
validated an LC–MS analytical technique for the characterization of circulating BSA
microheterogeneity in veal calves exposed to heat stress. The method was specifically tailored to the
structural characteristics of the BSA molecule as well as to the complexity of the biological samples,
allowing the identification of several BSA isoforms, each characterized by a specific structural defect.
The mass spectrometry based approach enabled the identification of BSA isoforms with reversible and
irreversible oxidation and/or glycation and the native BSA, the only isoform endowed with structural
and functional integrity. We found that, in veal calves, heat stress is associated to a significant
reduction of the native BSA and to a significant increment of the reversibly and irreversibly oxidized
BSA.
Molecular fingerprinting of principal neurons in the rodent hippocampus: A neuroinformatics
approach
D.J. Hamilton, C.M. White, C.L. Rees, D.W. Wheeler, G.A. Ascoli
ABSTRACT
Neurons are often classified by their morphological and molecular properties. The online knowledge
base Hippocampome.org primarily defines neuron types from the rodent hippocampal formation based
on their main neurotransmitter (glutamate or GABA) and the spatial distributions of their axons and
dendrites. For each neuron type, this open-access resource reports any and all published information
regarding the presence or absence of known molecular markers, including calcium-binding proteins,
neuropeptides, receptors, channels, transcription factors, and other molecules of biomedical relevance.
The resulting chemical profile is relatively sparse: even for the best studied neuron types, the
expression or lack thereof of fewer than 70 molecules has been firmly established to date. The mouse
genome-wide in situ hybridization mapping of the Allen Brain Atlas provides a wealth of data that,
when appropriately analyzed, can substantially augment the molecular marker knowledge in
Hippocampome.org. Here we focus on the principal cell layers of dentate gyrus (DG), CA3, CA2, and
CA1, which together contain approximately 90% of hippocampal neurons. These four anatomical
parcels are densely packed with somata of mostly excitatory projection neurons. Thus, gene expression
data for those layers can be justifiably linked to the respective principal neuron types: granule cells in
DG and pyramidal cells in CA3, CA2, and CA1. In order to enable consistent interpretation across
genes and regions, we screened the whole-genome dataset against known molecular markers of those
neuron types. The resulting threshold values allow over 6000 very-high confidence (>99.5%)
expressed/not-expressed assignments, expanding the biochemical information content of
Hippocampome.org more than five-fold. Many of these newly identified molecular markers are
potential pharmacological targets for major neurological and psychiatric conditions.
217
Volume 145 October 2017
Two-dimensional liquid chromatography in pharmaceutical analysis Instrumental aspects,
trends and applications
Marion Iguiniz, Sabine Heinisch
ABSTRACT
The interest in two-dimensional liquid chromatography (2D-LC) has been growing up since the last
decades. This promising technique appears as a relevant solution for various analytical challenges
encountered in pharmaceutical analysis. The objective of this review is to give an overview of past,
current and emerging trends in 2D-LC techniques applied to pharmaceutical compounds. The
referenced studies cover the late 1980s to the present. Information regarding the different aspects of
this analytical technique, including chromatographic conditions, instrumental setup and compounds of
interest, was compiled and summarized into a synoptic table. Particular attention is paid to key features
including (i) the interfaces used for coupling the two dimensions, (ii) the application fields, and (iii)
the chromatographic modes that can be combined together. Finally an attempt is made to predict future
advances in two-dimensional separation techniqes for pharmaceutical analysis.
Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A
generic method development approach
Balázs Bobály, Valentina D‘Atri, Alain Beck, Davy Guillarme, Szabolcs Fekete
ABSTRACT
Hydrophilic interaction liquid chromatography (HILIC) is a well-established technique for the
separation and analysis of small polar compounds. A recently introduced widepore stationary phase
expanded HILIC applications to larger molecules, such as therapeutic proteins. In this paper, we
present some generic HILIC conditions adapted for a wide range of FDA and EMA approved
recombinant monoclonal antibody (mAb) species and for an antibody-drug conjugate (ADC). Seven
approved mAbs possessing various isoelectric point (pI) and hydrophobicity as well as a cysteine
conjugated ADC were used in this study. Samples were digested by IdeS enzyme and digests were
further fragmented by chemical reduction. The resulting fragments were separated by HILIC. The
main benefit of HILIC was the separation of polar variants (glycovariants) in a reasonable analysis
time at the protein level, which is not feasible with other chromatographic modes. Three samples were
selected and chromatographic conditions were further optimized to maximize resolution. A
commercial software was used to build up retention models. Experimental and predicted
chromatograms showed good agreement and the average error of retention time prediction was less
than 2%. Recovery of various species and sample stability under the applied conditions were also
discussed.
218
A new platform for serological analysis based on porous 3-dimensional polyethylene sinter
bodies
Mohammed Alasel, Michael Keusgen
ABSTRACT
A new sensitive and selective platform, three-dimensional immunosensor, has been developed for a
rapid serological diagnosis; detection of a Borrelia infection was considered as a model assay. The
immunosensor is based on a 3-dimensional (3D) porous solid surface (sinter body) with dimensions of
2 × 2.5 mm where a recombinant variable lipoprotein surface-exposed protein (VlsE; Borrelia-antigen)
is immobilized by different techniques. The sinter body served as a robust and inexpensive carrier,
which facilitated a successful hydrophobic adsorption as well as covalent immobilization of the
antigen with sufficient amounts of on the surface. Because of sinter body‘s porosity, the detection
could be performed in an immune affinity flow system based on a little disposable plastic column. The
flow of reagents through the column is advantageous in terms of reducing the non-specific interaction
and shortening the test time. Furthermore, three labels were tested for a colorimetric detection: i) a
horseradish peroxidase (HRP) labeled secondary antibody, ii) nanoparticles based on Sudan IV, and
iii) gold nanoparticles modified with protein A. HRP secondary labeled antibody provides the most
sensitive test, 1000 fold dilution of serum sample can be clearly detected in only 20 min. Gold
nanoparticles modified with protein A were used as a direct label or as a catalyst for reduction of silver
ions. Direct detection with gold nanoparticles provides short time of analysis (5 min) while detection
of metallic silver required longer time (12 min) but with improved sensitivity. Nanoparticles based on
Sudan IV showed high background and were less favorable.
Designing a calibration set in spectral space for efficient development of an NIR method for
tablet analysis
Md Anik Alam, James Drennen, Carl Anderson
ABSTRACT
Designing a calibration set is the first step in developing a multivariate spectroscopic calibration
method for quantitative analysis of pharmaceutical tablets. This step is critical because successful
model development depends on the suitability of the calibration data. For spectroscopic-based
methods, traditional concentration based techniques for designing calibration sets are prone to have
redundant information while simultaneously lacking necessary information for a successful calibration
model. A method for designing a calibration set in spectral space was developed. The pure component
spectra of a tablet formulation were used to define the spectral space of that formulation. This method
maximizes the information content of measurements and minimizes sample requirements to provide an
efficient means for developing multivariate spectroscopic calibration. A comparative study was
conducted between a commonly employed full factorial approach to calibration development and the
newly developed technique. The comparison was based on a system to quantify a model drug,
acetaminophen, in pharmaceutical compacts using near infrared spectroscopy. A 2-factor full factorial
design (acetaminophen with 5 levels and MCC: Lactose with 3 levels) was used for calibration
development. Three replicates at each design point resulted in a total of 45 tablets for the calibration
set. Using the newly developed spectral based method, 11 tablets were prepared for the calibration set.
Partial least square (PLS) models were developed from respective calibration sets.
219
On-line prediction of the glucose concentration of CHO cell cultivations by NIR and Raman
spectroscopy: Comparative scalability test with a shake flask model system
Bence Kozma, Edit Hirsch, Szilveszter Gergely, László Párta, András Salgó
ABSTRACT
In this study, near-infrared (NIR) and Raman spectroscopy were compared in parallel to predict the
glucose concentration of Chinese hamster ovary cell cultivations. A shake flask model system was
used to quickly generate spectra similar to bioreactor cultivations therefore accelerating the
development of a working model prior to actual cultivations. Automated variable selection and several
pre-processing methods were tested iteratively during model development using spectra from six shake
flask cultivations. The target was to achieve the lowest error of prediction for the glucose
concentration in two independent shake flasks. The best model was then used to test the scalability of
the two techniques by predicting spectra of a 10 l and a 100 l scale bioreactor cultivation. The NIR
spectroscopy based model could follow the trend of the glucose concentration but it was not
sufficiently accurate for bioreactor monitoring. On the other hand, the Raman spectroscopy based
model predicted the concentration of glucose in both cultivation scales sufficiently accurately with an
error around 4 mM (0.72 g/l) that is satisfactory for the on-line bioreactor monitoring purposes of the
biopharma industry. Therefore, the shake flask model system was proven to be suitable for scalable
spectroscopic model development.
Conventional and accelerated-solvent extractions of green tea (camellia sinensis) for
metabolomics-based chemometrics
Joshua J. Kellogg, Emily D. Wallace, Tyler N. Graf, Nicholas H. Oberlies, Nadja B. Cech
ABSTRACT
Metabolomics has emerged as an important analytical technique for multiple applications. The value of
information obtained from metabolomics analysis depends on the degree to which the entire
metabolome is present and the reliability of sample treatment to ensure reproducibility across the
study. The purpose of this study was to compare methods of preparing complex botanical extract
samples prior to metabolomics profiling. Two extraction methodologies, accelerated solvent extraction
and a conventional solvent maceration, were compared using commercial green tea [Camellia sinensis
(L.) Kuntze (Theaceae)] products as test cases. The accelerated solvent protocol was first evaluated to
ascertain critical factors influencing extraction using a D-optimal experimental design study. The
accelerated solvent and conventional extraction methods yielded similar metabolite profiles for the
green tea samples studied. The accelerated solvent extraction yielded higher total amounts of extracted
catechins, was more reproducible, and required less active bench time to prepare the samples. This
study demonstrates the effectiveness of accelerated solvent as an efficient methodology for
metabolomics studies.
220
Phase separation of in situ forming poly (lactide-co-glycolide acid) implants investigated using a
hydrogel-based subcutaneous tissue surrogate and UV–vis imaging
Yu Sun, Henrik Jensen, Nickolaj J. Petersen, Susan W. Larsen, Jesper Østergaard
ABSTRACT
Phase separation of in situ forming poly (lactide-co-glycolide acid) (PLGA) implants with agarose
hydrogels as the provider of nonsolvent (water) mimicking subcutaneous tissue was investigated using
a novel UV–vis imaging-based analytical platform. In situ forming implants of PLGA-1-methyl-2-
pyrrolidinone and PLGA-triacetin representing fast and slow phase separating systems, respectively,
were evaluated using this platform. Upon contact with the agarose hydrogel, the phase separation of
the systems was followed by the study of changes in light transmission and absorbance as a function of
time and position. For the PLGA-1-methyl-2-pyrrolidinone system, the rate of spatial phase separation
was determined and found to decrease with increasing the PLGA concentration from 20% to 40%
(w/w). Hydrogels with different agarose concentrations (1% and 10% (w/v)) were prepared for
providing the nonsolvent, water, to the in situ forming PLGA implants simulating the injection site
environment. The resulting implant morphology depended on the stiffness of hydrogel matrix,
indicating that the matrix in which implants are formed is of importance. Overall, the work showed
that the UV–vis imaging-based platform with an agarose hydrogel mimicking the subcutaneous tissue
holds potential in providing bio-relevant and mechanistic information on the phase separation
processes of in situ forming implants.
Mid-infrared and near-infrared spectroscopy for rapid detection of Gardeniae Fructus by a
liquid-liquid extraction process
Lingyan Tao, Zhonglin Lin, Jiashan Chen, Yongjiang Wu, Xuesong Liu
ABSTRACT
Gardeniae Fructus is widely used in the pharmaceutical industry, and many studies have confirmed its
medical and economic value. In this study, samples collected from different liquid-liquid extraction
batches of Gardeniae Fructus were detected by mid-infrared (MIR) and near-infrared (NIR)
spectroscopy. Seven analytes, neochlorogenic acid (5-CQA), cryptochlorogenic acid (4-CQA),
chlorogenic acid (3-CQA), geniposidic acid (GEA), deacetyl-asperulosidic acid methyl ester
(DAAME), genipin-gentiobioside (GGB), and gardenoside (GA), were chosen as quality property
indexes of Gardeniae Fructus. The two kinds of spectra were each used to build models by single
partial least squares (PLS). Additionally, both spectral data were combined and modeled by multiblock
PLS. For single spectroscopy modeling results, NIR had a better prediction for high-concentration
analytes (3-CQA, DAAME, GGB, and GA) whereas MIR performed better for low-concentration
analytes (5-CQA, 4-CQA, and GEA). The multiblock methodology was found to be better compared
to single spectroscopy models for all seven analytes. Specifically, the coefficients of determination
(R2) of the NIR, MIR, and multiblock PLS calibration models of all seven components were higher
than 0.95. Relative standard errors of prediction (RSEP) were all less than 7%, except for models of
GGB, which were 10.36%, 13.24%, and 8.15% for the NIR-PLS, MIR-PLS, and multiblock models,
respectively. These results indicate that MIR and NIR spectrographic techniques could provide a new
choice for quality control in industrial production of Gardeniae Fructus.
221
Quantitative analysis of a biopharmaceutical protein in cell culture samples using automated
capillary electrophoresis (CE) western blot
Dong Xu, Kentaro Marchionni, Yunli Hu, Wei Zhang, Zoran Sosic
ABSTRACT
An effective control strategy is critical to ensure the safety, purity and potency of biopharmaceuticals.
Appropriate analytical tools are needed to realize such goals by providing information on product
quality at an early stage to help understanding and control of the manufacturing process. In this work,
a fully automated, multi-capillary instrument is utilized for size-based separation and western blot
analysis to provide an early readout on product quality in order to enable a more consistent
manufacturing process. This approach aims at measuring two important qualities of a
biopharmaceutical protein, titer and isoform distribution, in cell culture harvest samples. The acquired
data for isoform distribution can then be used to predict the corresponding values of the final drug
substance, and potentially provide information for remedy through timely adjustment of the
downstream purification process, should the expected values fall out of the accepted range.
Synchronous characterization of carbohydrates and ginsenosides yields deeper insights into the
processing chemistry of ginseng
Shan-Shan Zhou, Jun Xu, Ming Kong, Ka-Man Yip, Hu-Biao Chen
ABSTRACT
Carbohydrates and ginsenosides in ginseng are biologically interrelated. Their synchronous analysis is
therefore essential in chemical research on ginseng to characterize its ―holistic‖ quality. Here we
investigated the processing chemistry of red ginseng (RG), a ginseng product processed by water-
steaming, for which both carbohydrates and ginsenosides were qualitatively and quantitatively
determined through multiple analytical techniques. Results revealed that the steam-processing not only
qualitatively and quantitatively altered the ginsenosides but also affected the polymeric carbohydrates
via changing their physiochemical parameters, i.e. water-solubility, molecular size, types and ratios of
constituent monosaccharides. Potential mechanisms involved in the transformation of ginseng
chemicals are proposed and discussed, including hydrolysis (deglycosylation, demalonylation,
deacetylation), dehydration, polymerization, volatilization, reduction and the Maillard reaction. The
study strengthens the research on the processing chemistry of RG, and therefore should be helpful for
elucidating the scientific basis of RG preparation and application.
222
Development and validation of an ICP-MS method for the determination of elemental impurities
in TP-6076 active pharmaceutical ingredient (API) according to USP 〈232〉/〈233〉
Osama Chahrour, John Malone, Mark Collins, Vrushali Salmon, Nick Dunwoody
ABSTRACT
The new guidelines of the United States pharmacopeia (USP), European pharmacopeia (EP) and
international conference on harmonization (ICH) regulating elemental impurities limits in
pharmaceuticals signify the end of unspecific analysis of metals as outlined in USP 〈231〉. The new
guidelines specify both daily doses and concentration/limits of elemental impurities in pharmaceutical
final products, active pharmaceutical ingredients (API) and excipients. In chapter USP 〈233〉
method implementation, validation and quality control during the analytical process are described. We
herein report the use of a stabilising matrix that overcomes low spike recovery problem encountered
with Os and allows the determination of all USP required elemental impurities (As, Cd, Hg, Pb, V, Cr,
Ni, Mo, Cu, Pt, Pd, Ru, Rh, Os and Ir) in a single analysis. The matrix was used in the validation of a
method to determine elemental impurities in TP-6076 active pharmaceutical ingredient (API) by ICP-
MS according to the procedures defined in USP〈233〉 and to GMP requirements. This validation
will support the regulatory submission of TP-6076 which is a novel tetracycline analogue effective
against the most urgent multidrug-resistant gram-negative bacteria. Evaluation of TP-6076 in IND-
enabling toxicology studies has led to the initiation of a phase 1 clinical trial.
Comparison of SEC and CE-SDS methods for monitoring hinge fragmentation in IgG1
monoclonal antibodies
Oluwatosin O. Dada, Romesh Rao, Natalie Jones, Nomalie Jaya, Oscar Salas-Solano
ABSTRACT
Fragmentation of monoclonal antibodies is a critical quality attribute routinely monitored to assess the
purity and integrity of the product from development to commercialization. Cleavage in the upper
hinge region of IgG1 monoclonal antibodies is a common fragmentation pattern widely studied by size
exclusion chromatography (SEC). Capillary electrophoresis with sodium dodecylsulfate (CE-SDS) is a
well-established technique commonly used for monitoring antibody fragments as well, but its
comparability to SEC in monitoring hinge fragments has not been established until now. We report a
characterization strategy that establishes the correlation between hinge region fragments analyzed by
SEC and CE-SDS. Monoclonal antibodies with elevated hinge fragments were generated under low pH
stress conditions and analyzed by SEC and CE-SDS. The masses of the fragments generated were
determined by LC-MS. Electrophoretic migration of the hinge fragmentation products in CE-SDS
were determined based on their mass values. Comparative assessment of fragments by SEC, and CE-
SDS showed similar correlation with incubation time. This study demonstrates that CE-SDS can be
employed as a surrogate technique to SEC for monitoring hinge region fragments. Most importantly,
combination of these techniques can be used to obtain comprehensive understanding of fragment
related characteristics of therapeutic protein products.
223
Revisiting blood-brain barrier: A chromatographic approach
Xavier Subirats, Laura Muñoz-Pascual, Michael H. Abraham, Martí Rosés
ABSTRACT
Drugs designed to reach a pharmacological CNS target must be effectively transported across the
blood-brain barrier (BBB), a thin monolayer of endothelial cells tightly attached together between the
blood and the brain parenchyma. Because of the lipidic nature of the BBB, several physicochemical
partition models have been studied as surrogates for the passive permeation of potential drug
candidates across the BBB (octanol-water, alkane-water, PAMPA...). In the last years, biopartition
chromatography is gaining importance as a noncellular system for the estimation of biological
properties in early stages of drug development. Microemulsions (ME) are suitable mobile phases,
because of their ease of formulation, stability and adjustability to a large number of compositions
mimicking biological structures. In the present work, several microemulsion liquid chromatographic
(MELC) systems have been characterized by means of the Abraham‘s solvation parameter model, in
order to assess their suitability as BBB distribution or permeability surrogates. In terms of similarity
between BBB and MELC systems (dispersion forces arising from solute non-bonded electrons,
dipolarity/polarizability, hydrogen-bond acidity and basicity, and molecular volume), the passive
permeability surface area product (log PS) for neutral (including zwitterions), fully and partially
ionized drugs was found to be well correlated with the ME made of 3.3% SDS (w/v; surfactant) 0.8%
heptane (w/v; oil phase) and 6.6% 1-butanol (w/v; co-surfactant) in 50 mM aqueous phosphate buffer,
pH 7.4.
Liquid chromatographic enantioseparation of carbocyclic β-amino acids possessing limonene
skeleton on macrocyclic glycopeptide-based chiral stationary phases
Tímea Orosz, Nóra Grecsó, Gyula Lajkó, Zsolt Szakonyi, Antal Péter
ABSTRACT
Polar-ionic and reversed-phase high-performance liquid chromatographic separations of limonene-
based cyclic β-amino acid enantiomers were carried out by using macrocyclic glycopeptide-based
chiral selectors applying Chirobiotic T, TAG and R columns. The effects of additives, concentration of
the co- and counter-ions and the temperature in polar-ionic mobile phase systems were studied. The
influence of pH, MeOH content and alcohol additives were investigated in the reversed-phase mode.
The difference in the change in standard enthalpy Δ(ΔH°), entropy Δ(ΔS°), and free energy Δ(ΔG°)
was calculated from the linear van't Hoff plots derived from the ln α vs 1/T curves in the temperature
range 5–40 °C. Unusual temperature behavior was observed on Chirobiotic TAG for most of the
analytes: decreased retention times were accompanied with increased separation factors with
increasing temperature, and separation was entropically-driven. For two of the studied analytes
enthalpically-driven enantioseparations were observed. The elution sequence was determined in all
cases, but no general rule could be established.
224
On-line coupling of molecularly imprinted solid phase extraction with liquid chromatography
for the fast determination of coumarins from complex samples
Andrea Machyňáková, Ivona Lhotská, Katarína Hroboňová, Dalibor Ńatínský
ABSTRACT
In this work, an on-line SPE-HPLC method with spectrophotometric detection was developed for the
determination of coumarins in complex samples. For the on-line cleanup of samples, a molecularly
imprinted polymer was packed into the column cartridge and coupled directly with HPLC (MISPE-
HPLC) using a column switching system. The separation of coumarins was performed on a C18 core-
shell column (100 × 4.6 mm, 5 μm) with a mobile phase consisting of 0.3% acetic acid/acetonitrile
with gradient elution at a flow-rate of 1 mL min−1. The total time of the whole analytical run,
including the extraction step, was 13.25 min. The on-line MISPE-HPLC method was optimized and
validated. The results showed good linearity (0.10–100 μg mL−1) with correlation coefficients higher
than 0.99. The LOD values were from 0.03 to 0.15 μg mL−1. The proposed method was successfully
applied for analysis of real samples (Cassia cinnamon, chamomile tea, and Tokaj specialty wines) and
obtained recoveries varied from 78.7% to 112.2% with an RSD less than 9%.
Metabolic profiling analysis of Siraitia grosvenorii revealed different characteristics of green
fruit and saccharified yellow fruit
Chengnan Fang, Qingqing Wang, Xinyu Liu, Guowang Xu
ABSTRACT
Siraitia grosvenorii is an economic and medicinal plant, its fruit is considered to be good to health for
its diverse bioactive ingredients. However, the clarification of chemical composition and their changes
after saccharification procedure are not well performed. In present study, a metabolomics method
based on ultra-high-performance liquid chromatography tandem quadrupole time-of-flight mass
spectrometry was developed for metabolic profiling acquisition of Siraitia grosvenorii extract.
Furthermore, information dependent analysis (IDA) combined with self-constructed LC–MS/MS
identification system for metabolites were employed to identify primary and secondary metabolites in
Siraitia grosvenorii. A total of 126 metabolites were identified or tentatively identified. The obvious
differences of metabolic profiling between green fruit and saccharified yellow fruit were observed, and
metabolites showed their own distribution characteristics in peel, flesh and seed. The majority of the
nutrients and effective components were more distributed in flesh and peel, and saccharification was
conducive to accumulation of sweet glycosides. This study not only expanded metabolite composition
information of Siraitia grosvenorii, but also specified distribution characteristics of identified
metabolites.
225
Comparison of α-glucosidase inhibitory effect and bioactive constituents of Anemarrhenae
Rhizoma and Fibrous Roots
Si-Hui Nian, Hui-Jun Li, E-Hu Liu, Ping Li
ABSTRACT
Comprehensive utilization of medicinal plant resources is of great significance for sustainable
development of traditional Chinese medicines. In the present study, the α-glucosidase inhibitory
activities of the rhizome and fibrous root of Anemarrhena Asphodeloides Bunge, were compared
detailedly, and a high performance liquid chromatography coupled with electrospray ionization tandem
triple quadrupole mass spectrometry (HPLC-QQQ/MS) method was developed for simultaneous
quantification of seven bioactive constituents including neomangiferin, mangiferin, isomangiferin,
timosaponin BII, timosaponin B, timosaponin AIII, and timosaponin N in 40 batches of samples. The
results demonstrated that fibrous root extracts had more potent α-glucosidase inhibitory activity than
rhizome extracts. Mangiferin and isomangiferin were abundant in fibrous root, while the analyzed
saponins were rich in rhizome. Based on the chemometrics methods including principal component
analysis (PCA), orthogonal partial least square discriminant analysis (OPLS-DA), and partial least
square (PLS), mangiferin and isomangiferin might be mainly responsible for α-glucosidase inhibitory
activity of the genus. These findings indicate that the established HPLC-QQQ/MS method was proven
to be useful and efficient for quality control of Anemarrhena materials, and fibrous root had the
potential to be utilized as anti-diabetic medicinal resource.
Characterization of forced degradation products of torasemide through MS tools and
explanation of unusual losses observed during mass fragmentation of drug and degradation
products through density functional theory
Moolchand Kurmi, Neha Patel, Shalu Jhajra, Prasad V. Bharatam, Saranjit Singh
ABSTRACT
Mass spectrometry tools (HRMS/LC-HRMS, MSn, and/or on-line H/D exchange) were employed to
establish mass fragmentation pattern of torasemide and to characterize its degradation products.
During collision-induced dissociation, multiple rearrangement processes and unusual losses of sulfur
(S), sulfanyl (HS), sulfur dioxide (SO2), sulphinic acid radical (HSO2), sulfur monoxide (SO), carbon
monoxide (CO), formyl radical (CHO) and C5H3NOS were observed. The same were successfully
explained by study of energy profiles, established by application of density functional theory (DFT).
226
Metabolic profiling of the traditional Chinese medicine formulation Yu Ping Feng San for the
identification of constituents relevant for effects on expression of TNF-α, IFN-γ, IL-1β and IL-4
in U937 cells
Stefanie Nikles, Marlene Monschein, Huiqin Zou, Yong Liu, Rudolf Bauer
ABSTRACT
Yu Ping Feng San (YPFS) is a classical TCM formulation which has been traditionally used for
treatment of immune system related diseases such as chronic bronchitis, allergic rhinitis and asthma.
The formula is a mixture of Radix Saposhnikoviae (Fangfeng), Radix Astragali (Huangqi), and
Rhizoma Atractylodis macrocephalae (Baizhu). TLC- and LC-DAD-ESI-MS/MS methods have been
developed for the analysis of the metabolic profiles of the single herbs and of the formula. Decoctions
and ASE extracts were analyzed in order to trace components of the individual herbs in YPFS. Nine
constituents of Radix Saposhnikoviae, ten constituents of Radix Astragali and five constituents of
Rhizoma Atractylodis macrocephalae have been assigned in the chemical profiles of the formula,
which now allow the standardisation of YPFS. The pharmacological testing showed that all extracts
significantly inhibited expression of TNF-α, IFN-γ, and IL-1β in U937 cells, while the inhibition of IL-
4 was consistently low. Compared to conventional analyses which are focused on a limited set of
compounds, metabolomics approaches, together with novel data processing tools, enable a more
holistic comparison of the herbal extracts. In order to identify the constituents which are relevant for
the immunomodulatory effects of the formula, metabolomics studies (PCA, OPLS-DA) have been
performed using UPLC/QTOF MS data.
A comprehensive stability-indicating HPLC method for determination of chloroquine in active
pharmaceutical ingredient and tablets: Identification of oxidation impurities
Ana Silva Coelho, Clara Elisa Pontes Chagas, Rodrigo Maia de Pádua, Gerson Antônio Pianetti,
Christian Fernandes
ABSTRACT
Malaria is the most common parasitic disease in humans. It is estimated that 3 billion people live under
the risk of contracting this disease in the world. Chloroquine (CQ) is the drug of choice to treat cases
of non-complicated malaria. Forced degradation studies are important to know the drug‘s potentials
degradation products and to develop a stability indicating method. Thus, chloroquine active
pharmaceutical ingredient (API), chloroquine tablets and placebo were submitted to a detailed forced
degradation study, using several stressing agents. The results were used on the development of a
stability indicating method, using high performance liquid chromatography. The method was validated
showing selectivity, precision, accuracy, robustness and linearity in the range of 30–360 μg/mL of
chloroquine. Chloroquine API and tablets were susceptible to alkaline hydrolysis with NaOH 1 mol/L,
and to oxidation with H2O2 3.0%. Two degradation products were formed in oxidative test. Kinetics
of chloroquine degradation in alkaline hydrolysis was performed for both API and tablets. The
calculated decay constant (k1) was 0.223 days−1 for API and 0.182 days−1 for tablets, while the half-
life (t1/2) was 3.1 days for API and 3.8 days for tablets. Chemical structures have been proposed for
the two degradation products formed in the presence of H2O2, using an UHPLC-UV-MS/MS
approach.
227
Identification of impurities in macrolides by liquid chromatography–mass spectrometric
detection and prediction of retention times of impurities by constructing quantitative structure–
retention relationship (QSRR)
Xia Zhang, Jin Li, Chen Wang, Danqing Song, Changqin Hu
ABSTRACT
Macrolides are multicomponent drugs whose impurity control is always a challenge demanding
analysis method with good sensitivity and selectivity. Three separate, sensitive, accurate liquid
chromatography tandem mass spectrometry methods (LC–MS) were developed for the measurement
of three 16-membered ring macrolides (josamycin, josamycin propionate and midecamycin acetate)
and related substances in commercial samples. The characteristics of impurities in macrolides were
summarized as useful guidance for the impurity analysis of this class of drugs. For each drug, a large
number of unknown components have been detected with the high-sensitive MS detector and possible
structures of the majority of them were postulated based on the summarized fragmentation rules of 16-
membered ring macrolides. A QSRR model was constructed by multilinear regression to predict the
retention times of identified impurities which were not detected by the LC–MS methods, without
obtaining their reference standards. Satisfactory performance was obtained during leave-one-out cross-
validation with a predictive ability (Q2) of 0.95. The generalisation ability of the model was further
confirmed by an average error of 2.3% in external prediction. The best QSRR model, based on eight
molecular descriptors, exhibited a promising predictive performance and robustness.
The impact of ZnO and TiO2 on the stability of clotrimazole under UVA irradiation:
Identification of photocatalytic degradation products and in vitro cytotoxicity assessment
Agata Kryczyk, Paweł Żmudzki, Paulina Koczurkiewicz, Joanna Piotrowska, Urszula Hubicka
ABSTRACT
In order to ensure the safe and effective use of pharmaceutical products especially for topical
administration photostability testing is necessary. The current paper presents an in-depth analysis of
the stability of one of the most common antifungal agents, namely clotrimazole. Clotrimazole has
proven to be stable under UVA irradiation in applied experimental conditions, but the presence of
catalysts such as ZnO and TiO2 has contributed significantly to the degradation of this compound. The
findings indicate that its photocatalytic degradation reactions followed the pseudo first-order kinetics
with rate constant depending on the pH and the used solvent. Using LC–MS/MS, 14 presumable
degradation products of clotrimazole were identified and the plausible transformation pathways were
proposed. The in vitro cytotoxicity risk evaluation based on photostability of clotrimazole was also
performed using the Human skin fibroblast cell line (BJ) ATCC™ CRL-2522. There was no
statistically significant difference between cells viability in all analyzed combinations of clotrimazole,
TiO2/ZnO, and UVA irradiation (p < 0.05).
228
Chemometrically assisted development and validation of LC–MS/MS method for the analysis of
potential genotoxic impurities in meropenem active pharmaceutical ingredient
Katerina Grigori, Yannis L. Loukas, AnĎelija Malenović, Vicky Samara, Yannis Dotsikas
ABSTRACT
A sensitive Liquid Chromatography tandem mass spectrometry (LC–MS/MS) method was developed
and validated for the quantitative analysis of three potential genotoxic impurities (318BP, M9, S5) in
meropenem Active Pharmaceutical Ingredient (API). Due to the requirement for LOD values in ppb
range, a high concentration of meropenem API (30 mg/mL) had to be injected. Therefore, efficient
determination of meropenem from its impurities became a critical aim of this study, in order to divert
meropenem to waste, via a switching valve. After the selection of the important factors affecting
analytes‘ elution, a Box-Behnken design was utilized to set the plan of experiments conducted with
UV detector. As responses, the separation factor s between the last eluting impurity and meropenem,
as well as meropenem retention factor k were used. Grid point search methodology was implemented
aiming to obtain the optimal conditions that simultaneously comply to the conflicted criteria. Optimal
mobile phase consisted of ACN, methanol and 0.09% HCOOH at a ratio 71/3.5/15.5 v/v. All
impurities and internal standard omeprazole were eluted before 7.5 min and at 8.0 min the eluents were
directed to waste. The protocol was transferred to LC–MS/MS and validated according to ICH
guidelines.
Identification and interconversion of isomeric 4,5-functionalized 1,2,3-thiadiazoles and 1,2,3-
triazoles in conditions of electrospray ionization
D.M. Mazur, M.E. Zimens, V.A. Bakulev, A.T. Lebedev
ABSTRACT
1,2,3-Triazoles and 1,2,3-thiadiazoles have been receiving permanent interest due to their exciting
chemical reactivity and interesting biological properties including antibacterial, anticancer and
antiviral activities. There are four compounds bearing 1H-1,2,3-triazole core in clinical studies which
may appear in the market of drugs in nearest future. Definitely reliable methods of their identification
and quantification should be developed by that time. Mass spectrometry showed itself as the most
reliable method of analysis when dealing with trace levels of organic compounds in the mixtures and
in the most complex matrices, including biological ones. In the present study tandem mass
spectrometry was used to study fragmentation pathways of protonated and deprotonated molecules of
isomeric 4,5-functionalized 1,2,3-thiadiazoles and 1,2,3-triazoles in conditions of electrospray
ionization (ESI). A group of marker ions allowing differentiation between the targeted isomeric
compounds was established. Besides, interconversion of these isomers into one another was studied in
the gas phase in conditions mimicking these processes in solution.
229
Dissolution assessment of allopurinol immediate release tablets by near infrared spectroscopy
Jelena Smetińko, Sneņana Miljanić
ABSTRACT
The purpose of this study was to develop a NIR spectroscopic method for assessment of drug
dissolution from allopurinol immediate release tablets. Thirty three different batches of allopurinol
immediate release tablets containing constant amount of the active ingredient, but varying in excipients
content and physical properties were introduced in a PLS calibration model. Correlating allopurinol
dissolution reference values measured by the routinely used UV/Vis method, with the data extracted
from the NIR spectra, values of correlation coefficient, bias, slope, residual prediction determination
and root mean square error of prediction (0.9632, 0.328%, 1.001, 3.58, 3.75%) were evaluated. The
obtained values implied that the NIR diffuse reflectance spectroscopy could serve as a faster and
simpler alternative to the conventional dissolution procedure, even for the tablets with a very fast
dissolution rate (>85% in 15 minutes). Apart from the possibility of prediction of the allopurinol
dissolution rate, the other multivariate technique, PCA, provided additional data on the non-chemical
characteristics of the product, which could not be obtained from the reference dissolution values.
Analysis on an independent set of samples confirmed that a difference between the UV/Vis reference
method and the proposed NIR method was not significant. According to the presented results, the
proposed NIR method may be suitable for practical application in routine analysis and for continuously
monitoring the product's chemical and physical properties responsible for expected quality.
Enhanced and green extraction polyphenols and furanocoumarins from Fig (Ficus carica L.)
leave using deep eutectic solvents
Tong Wang, Jiao Jiao, Qing-Yan Gai, Peng Wang, Yu-Jie Fu
ABSTRACT
Nowadays, green extraction of bioactive compounds from medicinal plants has gained increasing
attention. As green solvent, deep eutectic solvent (DES) have been highly rated to replace toxic
organic solvents in extraction process. In present study, to simultaneous extraction five main bioactive
compounds from fig leaves, DES was tailor-made. The tailor-made DES composed of a 3:3:3 molar
ratio of glycerol, xylitol and D-(−)-Fructose showed enhanced extraction yields for five target
compounds simultaneously compared with traditional methanol and non-tailor DESs. Then, the tailor-
made DES based extraction methods have compared and microwave-assisted extraction was selected
and optimized due to its high extraction yields with lower time consumption. The influencing
parameters including extraction temperature, liquid-solid ratio, and extraction time were optimized
using response surface methodology (RSM). Under optimal conditions the extraction yield of
caffeoylmalic acid, psoralic acid-glucoside, rutin, psoralen and bergapten was 6.482 mg/g, 16.34 mg/g,
5.207 mg/g, 15.22 mg/g and 2.475 mg/g, respectively. Macroporous resin D101 has been used to
recovery target compounds with recovery yields of 79.2%, 83.4%, 85.5%, 81.2% and 75.3% for
caffeoylmalic acid, psoralic acid-glucoside, rutin, psoralen and bergapten, respectively. The present
study suggests that DESs are truly designer and efficient solvents and the method we developed was
efficient and sustainable for extraction main compounds from Fig leaves.mg/g
230
Site- and species-specific hydrolysis rates of cocaine
Levente Szöcs, Gergely Völgyi, Ákos Urai, Sándor Hosztafi, Béla Noszál
ABSTRACT
The hydroxide-catalyzed non-enzymatic hydrolysis of cocaine is quantified in terms of ten site- and
species-specific rate constants in connection with also ten site- and species-specific acid-base
equilibrium constants, comprising all the twelve coexisting species in solution. This characterization
involves the major and minor decomposition pathways via benzoylecgonine and ecgonine methyl
ester, respectively, leading to ecgonine, the final product. Hydrolysis has been found to be 10–330
times faster at site 2 than at site 3, depending on the ionization status of the amino moiety and the rest
of the molecule. Nitrogen protonation accelerates the hydrolyses approximately ten times both at site 2
and site 3.
Comparative stability-indicating chromatographic methods for determination of 4-
hexylresorcinol in pharmaceutical formulation and shrimps
Rafeek F. Shokry, Lories I. Bebawy, Mohamed R. Elghobashy, Samah S. Abbas
ABSTRACT
Three chromatographic stability-indicating methods were developed for determination of 4-
hexylresorcinol in pure form and in a pharmaceutical formulation. Method A was based on a gradient
elution liquid chromatographic HPLC determination of 4-hexylresorcinol, its related impurities and in
presence of its degradation products. UPLC–MS/MS (Method B) was described for determination of
the cited drug in presence of its degradation products. Method C was a thin- layer chromatography
(TLC)-densitometry method for the separation and determination of the active ingredient, one of its
related impurities and in presence of its degradation products. The mechanism of alkali, oxidative and
photodegradation of 4-hexylresorcinol was studied according to ICH guidelines. The degradation
products were characterized by the LC–MS/MS method. Methods A and B were applicable for
determination of 4-hexylresorcinol residues in shrimp meat. The studied drug was easily degraded in
alkali medium giving toxic compounds. The results obtained by the proposed methods were
statistically analyzed and compared with those obtained by applying a reported method.
231
Enantiomeric separation of seven β-agonists by NACE—Study of chiral selectivity with
diacetone-d-mannitol–boric acid complex
Lili Lv, Lijuan Wang, Jun Li, Yajun Jiao, Hongyuan Yan
ABSTRACT
A rapid and effective nonaqueous capillary electrophoresis (NACE)–ultraviolet (UV) method was
developed for the enantiomeric separation of seven β-agonists. Diacetone-d-mannitol–boric acid
complex was used as a new chiral selector. It was in situ synthesized by the reaction of diacetone-d-
mannitol and boric acid in methanol medium containing triethylamine. The effects of diacetone-d-
mannitol, boric acid, and triethylamine concentrations on the enantioseparation were carefully
investigated. Under the optimized conditions, baseline enantioseparation could be obtained for six of
the tested β-agonists within 12 min. These results were better than that obtained with d-mannitol–boric
acid complex in previous work. 11B nuclear magnetic resonance (11B NMR) was applied to determine
the fraction of boron species and confirm the formation of diacetone-d-mannitol–boric acid complex.
Validation of the established NACE method was also carried out according to ICH guidelines.
Calibration curves showed good linearity with correlation coefficients (r) ≥ 0.9992 over a certain
concentration range for each enantiomer of the tested five β-agonists. The relative standard deviations
(RSDs) of intra-day precisions and inter-day precisions of migration times were ≤1.4% (n = 6), and
≤6.3% (n = 10), respectively. That of peak areas were ≤3.7% (n = 6), and ≤5.6% (n = 10), respectively.
The limits of detection (LODs) and the limits of quantitation (LOQs) based on the signal-to-noise
ratios of 3 and 10 were found below 1.25 μg mL−1 and 5.00 μg mL−1, respectively.
An optimized HPLC-UV method for quantitatively determining sesquiterpenes in
Nardostachyos Radix et Rhizoma
Vu Ngoc Han Le, Trong Quan Khong, Min Kyun Na, Kyung Tae Kim, Jong Seong Kang
ABSTRACT
Nardostachyos Radix et Rhizoma (NR), the root and rhizome from either Nardostachys jatamansi
Batal or Nardostachys jatamansi DC, is known to have biological functions including neuro-protective
and anti-pancreatitis activity. The main bioactive compounds within NR are all classified as
sesquiterpenes, and include desoxo-narchinol A, nardosinonediol, and nardosinone. Although NR is a
valuable herb that is widely used in many Asian countries, robust quality control protocols for NR are
still in question, especially those that can analyze the three main active compounds. Current
quantitative methods within the Chinese Pharmacopoeic use nardosinone as a marker compounds. One
compound cannot represent a complicated matrix, and is thus insufficient to control the quality of this
herbal medicine. Moreover, there are no high-performance liquid chromatography (HPLC) methods
that can simultaneously analyze desoxo-narchinol A (DA), nardosinonediol (NE), and nardosinone
(ND) within NR. This study aimed to establish an efficient quality control protocol by developing an
analytical method that simultaneously detects the three sesquiterpenes with HPLC using response
surface methodology (RSM) to optimize sample preparation. Optimized HPLC conditions included a
mobile phase of 0.1% formic acid in water (A), and a 0.1% formic acid in acetonitrile (B) under an
elution program of 20% B–80% B for 30 min at 254 nm. The method was well validated,
demonstrating satisfactory linearity, accuracy, precision, recovery, repeatability, and stability.
Optimized conditions for creating the analytical sample were predicted by RSM using a Box-Behnken
design.
232
Photostability testing using online reactor HPLC hyphenation and mass spectrometric
compound identification illustrated by ketoprofen as model compound
Jaber Assaf, Diego Zulkiewicz Gomes, Bernhard Wuest, Maria Kristina Parr
ABSTRACT
Investigations on the photochemical stability of pharmaceutical substances are mandatory in drug
development and licensing as photo-induced degradation of an active pharmaceutical ingredient (API)
may not only lead to decreased API concentrations but also to toxic or reactive products. Thus, the US
Food and Drug Administration (FDA) and the International Council for Harmonisation of Technical
Requirements for Pharmaceuticals for Human Use (ICH) issued Guidance for Industry Q1B
―Photostability Testing of New Drug Substances and Products‖ for testing of pure but also packed
drugs. However, photoproducts are also known to be generated in vivo under sunlight exposure of the
skin and lead to considerable amounts of adverse drug effects. Herein we present an alternative system
that may be used for photostability testing mimicking both situations. It combines a tailored
photoreactor with an exchangeable pen light source and a modified HPLC system with online-SPE.
Identification of photoproducts may be performed using mass spectrometry. The potential of accurate
mass spectrometry as a tool for identification of photoproducts was demonstrated as well. A
comparison of the online photoreactor system and the traditional photochamber irradiation was
performed using ketoprofen for proof of concept. In both designs acetylbenzophenone and
ethylbenzophenone were detected as main photoproducts. The new device allows for fast and easy
photostability studies that may help to reduce time consuming in vitro experiments and animal trials.
Using state of the art instruments kinetic studies could also easily be performed with qualitative and
quantitative perspectives combined into one experimental design with only very low amounts of API
needed. This may be useful in early drug development, where only small amounts of API are available.
Scale-up may also be easily realized for the generation of reference material for quantification and
quality control (QC) processes as well as for toxicity testing.
Optoelectronic iron detectors for pharmaceutical flow analysis
Natalia Rybkowska, Robert Koncki, Kamil Strzelak
ABSTRACT
Compact flow-through optoelectronic detectors fabricated by pairing of light emitting diodes have
been applied for development of economic flow analysis systems dedicated for iron ions
determination. Three analytical methods with different chromogens selectively recognizing iron ions
have been compared. Ferrozine and ferene S based methods offer higher sensitivity and slightly lower
detection limits than method with 1,10‐phenantroline, but narrower ranges of linear response. Each
system allows detection of iron in micromolar range of concentration with comparable sample
throughput (20 injections per hour). The developed flow analysis systems have been successfully
applied for determination of iron in diet supplements. The utility of developed analytical systems for
iron release studies from drug formulations has also been demonstrated.
233
Isoconversional approach for non-isothermal decomposition of un-irradiated and photon-
irradiated 5-fluorouracil
Hala Sh. Mohamed, AbdelRahman A. Dahy, Refaat M. Mahfouz
ABSTRACT
Kinetic analysis for the non-isothermal decomposition of un-irradiated and photon-beam-irradiated 5-
fluorouracil (5-FU) as anti-cancer drug, was carried out in static air. Thermal decomposition of 5-FU
proceed in two steps. One minor step in the temperature range of (270–283 °C) followed by the major
step in the temperature range of (285–360 °C). The non-isothermal data for un-irradiated and photon-
irradiated 5-FU was analyzed using linear (Tang) and non-linear (Vyazovkin) isoconversional
methods. The results of the application of these free models on the present kinetic data showed quite a
dependence of the activation energy on the extent of conversion. For un-irradiated 5-FU, the non-
isothermal data analysis indicates that the decomposition is generally described by A3 and A4 modeles
for the minor and major decomposition steps, respectively. For a photon-irradiated sample of 5-FU
with total absorbed dose of 10 Gy, the decomposition is controlled by A2 model throughout the
coversion range. The activation energies calculated in case of photon-irradiated 5-FU were found to be
lower compared to the values obtained from the thermal decomposition of the un-irradiated sample
probably due to the formation of additional nucleation sites created by a photon-irradiation. The
decomposition path was investigated by intrinsic reaction coordinate (IRC) at the B3LYP/6-
311++G(d,p) level of DFT. Two transition states were involved in the process by homolytic rupture of
NH bond and ring secession, respectively.
Characterization of impurities in sodium cromoglycate drug substance and eye drops using LC-
ESI-ion trap MS and LC-ESI-QTOF MS
Peixi Zhu, Jingxian Lu, Zhijian Wang, Weike Su, Erwin Adams
ABSTRACT
As requested by regulatory authorities, impurity profiling is an important issue of quality control. In
this work, a simple and sensitive liquid chromatographic (LC) method compatible with mass
spectrometry (MS) was developed to study related substances and degradation products in sodium
cromoglycate drug substance and eye drops. The method used a Sunfire column (4.6 mm × 150 mm,
3.5 μm). Mobile phase A consisted of 10 mM ammonium formate and mobile phase B were
acetonitrile. Linear gradient elution with a post-run time of 8 min was performed as follows: 0–30 min,
3% B to 50% B; 30–35 min, 50% B. The flow rate was set at 1.0 mL/min. Degradation experiments
were performed to check the stability indicating properties of the developed method. Based on MSn
spectral data and exact mass measurements, the chemical structures of 2 unknown impurities and 6
unknown degradation products were characterized, including impurity C listed in the European
Pharmacopoeia as unknown structure. In addition, a plausible mechanism for the formation of the
degradation products was also proposed.
234
Method validation and nanoparticle characterization assays for an innovative amphothericin B
formulation to reach increased stability and safety in infectious diseases
Maraine Catarina Tadini, Ana Maria de Freitas Pinheiro, Daniel Blascke Carrão, Ana Luiza Scarano
Aguillera Forte, Franciane Marquele-Oliveira
ABSTRACT
Drug Delivery Systems (DDS) of known drugs are prominent candidates towards new and more-
effective treatments of various infectious diseases as they may increase drug bioavailability, control
drug delivery and target the site of action. In this sense, the encapsulation of Amphotericin B (AmB) in
Nanostructured Lipid Carriers (NLCs) designed with pH-sensible phospholipids to target infectious
tissues was proposed and suitable analytical methods were validated, as well as, proper nanoparticle
characterization were conducted. Characterization assays by Dinamic Light Scattering (DLS) and
Atomic Force Microscopy demonstrated spherical particles with nanometric size 268.0 ± 11.8 nm and
Zeta Potential −42.5 ± 1.5 mV suggestive of important stability. DSC/TGA and FT-IR assessments
suggested mechanical encapsulation of AmB. The AmB aggregation study indicated that the
encapsulation provided AmB at the lowest cytotoxic form, polyaggregate. Analytical methods were
developed and validated according to regulatory agencies in order to fast and assertively determine
AmB in nanoparticle suspension and, in Drug Encapsulation Efficiency (EE%), release and stability
studies. The quantification method for AmB in NLC suspension presented linearity in 5.05–60.60 μg
mL−1 range (y = 0.07659x + 0.05344) and for AmB in receptor solution presented linearity in 0.15–
10.00 μg mL−1 range (y = 54609x + 263.1), both with r ≥ 0.999. EE% was approximately 100% and
according to the release results, at pH 7.4, a sustained controlled profile was observed for up 46 h.
Surfactants enhance recovery of poorly soluble drugs during microdialysis sampling:
Implications for in vitro dissolution-/permeation-studies
Sebastian Koplin, Mont Kumpugdee-Vollrath, Annette Bauer-Brandl, Martin Brandl
ABSTRACT
Aim of this project was to investigate the applicability of a recently developed in vitro microdialysis-
sampling approach in connection with a dissolution-/permeation (D/P) system, especially the impact of
surfactants within the perfusion fluid. The D/P-system is based on side-by-side chambers, separated by
a barrier that simulates the intestinal barrier. Here, in contrast to conventional D/P-systems, the
dissolution of the drug (donor chamber concentration) is followed by microdialysis sampling. This
approach appears promising, because it is expected not to disturb the dynamic interplay between drug-
dissolution (-release) and drug permeation. Furthermore, it should allow quantification of the unbound
(free) drug concentration. In the first step, it was assessed, if the addition of the anionic surfactant
sodium dodecyl sulphate (SDS) to the perfusate of the microdialysis system affects the recovery of the
(slightly) water-soluble model drug acyclovir and the poorly water soluble model drug celecoxib
(CXB). SDS had no influence on acyclovir-recovery, but substantially enhanced CXB-recovery, partly
due to improved extraction efficiency, partly due to inhibition of loss of CXB due to non-specific
binding to surfaces and the probe. The fraction of CXB recovered from aqueous CXB-solutions by
microdialysis sampling using SDS-containing perfusates correlated well with the celecoxib
concentration in the samples, but was found independent of the SDS-concentrations (above critical
micelle concentration).
235
Simultaneous identification and quantification of polymethoxyflavones, coumarin and phenolic
acids in Ageratum conyzoides by UPLC-ESI-QToF-MS and UPLC-PDA
Larissa G. Faqueti, Louis P. Sandjo, Maique W. Biavatti
ABSTRACT
Ageratum conyzoides L. is a plant widely used in traditional medicine of tropical and subtropical
regions for its anti-inflammatory and antinociceptive properties. Nevertheless, the chemical
composition of its medicinal preparation has not been yet accurately established. In this study,
chromatographic methods were developed for the simultaneous identification and quantification of the
main compounds in A. conyzoides aqueous extract, using UPLC-PDA-ESI-QToF-MS. The qualitative
analyses defined by MS/MS analysis allowed the identification of 27 compounds in the aqueous
extract, including the toxic pyrrolizidine alkaloids, phenolic acids, coumarin and
polymethoxyflavones. Among the metabolites, twelve were detected for the first time in the species.
The quantitative method was validated according to the official guidelines, demonstrating to be
specific, linear, precise, accurate and sensitive for the quantification of chlorogenic acid, coumaric
acid, coumarin (1,2-benzopyranone), 5,6,7,3′,4′,5′-hexamethoxyflavone, nobiletin, 5′-methoxynobiletin
and eupalestin.
Degradation and metabolite formation of estrogen conjugates in an agricultural soil
Li Ma, Scott R. Yates
ABSTRACT
Estrogen conjugates are precursors of free estrogens such as 17ß-estradiol (E2) and estrone (E1),
which cause potent endocrine disrupting effects on aquatic organisms. In this study, microcosm
laboratory experiments were conducted at 25 °C in an agricultural soil to investigate the aerobic
degradation and metabolite formation kinetics of 17ß-estradiol-3-glucuronide (E2-3G) and 17ß-
estradiol-3-sulfate (E2-3S). The aerobic degradation of E2-3G and E2-3S followed first-order kinetics
and the degradation rates were inversely related to their initial concentrations. The degradation of E2-
3G and E2-3S was extraordinarily rapid with half of mass lost within hours. Considerable quantities of
E2-3G (7.68 ng/g) and E2-3S (4.84 ng/g) were detected at the end of the 20-d experiment, particularly
for high initial concentrations. The major degradation pathway of E2-3G and E2-3S was oxidation,
yielding the primary metabolites 17ß-estrone-3-glucuronide and 17ß-estrone-3-sulfate, respectively.
Common metabolites were E2, the second primary metabolite, and E1, the secondary metabolite.
Additionally, ring B unsaturated estrogens and their sulfate conjugates were tentatively proposed as
minor metabolites. The persistence of E2-3G and E2-3S (up to 20 d) suggests that the high rate of
application of conjugated estrogen-containing substances could be responsible for the frequent
detection of free estrogens in surface and subsurface water.
236
Chemical analysis and potential endocrine activities of aluminium coatings intended to be in
contact with cosmetic water
Elias Bou-Maroun, Laurence Dahbi, Marie-Pierre Gomez-Berrada, Philippine Pierre, Marie-Christine
Chagnon
ABSTRACT
The objective of the work was to check the presence of Non-Intended Added Substances (NIAS) with
hormonal activities in aluminium coatings extracts coded: AA, BBF, MC and RR, furnished by four
different suppliers. Water samples were prepared at room temperature or at 40 °C for three months to
verify the storage effect on the coatings. Solid phase extraction was used to concentrate and to extract
coating substances. Hormonal activities were checked in vitro using reporter gene bioassays. Except
BBF, all extracts induced a weak but significant estrogenic agonist activity in the human cell line.
Using an estrogenic antagonist (ICI-182, 780), the answer was demonstrated specific in the bioassay.
RR was the only extract to induce a concentration dependent anti-androgenic response in the MDA-
KB2 cell line. Analysis performed using GC–MS and HPLC–MS detected 12 substances in most of the
extracts. 8 NIAS were present. Among them, 4 were identified with certainty: HMBT, BGA, DCU and
BPA. Estrogenic potency was BPA > DCU > BGA > HMBT. HMBT was also anti-androgenic at high
concentration. Combining chemical analysis and bioassays data, we demonstrated that in the RR and
the RR40 extracts, the observed estrogenic response was mainly due to BPA, the anti-androgenic
activity of RR could be due to a synergism between HMBT and BPA. For MC and AA, estrogenic
responses appear to be due to the presence of DCU. Except BBF, storage conditions tended to increase
estrogenic activities in all extracts.
Identification and determination of related substances of ceftaroline fosamil in medicinal
product by high performance liquid chromatography with diode array detection and tandem
mass spectrometry
Izabela Karpiuk, Katarzyna Michalska, Katarzyna Bus, Monika Kiljan, Stefan Tyski
ABSTRACT
Ceftaroline fosamil, the prodrug of ceftaroline, is an advanced-generation cephalosporin antibacterial
agent approved for treatment in the European Union in 2012. The drug is dedicated to curing
complicated skin and soft tissue infections and community-acquired pneumonia. The developed
analytical method of high performance liquid chromatography (HPLC) followed by diode array
detector (DAD) set at 243 nm was used to identify and determine degradation products of ceftaroline
fosamil. The elaborated method demonstrated good selectivity and linearity [r > 0.998 for the range
0.8–1.2 mg/mL (80–120%) and r > 0.997 for the range 0.005–0.015 mg/mL (0.5–1.5%)]. Limit of
detection (LOD) and limit of quantification (LOQ) values were equal to 0.15 μg/mL and 0.5 μg/mL,
respectively. The forced decomposition of ceftaroline fosamil carried out under acidic, basic,
oxidative, photolytic and thermal conditions revealed the high sensitivity of ceftaroline on
photodegradation and thermolysis processes. Representative Ceftaroline samples were selected and
analysed by LC coupled with electrospray ionisation time-of-flight mass spectrometry (LC-ESI-Q-
TOF-MS) to identify the related substances that appeared under stress conditions. The LC-DAD
method was transferred without any modification to LC–MS/MS system, allowing it to correlate
results back to the LC-DAD method.
237
Quantification of concentrated Chinese medicine granules by quantitative polymerase chain
reaction
Yat-Tung Lo, Pang-Chui Shaw
ABSTRACT
Determination of the amount of constituent in a multi-herb product is important for quality control. In
concentrated Chinese medicine granules (CCMG), no dregs are left after dissolution of the CCMG.
This study is the first to examine the feasibility of using quantitative polymerase chain reaction
(qPCR) to find the amount of CCMG in solution form. DNA was extracted from Hirudo and Zaocys
CCMG mixed at different ratios and amplified in qPCR using species-specific primers. The threshold
cycle (CT) obtained was compared with the respective standard curves. Results showed that
reproducible quantification results could be obtained (1) for 5–50 mg CCMG using a modified DNA
extraction protocol, (2) amongst DNA extracted from the same batch of CCMG and (3) amongst
different batches of CCMG from the same company. This study demonstrated the constitute amount of
CCMG in a mixture could be determined using qPCR. This work has extended the application of DNA
techniques for the quantification of herbal products and this approach may be developed for quality
assurance in the CCMG industry.
Chiral separation of terbutaline and non-steroidal anti-inflammatory drugs by using a new
lysine–bridged hemispherodextrin in capillary electrophoresis
V. Cucinotta, M. Messina, A. Contino, G. Maccarrone, A. Giuffrida
ABSTRACT
A method for the separation of a mixture of terbutaline and non-steroidal anti-inflammatory drugs was
developed using capillary electrophoresis with a new hemispherodextrin, ad hoc designed, the lysine −
bridged hemispherodextrin (THLYSH). The use of lysine residues to bridge the trehalose capping unit
moiety to the cyclodextrin cavity gives rise to a receptor with two long chains with amine nitrogen
atoms, whose charge can be easily tuned as a function of the solution pH. The new hemispherodextrin
was accurately characterised by ESI–MS and NMR spectroscopy, also highlighting its protonation
behaviour. Circular dichroism and ESR spectroscopy measurements were also carried out to test its
inclusion ability towards anthraquinone-3-sulfonate and its metal coordination ability towards
copper(II) ion, respectively. Analogously to the other hemispherodextrins, the main skill of this new
derivative lies in its chiral selector properties, as shown by the separation of the enantiomeric pairs of
terbutaline and ibuprofen, flurbiprofen, suprofen and tiaprofenic acid by capillary electrophoresis. The
focused use of the solution equilibria involved in the separations made it possible to understand the
phenomena occurring in solution, and to finely tune the charge status of the receptor. In this way the
chiral separation of the racemic mixture was successfully obtained, even if the receptor was
individually used, differently by the other hemispherodextrins previously studied whose chiral
separation capabilities are present only if used as binary mixtures.
238
Separation and characterization of allergic polymerized impurities in cephalosporins by 2D-
HPSEC × LC-IT-TOF MS
Yu Xu, DanDan Wang, Lan Tang, Jian Wang
ABSTRACT
Eleven unknown allergic impurities in cefodizime, cefmenoxime and cefonicid were separated and
characterized by a trap-free two-dimensional high performance size exclusion chromatography
(HPSEC) and reversed phase liquid chromatography (RP-HPLC) coupled to high resolution ion
trap/time-of-flight mass spectrometry (2D-HPSEC × LC-IT-TOF MS) with positive and negative
modes of electrospray ionization method. Separation and characterization the allergic polymerized
impurities in β-lactam antibiotics were on the basis of column-switching technique which effectively
combined the advantages of HPSEC and the ability of RP-HPLC to identify the special impurities. In
the first dimension HPSEC, the column was Xtimate SEC-120 analytical column (7.8 mm × 30 cm, 5
μm), and the gradient elution used pH 7.0 buffer-acetonitrile as mobile phase And the second
dimension analytical column was ZORBAX SB-C18 (4.6 × 150 mm, 3.5 μm) with ammonium formate
solution (10 mM) and ammonium formate (8 mM) in [acetonitrile-water (4:1, v/v)] solution as mobile
phase. Structures of eleven unknown impurities were deduced based on the high resolution MSn data
with both positive and negative modes, in which nine impurities were polymerized impurities. The
forming mechanism of β-lactam antibiotic polymerization in cephalosporins was also studied. The
question on incompatibility between non-volatile salt mobile phase and mass spectrometry was solved
completely by multidimensional heart-cutting approaches and online demineralization technique,
which was worthy of widespread use and application for the advantages of stability and repeatability.
Phytochemical analysis and anti-inflammatory evaluation of compounds from an aqueous
extract of Croton cajucara Benth
Adamara M. Nascimento, Daniele Maria-Ferreira, Fernando T. Dal Lin, Alexandre Kimura, Lauro M.
de Souza
ABSTRACT
Croton cajucara Benth is a medicinal plant popularly used in the Brazilian Amazonia, where it is
known as sacaca, being consumed as tea, decoction or infusion of the leaves and stem bark. From a
decoction of the leaves, a comprehensive phytochemical analysis was developed by liquid
chromatography-mass spectrometry. Many compounds were identified for the first time in C. cajucara,
such as O-glycosides of kaempferol and quercetin, flavonoid-C-glycosides, tannins and cinnamic acid
derivatives. These compounds were fractionated by polarity and assayed for their anti-inflammatory
activity, using a model of mice edema, induced by an intraplantar injection of carrageenan. All
fractions exhibited anti-inflammatory properties.
239
Supercritical fluid chromatography approach for a sustainable manufacture of new
stereoisomeric anticancer agent
Alina Ghinet, Yasmine Zehani, Emmanuelle Lipka
ABSTRACT
Two routes aimed at the manufacture of unprecedented stereoisomeric combretastatin A-4 analogue
were described: flash chromatography vs supercritical fluid chromatography. The latter has many
advantages over liquid chromatography and was therefore chosen for the small scale separation of
methyl 1-[(3-hydroxy-4-methoxyphenyl) (3,4,5-trimethoxyphenyl)methyl]-5-oxo-l-prolinate 5, with
potential antitumoral activity. After a screening of six different polysaccharide based chiral stationary
phases and four co-solvents, the percentage of co-solvent, the flow-rate and the outlet pressure were
optimized through a design of experiments (DoE). The preparation of 50 mg of each stereoisomer was
achieved successfully on a Chiralpak AD-H with isopropanol as a co-solvent. Productivity (kkd),
solvent usage and environmental factor (E Factor) were calculated. Flash chromatography and
supercritical fluid chromatography approaches were compared in terms of yield and purity of each
stereoisomer manufactured.
Use of ionic liquids as headspace gas chromatography diluents for the analysis of residual
solvents in pharmaceuticals
Omprakash Nacham, Tien D. Ho, Jared L. Anderson, Gregory K. Webster
ABSTRACT
In this study, two ionic liquids (ILs), 1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide
([BMIM][NTf2]) and trihexyltetradecylphosphonium bis[(trifluoromethyl)sulfonyl]imide ([P66614]
[NTf2]) were examined as contemporary diluents for residual solvent analysis using static headspace
gas chromatography (SHS-GC) coupled with flame ionization detection (FID). ILs are a class of non-
molecular solvents featuring negligible vapor pressure and high thermal stabilities. Owing to these
favorable properties, ILs have potential to enable superior sensitivity and reduced interference,
compared to conventional organic diluents, at high headspace incubation temperatures. By employing
the [BMIM][NTf2] IL as a diluent, a 25-fold improvement in limit of detection (LOD) was observed
with respect to traditional HS-GC diluents, such as N-methylpyrrolidone (NMP). The established IL-
based method demonstrated LODs ranging from 5.8 parts-per-million (ppm) to 20 ppm of residual
solvents in drug substances. The optimization of headspace extraction conditions was performed prior
to method validation. An incubation temperature of 140 °C and a 15 min incubation time provided the
best sensitivity for the analysis. Under optimized experimental conditions, the mass of residual
solvents partitioned in the headspace was higher when using [BMIM][NTf2] than NMP as a diluent.
The analytical performance was demonstrated by determining the repeatability, accuracy, and linearity
of the method. Linear ranges of up to two orders of magnitude were obtained for class 3 solvents.
Excellent analyte recoveries were obtained in the presence of three different active pharmaceutical
ingredients. Owing to its robustness, high throughput, and superior sensitivity, the HS-GC IL-based
method can be used as an alternative to existing residual solvent methods.
240
Adduct ion-targeted qualitative and quantitative analysis of polyoxypregnanes by ultra-high
pressure liquid chromatography coupled with triple quadrupole mass spectrometry
Xu Wu, Lin Zhu, Jiang Ma, Yang Ye, Ge Lin
ABSTRACT
Polyoxypregnane and its glycosides (POPs) are frequently present in plants of Asclepiadaceae family,
and have a variety of biological activities. There is a great need to comprehensively profile these
phytochemicals and to quantify them for monitoring their contents in the herbs and the biological
samples. However, POPs undergo extensive adduct ion formation in ESI-MS, which has posed a
challenge for qualitative and quantitative analysis of POPs. In the present study, we took the advantage
of such extensive adduct ion formation to investigate the suitability of adduct ion-targeted analysis of
POPs. For the qualitative analysis, we firstly demonstrated that the sodium and ammonium adduct ion-
targeted product ion scans (PIS) provided adequate MS/MS fragmentations for structural
characterization of POPs. Aided with precursor ion (PI) scans, which showed high selectivity and
sensitivity and improved peak assignment confidence in conjunction with full scan (FS), the
informative adduct ion-targeted PIS enabled rapid POPs profiling. For the quantification, we used
formic acid rather than ammonium acetate as an additive in the mobile phase to avoid simultaneous
formation of sodium and ammonium adduct ions, and greatly improved reproducibility of MS response
of POPs. By monitoring the solely formed sodium adduct ions [M+Na]+, a method for simultaneous
quantification of 25 POPs in the dynamic multiple reaction monitoring mode was then developed and
validated. Finally, the aforementioned methods were applied to qualitative and quantitative analysis of
POPs in the extract of a traditional Chinses medicinal herb, Marsdenia tenacissima (Roxb.) Wight et
Arn., and in the plasma obtained from the rats treated with this herb.
Quantitative determination of dobutamine in newborn pig plasma samples by HPLC–MS/MS
O.E. Albóniga, M.L. Alonso, M.E. Blanco, O. González, R.M. Alonso
ABSTRACT
A novel gradient reverse phase high performance liquid chromatography tandem mass spectrometry
(HPLC/MS-MS) was performed as a method for the determination of dobutamine hydrochloride
(DOB) in newborn pig plasma samples. It was developed and validated after optimization of sample
treatment and various chromatographic and mass spectrometric conditions. Trimethoxydobutamine
(TMD) was used as internal standard. Heptafluorobutyric acid (HFBA) and ethyl acetate were used for
the treatment of plasma samples. The separation of dobutamine and internal standard was done using a
Kinetex F5 (50 × 2.1 mm, 2.6 μm, 100 Å) analytical column. The mobile phase was a mixture of
acetonitrile and HCOOH 0.01%. The column oven temperature was optimized at 40° C and the flow
rate was 0.25 mL/min. DOB and TMD were detected by multiple reaction monitoring (MRM) mode in
ESI+, using a cone voltage (CV) of 25 V and a collision energy (CE) of 25 eV. The weighted
calibration curve (1/x2) was found to be linear over the concentration range of 1–100 ng/mL (r2 >
0.999). The limit of quantification (LLOQ) of the method was 1 ng/mL. The values of selectivity,
carryover, LLOQ, linearity, accuracy, precision, matrix effect, stability and recovery obtained meet the
acceptable range according to European Medicines Agency (EMA) and Food and Drug Administration
(FDA) guidelines. The method was efficiently applied to quantify DOB in plasma samples from a
pharmacokinetic/pharmacodynamic study in a disease model of newborn piglet.
241
An integrative investigation of the toxicity of Aconiti kusnezoffii radix and the attenuation effect
of its processed drug using a UHPLC-Q-TOF based rat serum and urine metabolomics strategy
Zhenyu Sui, Qing Li, Lin Zhu, Zhenru Wang, Kaishun Bi
ABSTRACT
Aconiti kusnezoffii radix (AKR), the root of Aconitum kusnezoffii Reichb., is commonly used in the
treatment of the rheumatoid arthritis. However, the clinical application is limited due to its potential
toxicity. Therefore, to investigate the mechanism of its potential neurotoxicity and nephrotoxicity, a
comprehensive metabolomics study combined with serum biochemistry and histopathology
measurements was carried out. A UHPLC-Q-TOF mass spectrometry based metabolomics approach
was applied to characterize the AKR toxicity, while the toxicity attenuation effects of Aconiti
kusnezoffii radix cocta (AKRC) on Wistar rats were also investigated. Two chromatographic
techniques involving reversed-phase chromatography and hydrophilic interaction chromatography
were combined for the serum and urine detection, which balanced the integrity and selectivity of the
two matrices. Principal component analysis was used to determine the groups, and principal
component analysis discriminant analysis was carried out to confirm the important variables. Then, the
developed integrative toxicity evaluation method was applied to assess the toxicity of AKR and the
attenuation effect of AKRC. The highly sensitive and specific toxic biomarkers, which can provide
practical bases were identified for the diagnosis of the neurotoxicity and nephrotoxicity induced by
AKR. In all, a total of 19 putative biomarkers were characterized, and related metabolic pathways were
identified. The study demonstrated that the established metabolomics strategy is a powerful approach
for investigating the mechanisms of herbal toxicity and the attenuation effect of a processing method
and would provide medical solutions for other toxic herbal medications and further clinical evidence
on how AKR improves symptoms of rheumatoid arthritis patients.
Calibration and validation of a MCC/IMS prototype for exhaled propofol online measurement
Felix Maurer, Larissa Walter, Martin Geiger, Jörg Ingo Baumbach, Sascha Kreuer
ABSTRACT
Propofol is a commonly used intravenous general anesthetic. Multi-capillary column (MCC) coupled
Ion-mobility spectrometry (IMS) can be used to quantify exhaled propofol, and thus estimate plasma
drug concentration. Here, we present results of the calibration and analytical validation of a MCC/IMS
pre-market prototype for propofol quantification in exhaled air. Calibration with a reference gas
generator yielded an R2 ≥ 0.99 with a linear array for the calibration curve from 0 to 20 ppbv. The
limit of quantification was 0.3 ppbv and the limit of detection was 0.1 ppbv. The device is able to
distinguish concentration differences > 0.5 ppbv for the concentration range between 2 and 4 ppbv and
> 0.9 ppbv for the range between 28 and 30 ppbv. The imprecision at 20 ppbv is 11.3% whereas it is
3.5% at a concentration of 40 ppbv. The carry-over duration is 3 min. The MCC/IMS we tested
provided online quantification of gaseous propofol over the clinically relevant range at measurement
frequencies of one measurement each minute.
242
Biochemical and functional analysis of corticotropin releasing factor purified from an aqueous
extract of human placenta used as wound healer
Namrata Singh, Debasish Bhattacharyya
ABSTRACT
Human placental extract constitutes of innumerable therapeutically important components mostly used
in wound healing arising from the skin and burn injuries. However, there is still some bioactive present
in the placental extracts yet to be characterized to better under the complex process of wound healing
mediated by the placental extract. In this study, the presence of corticotropin releasing factor (CRF) in
an aqueous extract of human placenta was detected and quantified by dot blot and CRF-ELISA
immunoassay kit respectively. Subsequently, it was purified by immuno-affinity chromatography and
quantified as 0.45 ± 0.05 μg of CRF per ml of placental extract where its molecular weight found to be
4.78 kDa by MALDI-TOF. To study functional analysis of CRF, an in vitro WI-38 lung fibroblast cell
scratch wound model was used which indicated proliferation, motility of cells after treatment with
purified CRF. Moreover, reduction in apoptosis rate of cells during closure of wound was observed
from microscopy studies and FACS analysis. Also, Antalarmin, an antagonist of CRF type 1 receptor
inhibited the wound closure potency of the purified component. Faster healing of wound with an
elevation of IL-6 and TGF-β during early stages of repair by placental CRF was observed on excision
rat model. The process of healing was accompanied by the decrease in the level of TNF-α and IFN-γ.
Toxic compounds from tobacco in placenta samples analyzed by UPLC-QTOF-MS
Somayeh Mohammadi, Celia Domeno, Isabel Nerin, Margarita Aznar, Cristina Nerin
ABSTRACT
Polycyclic aromatic hydrocarbons (PAHs), tobacco-specific nitrosamines (TSNAs) and aromatic
amines are carcinogens present in cigarette smoke. These compounds are distributed in the human
body and they could be transferred to the foetus during the pregnancy. Placenta is the main barrier to
these toxic compounds and its study is the objective of this work. A method based on solid-phase
extraction (SPE) with ultra-performance liquid chromatography−tandem quadrupole-time-of-flight
mass spectrometry (UPLC-QTOF-MS) has been examined and optimized for the analysis of 9 target
analytes (4 tobacco-specific nitrosamines and some of their metabolites, 3 aromatic amines, nicotine
and cotinine) in 26 placenta samples from smoking and non-smoking women. Limits of detection
(LODs) were in the range of 3–27 ng/g of placenta. Nicotine, cotinine, N-nitrosoanatabine (NAT) and
4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone (NNK) metabolite, 4-(methylnitrosamino)-1-(3-
pyridyl)-1-butanol (NNAL) were detected in the placenta samples of smoking woman. Nicotine was
detected in 3 out of 8 placentas from smoking women, always below the limit of quantification (88
ng/g). This could be expected, as the half-life of nicotine in the body is limited to about 0.5–3 h.
Cotinine, the main metabolite from nicotine, was detected in all placentas from smoking women at
concentrations between 17.2 and 61.8 ng/g, reaching the highest values for those women that smoked
the highest number of cigarettes. NAT and NNAL were detected in all placentas from smoking
women, always below the limit of quantification (40 ng/g and 33 ng/g respectively).
243
HPLC-HG-AFS determination of arsenic species in acute promyelocytic leukemia (APL) plasma
and blood cells
Meihua Guo, Wenjing Wang, Xin Hai, Jin Zhou
ABSTRACT
Arsenic trioxide (ATO) has been successfully used in the treatment of acute promyelocytic leukemia
(APL). To clarify the arsenic species in APL patients, high performance liquid chromatography-
hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) and HG-AFS methods were
developed and validated to quantify the plasma concentrations of inorganic arsenic (As(III) and As(V))
and methylated metabolites (MMA and DMA), and the total amounts of arsenic in blood cells and
plasma. Blood cells and plasma were digested with mixtures of HNO3H2O2 and analyzed by HG-
AFS. For arsenic speciation, plasma samples were prepared with perchloric acid to precipitate protein.
The supernatant was separated on an anion-exchange column within 6 min with isocratic elution using
13 mM CH3COONa, 3 mM NaH2PO4, 4 mM KNO3 and 0.2 mM EDTA-2Na. The methods provided
linearity range of 0.2–20 ng/mL for total arsenic and 2.0–50 ng/mL for four arsenic species. The
developed methods for total arsenic and arsenic species determination were precise and accurate. The
spiked recoveries ranged from 81.2%-108.6% and the coefficients of variation for intra- and inter-
batch precision were less than 9.3% and 12.5%, respectively. The developed methods were applied
successfully for the assay of total arsenic and arsenic species in 5 APL patients.
Quantification of IDP-73152, a novel antibiotic, in plasma from mice, rats and humans using an
ultra-high performance liquid chromatography/tandem mass spectrometry method for use in
pharmacokinetic studies
Myongjae Lee, Dohee Kim, Jeongcheol Shin, Hee-Yeol Lee, Suk-Jae Chung
ABSTRACT
IDP-73152, a novel inhibitor of a bacterial peptide deformylase, was recently approved as a new,
investigational drug in Korea for the clinical management of infections caused by Gram positive
bacteria. The objective of this study was to develop/validate a simple and robust analytical method for
the determination of IDP-73152 in plasma samples from rodents and humans, and to assess the
feasibility of the assay for use in pharmacokinetic studies using animal models. Plasma samples were
processed using a standard method for protein precipitation and an aliquot of the extract then injected
onto an UHPLC–MS/MS system. The drug and IDP-117293, an internal standard, were analyzed in
the positive ion-mode by electrospray ionization and quantified by monitoring the transition at m/z
555.2 → 245.2 for IDP-73152 and 563.3 → 253.1 for the internal standard, respectively. The lower
and upper limit of the assay was determined to be 5 and 10000 ng/ml, respectively, with an acceptable
linearity (R > 0.999) in the response-concentration relationship. Validation parameters, including
accuracy, precision, dilution, recovery, matrix effect and stability were found to be within the
acceptable ranges recommended by the assay validation guidelines of the United States FDA. The
method was successfully applied to the quantification of IDP-73152 in plasma from mice/rats that had
received a single oral administration of 80 mg/kg IDP-73152, in the form of the mesylate salt. These
findings suggest that the validated assay can be used in preclinical and clinical pharmacokinetic studies
of IDP-73152.
244
New approach for the diagnosis of histamine intolerance based on the determination of
histamine and methylhistamine in urine
Oriol Comas-Basté, M.Luz Latorre-Moratalla, Roberta Bernacchia, M.Teresa Veciana-Nogués,
M.Carmen Vidal-Carou
ABSTRACT
Histamine intolerance is a disorder in the homeostasis of histamine due to a reduced intestinal
degradation of this amine, mainly caused by diamine oxidase (DAO) enzyme deficiency, which
provokes its accumulation in plasma and the appearance of adverse health affects. A new approach for
the diagnosis of this intolerance could be the determination of histamine and its metabolites in urine.
The aim of this work was to develop and validate a rapid method to determine histamine and
methylhistamine in human urine by Ultra High Performance Liquid Chromatography and Fluorimetric
detection (UHPLC-FL). The proposed method is a consistent procedure to determine histamine and
methylhistamine in less than 11 min with adequate linearity and sensitivity. Relative standard
deviation was always lower than 5.5%, ensuring method precision; and mean recovery was greater
than 99% for both analytes. The structure of histamine and methylhistamine conjugated with OPA
were confirmed by UHPLC-ITD-FTMS which enabled to unequivocally identify both analytes in
standards and also in urine samples. The analysis of histamine and methylhistamine in urine samples
could be a potential new approach for the routine diagnosis of histamine intolerance, more patient-
friendly and with clear advantages in terms of equipment and personnel demand for sample collection
in comparison with current plasmatic DAO activity determination.
Development and validation of sensitive LC–MS/MS method for the quantification of SUVN-502
and its metabolite and its application for first in human pharmacokinetic study
Ramakrishna Nirogi, Devender Reddy Ajjala, Raghupathi Aleti, Lakshmiprasanna Rayapati, Naga
Surya Prakash Padala
ABSTRACT
A sensitive and rapid LC–MS/MS method was developed and validated for the quantification of
SUVN-502 and M1 of SUVN-502, a 5-HT6 receptor antagonist for the treatment of dementia
associated with Alzheimer‘s disease. Following solid-phase extraction, SUVN-502 and M1 of SUVN-
502 and IS were eluted with 10 mM ammonium acetate (pH 4.0) and acetonitrile using a rapid gradient
program on reverse phase column. Multiple reaction monitoring mode was used to monitor the
respective transitions of m/z 478.2 → 377.7 for SUVN-502 and m/z 464.1 → 377.7 for M1 of SUVN-
502. The assay exhibited a linear dynamic range of 10–10000 pg/mL for SUVN-502 and 20–20000
pg/mL for M1 of SUVN-502 in human plasma. Acceptable precision and accuracy were obtained for
concentrations over the standard curve range. The within batch accuracy and precision were within
acceptable limits. All the other validation parameters were within the acceptable limits. The validated
method was applied to analyze human plasma samples obtained from a human pharmacokinetic study
consisting single and multiple ascending doses.
245
Glycosylation patterns of selected proteins in individual serum and cerebrospinal fluid samples
Isabella Karlsson, Lorena Ndreu, Alessandro Quaranta, Gunnar Thorsén
ABSTRACT
A method we previously developed has been applied to the determination of the glycosylation pattern
of specific proteins in biological samples. Six proteins (alpha-1-antitrypsin, transferrin, haptoglobin,
C1 inhibitor, alpha-1 acid glycoprotein, and immunoglobulin G) were studied in serum samples from
five individuals and cerebrospinal fluid (CSF) samples from three individuals, to investigate the
expected normal distribution of glycosylation patterns and to assess whether this methodology can be
used to discriminate between samples from different individuals. For serum samples, the differences
were shown to be small, while much larger differences were found for the CSF samples, with a greater
number of glycoforms present. This can be linked to the occurrence of differential glycosylation in
proteins expressed in the brain compared with proteins expressed elsewhere in the body. The
developed method could distinguish differences in the glycosylation pattern of specific proteins in the
individual samples, which was not reflected in the glycan content of total CSF. This is the first time
that the glycoforms of several of these proteins have been investigated in CSF.
MIL-101(Cr)@GO for dispersive micro-solid-phase extraction of pharmaceutical residue in
chicken breast used in microwave-assisted coupling with HPLC–MS/MS detection
Yudan Wang, Xinpeng Dai, Xi He, Lin Chen, Xiaohong Hou
ABSTRACT
In this work, MIL-101(Cr)@GO (Graphite Oxide) was synthesized using a hydrothermal synthesis
method and was applied as a dispersive micro-solid-phase extraction (D-μ-SPE) sorbent for the
efficient concentration of four residual drugs (metronidazole, MNZ; tinidazole, TNZ;
chloramphenicol, CAP; sulfamethoxazole, SMX). Meanwhile, the extraction process was optimized by
combining it with microwave-assisted extraction. Factors affecting the D-μ-SPE efficiency, such as
selection of sorbent materials, pH of the sample solution, salting-out effect, amount of used material,
extraction time, desorption solvent and desorption time, were studied. Under the optimal extraction
conditions, the linearity ranged from 10 to 1000 ng kg−1 and 1–100 ng kg−1 (r2 ≥ 0.9928) for the
target analytes. The limits of detection were between 0.08 and 1.02 ng kg−1, and the limits of
quantitation were between 0.26 and 3.40 ng kg−1. Additionally, the developed method also exhibited
good precision (RSD ≤ 2.5%), repeatability (RSD ≤ 4.3%), high recoveries (88.9%–102.3%) and low
matrix effects (78.2%–95.1%). The proposed method proved to be an efficient and reliable approach
for the determination of the analytes. Finally, we successfully detected the four drugs in chicken
breast.
246
Development of a robust reporter gene assay to measure the bioactivity of anti-PD-1/anti-PD-L1
therapeutic antibodies
Lan Wang, Chuanfei Yu, Yalan Yang, Kai Gao, Junzhi Wang
ABSTRACT
Being regarded as the ‗cancer panacea‘, the anti-PD-1/anti-PD-L1 monoclonal antibodies (mAbs) have
become the R&D focus of biopharmaceutical industries. Several marketed such mAbs have been
proved particularly effective in treating various cancers. However, the cell-based bioassay to measure
the biological activities of the anti-PD-1/anti-PD-L1 mAbs as the lot release or stability test has been a
great challenge to quality control laboratories due to the immunomodulating nature of the mAbs. Here,
we describe the development and validation of a reporter gene assay consisting of two-cell systems to
measure the bioactivity of the anti-PD-1/anti-PD-L1 mAbs. We have generated two cell lines, the
CHO-PD-L1-CD3L cell line that stably expresses PD-L1 and the membrane-anchored anti-CD3 single
chain antibody fragment (scFv) named CD3L and the Jurkat-PD-1-NFAT cell line that stably
expresses PD-1 and the luciferase gene under the control of the NFAT response elements from the IL-
2 promoter. The results show good dose-dependent responsiveness to the mAbs and excellent
performance characteristics including specificity, accuracy and precision. The biological relevance of
the assay, the passage stability of the two cell lines, and the capability of measuring various anti-PD-
1/anti-PD-L1 mAbs render this assay applicable not only in lot release and stability test but also in
characterization and development of new anti-PD1/anti-PD-L1 mAbs.
LC–MS/MS-ESI method for simultaneous quantification of darolutamide and its active
metabolite, ORM-15341 in mice plasma and its application to a pharmacokinetic study
Sreekanth Dittakavi, Pavan Kumar V.S.P. Nagasuri, Suresh P. Sulochana, Syed Mohd Saim, Ramesh
Mullangi
ABSTRACT
A sensitive and rapid LC–MS/MS method was developed and validated for the simultaneous
quantitation of darolutamide and its active metabolite i.e. ORM-15341 in 50 μL mice plasma using
bicalutamide as an internal standard (I.S.) as per regulatory guidelines. Sample processing was
accomplished through liquid-liquid extraction. Chromatographic separation was achieved using an
Atlantis C18 column with an isocratic mobile phase comprising 0.2% formic acid:acetonitrile (35:65,
v/v) at a flow rate of 0.8 mL/min within 2.5 min. Detection and quantitation were done by multiple
reaction monitoring on a triple quadrupole mass spectrometer following the transitions: m/z 397 →
202, 395 → 202 and 429 → 255 for darolutamide, ORM-15341 and I.S, respectively in the negative
ionization mode. The calibration curve was linear from 0.61–1097 ng/mL for both darolutamide and
ORM-15341. The intra- and inter-day precisions were in the range of 1.34–13.8 and 4.85–12.9 and
3.91–13.7 and 6.54–14.2%, for darolutamide and ORM-15341, respectively. Darolutamide and ORM-
15341 were found to be stable under different stability conditions. The validated method was applied
to a pharmacokinetic study in mice.
247
Metabolite identification of AZD8055 in Sprague-Dawley rats after a single oral administration
using ultra-performance liquid chromatography and mass spectrometry
Md Mamunur Rashid, Hyun-A Oh, Hyunbeom Lee, Byung Hwa Jung
ABSTRACT
AZD8055 is an ATP-competitive specific dual mTOR inhibitor and exhibited potent antitumor activity
on several types of solid tumors. However, the metabolism of AZD8055 in the body still remains
unknown. In this study, metabolite identification of AZD8055 was performed using ultra high-
performance liquid chromatography-ion trap mass spectrometry (UHPLC-IT-MS) through both in
vitro and in vivo approaches using rat liver microsomes (RLMs) and rat plasma, urine and feces,
respectively. A total of eight putative metabolites (five phase I and three phase II) were identified, and
a tentative metabolic pathway was suggested for the first time. Considering the accurate mass and
mass fragmentations of the detected metabolites, their plausible structures were suggested.
Demethylation, hydroxylation, oxidation and morpholine ring opening were the major
biotransformation processes for the phase-I metabolism, while phase-II metabolites were merely
generated by the glucuronide conjugation reaction. The cumulative excretion of AZD8055 in urine and
feces was 0.13% and 1.11% of the dose, respectively. When the semi-quantitative analysis of the
metabolites was performed using UHPLC–MS/MS (ultra-performance liquid chromatography tandem
mass spectrometry) to evaluate the overall trend of metabolites formation and excretion, AZD8055
was excreted more in the form of the metabolites than itself and their formation was very fast.
Therefore it was presumed that biotransformation was playing a crucial role in its elimination.
Ultimately, this study provides novel insights regarding the in vitro and in vivo biotransformations of
AZD8055. Further investigations of metabolites of this potent anti-cancer compound could be
beneficial for the antitumor drug design and development process.
Quantification of 16 β-lactams in chicken muscle by QuEChERS extraction and UPLC-Q-
Orbitrap-MS with parallel reaction monitoring
Qing Chen, Xiao-Dong Pan, Bai-Fen Huang, Jian-Long Han
ABSTRACT
A method is described for the analysis of 16 β-lactams in chicken muscle by UPLC-quadrupole(Q)-
Orbitrap-MS with parallel reaction monitoring (PRM). QuEChERS approach includes clean-up step by
sorbent of primary-secondary amine (PSA) and C18 was adopted for sample preparation. Q-Orbitrap
with PRM showed high sensitivity with limits of detection (LODs) ranged from 0.01 μg kg−1 to 0.35
μg kg−1. The method was further validated by intra- and inter-day test with spiking levels less than
MRLs (maximum residue limits, the European Union). Recovery (83–112%) and precision values
(RSDs <15%) for all studied analytes were obtained. The result indicates that UPLC-Q-Orbitrap
coupled with QuEChERS preparation can serve as a routine quantification method for β-lactam
residues in chicken muscles.
248
Characterization of the phase I and phase II metabolic profile of tolvaptan by in vitro studies
and liquid chromatography–mass spectrometry profiling: Relevance to doping control analysis
Monica Mazzarino, Valeria Buccilli, Xavier de la Torre, Ilaria Fiacco, Francesco Botrè
ABSTRACT
Phase I and phase II biochemical reactions involved in the biotransformation pathways of tolvaptan
were characterized by LC–MS-based techniques and in vitro models to identify the most appropriate
marker(s) of intake. The effects of physiological and non-physiological factors on the metabolic profile
of tolvaptan were also evaluated. In vitro approaches were based on the use of pooled human liver
microsomes and recombinant isoforms of cytochrome P450 and uridine diphospho glucuronosyl-
transferase. Sample preparation included liquid/liquid extraction at neutral pH with tert-butyl methyl-
ether. In the case of the study of phase II metabolism an additional enzymatic hydrolysis step was
performed. The chromatographic separation was carried out using reversed-phase chromatography,
whereas detection was performed by either triple-quadrupole or time-of-flight analyzers in positive
electrospray ionization and different acquisition modes. Our data show that tolvaptan is metabolized to
at least 20 phase I metabolites, the biotransformation reactions being catalyzed mainly by CYP3A4
and CYP3A5 isoforms. The phase-I reactions include hydroxylation (in different positions),
carboxylation, oxidation, hydrogenation, dealkylation, isomerization and a combination of the above.
Most of the phase I metabolites undergo glucuronidation, carried out mostly by UGT2B7 and
UGT2B17 isoforms. Dealkylated, mono-hydroxylated and carboxylated metabolites both in the free
and in the glucuronidated form appear to be the most suitable urinary diagnostic markers for the
detection of tolvaptan intake in doping control.
An LC–MS/MS method for simultaneous determination of nine steroidal saponins from Paris
polyphylla var. in rat plasma and its application to pharmacokinetic study
Guangyi Yang, Wei Lu, Meng Pan, Chenning Zhang, Gao Song
ABSTRACT
Paris polyphylla var is an herbal plant herb widely used in Traditional Chinese Medicine. The purpose
of this study is to develop an Ultra Performance Liquid Chromatography-tandem mass spectrometer
(UPLC–MS) method to quantify the major components (i.e., nine saponins) from P. polyphylla in
plasma samples. A UItra BiPh column (100 × 2.1 mm, 5 μm) was used with acetonitrile/0.1% formic
acid in water as mobile phases. The analytes were quantified using a Waters XEVO TQ mass
spectrometer via multiple reaction monitoring (MRM) with positive scan mode. A protein precipitation
method was used to extract the analytes from rat plasma. The inter/intra-day precision, accuracy,
recovery, matrix effect, and stability were evaluated per the FDA guidance. The method showed
linearity in the concentration ranges of 2.4–1250 ng/mL. The intra-day and inter-day precisions (RSD)
of these analytes at three different levels were less than 15.0%. The extraction recoveries of these
analytes were from 83.8% to 109.4% and the matrix effects ranged from 87.4% to 105.4%. The
stabilities of these compounds in plasma were evaluated by analyzing three different concentrations
following storage at 25 °C for 6 h, and −80 °C for 30 days. All the samples displayed less than 15.0%
variations. The validated method was successfully used to a pharmacokinetic (PK) study using
Sprague Dawley (SD) rats with intravenous (i.v.) and oral (p.o.) administration of P. polyphylla
extract.
249
N-glucuronidation catalyzed by UGT1A4 and UGT2B10 in human liver microsomes: Assay
optimization and substrate identification
Danyi Lu, Qian Xie, Baojian Wu
ABSTRACT
N-glucuronidation is an important pathway for metabolism and disposition of tertiary amines in
humans. This reaction is mainly catalyzed by the enzymes UGT1A4 and UGT2B10. However, the
metabolic patterns of UGT1A4- and UGT2B10-mediated N-glucuronidation are not fully clear. In this
study, we first optimized in vitro reaction conditions for N-glucuronidation by using specific substrates
(i.e., trifluoperazine for UGT1A4, cotinine and amitriptyline for UGT2B10). Furthermore, we found
that hepatic N-glucuronidation showed significant species differences. In addition, UGT1A4 and
UGT2B10 were primarily responsible for N-glucuronidation of many tertiary amines, including
asenapine, loxapine, clozapine, chlorpromazine, dothiepin, doxepin, mirtazapine, mianserin,
chlorcyclizine, cyclizine, promethazine, cyclobenzaprine, imatinib, retrorsine, strychnine and brucine.
In conclusion, this study provides an in vitro assay system for evaluating N-glucuronidation of amines.
Also, UGT1A4- and UGT2B10-mediated N-glucuronidation might play significant roles in
metabolism and detoxification of tertiary amines in humans.
A simple high performance liquid chromatography–mass spectrometry method for Therapeutic
Drug Monitoring of isavuconazole and four other antifungal drugs in human plasma samples
Giovanna Fatiguso, Fabio Favata, Ilaria Zedda, Amedeo De Nicolò, Antonio D‘Avolio
ABSTRACT
Triazoles chanced the prevention and treatment of invasive fungal infections, but their
pharmacokinetic properties are still unclear. In particular, isavuconazole (ISC) is a new broad-
spectrum antifungal triazole approved in 2015 as first-line treatment for intravenous and oral use
against invasive aspergillosis and for mucormycosis. Nowadays, the optimal management of the
treatments with triazoles requires the use of Therapeutic Drug Monitoring (TDM), in order to prevent
sub-therapeutic or toxic concentrations. In turn, the routine use of TDM requires reliable quantification
methods The aim of this work was the development and full validation of a HPLC-mass spectrometry
assay for the simultaneous quantification of fluconazole, itraconazole, isavuconazole, posaconazole
and voriconazole in human samples. Both standards and quality controls were prepared in human
plasma. After the addition of internal standard (6,7-dimethyl-2,3-di(2-pyridyl)quinaxoline for
voriconazole, posaconazole and itraconazole; stable isotope labeled compounds for fluconazole and
isavuconazole), protein precipitation with acetonitrile and dilution with water were performed.
Chromatographic separation was performed on Atlantis® T3 5 μm 4.6 × 150 mm column, with a
gradient of water and acetonitrile, both added with 0.05% formic acid. Accuracy, intra-day and inter-
day imprecision fitted FDA and EMA guidelines, while matrix effects and recoveries resulted stable
between samples for each analyte. Stability results were in accordance with previously published data.
Finally, we tested this method by monitoring plasma concentrations in real patients and using external
quality controls with good results. This method resulted very simple, fast, cheap and very useful for
TDM application, to improve clinical management of antifungal therapy in critically ill patients.
250
Metabolic profiling of dehydrodiisoeugenol using xenobiotic metabolomics
Qian-Qian Lv, Xiao-Nan Yang, Dong-Mei Yan, Wei-Qing Liang, Fei Li
ABSTRACT
Dehydrodiisoeugenol (DDIE), a representative and major benzofuran-type neolignan in Myristica
fragrans Houtt., shows anti-inflammatory and anti-bacterial actions. In order to better understand its
pharmacological properties, xenobiotic metabolomics was used to determine the metabolic map of
DDIE and its influence on endogenous metabolites. Total thirteen metabolites of DDIE were identified
through in vivo and in vitro metabolism, and seven of them were reported for the first time in the
present study. The identity of DDIE metabolites was achieved by comparison of the MS/MS
fragmentation pattern with DDIE using ultra-performance chromatography electrospray ionization
quadrupole time-of-flight mass spectrometry (UPLC-ESI- QTOFMS). Demethylation and ring-
opening reaction were the major metabolic pathways for in vivo metabolism of DDIE. Recombinant
cytochrome P450 s (CYPs) screening revealed that CYP1A1 is a primary enzyme contributing to the
formation of metabolites D1-D4. More importantly, the levels of two endogenous metabolites 2,8-
dihydroxyquinoline and its glucuronide were significantly elevated in mouse urine after DDIE
exposure, which explains in part its modulatory effects on gut microbiota. Taken together, these data
contribute to the understanding of the disposition and pharmacological activities of DDIE in vivo.
Development and validation of a reliable method for thiopurine methyltransferase (TPMT)
enzyme activity in human whole blood by LC–MS/MS: An application for phenotypic and
genotypic correlations
Supaporn Wiwattanakul, Santirhat Prommas, Nuttawut Jenjirattithigarn, Siwalee Santon, Chonlaphat
Sukasem
ABSTRACT
A liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed for the
determination of thiopurine methyltransferase (TPMT) activity in human whole blood lysate, based on
conversion of 6-mercaptopurine (6-MP) by TPMT to 6-methylmercaptopurine (6-MMP) using S-
adenosyl-l-methionine (SAM) as the methyl donor. This method was improved from the previous
laborious method for washing of red cell lysate preparation to develop whole blood EDTA lysate. In
addition, the TPMT incubation was optimized and the chromatography was performed in a short
runtime of 7 min on a C18-column by detection via triple quadrupole mass spectrometry. The MS/MS
was optimally tuned to monitor mass to charge a ratio (m/z) for 6-MMP 167.2 → 151.9 and the isotope
6-MMP-d3 with m/z of 170.5 → 152.2 were applied as an internal standard. The calibration curve
covered the range of 2.5–360 ng/ml and the correlation coefficient was greater than 0.999. The
accuracy of this method was determined in four concentrations of control of quality that ranged
between 99.33 and 106.33%. The intra-assay coefficient of variation (CV) was less than 4.41% and the
inter-assay was less than 5.43%. This method developed for measuring TPMT by LC–MS/MS is a
reliable, safe, and simple with a small volume requirement (100 μl of whole blood EDTA). The assay
was used to study TPMT activity in 132 Thai children with a range from 29.0 to 89.1 nmol 6-MMP/g
Hb/h with means and median values of TPMT activity 55.9 ± 12.47 nmol 6-MMP/g Hb/h and 54.2
nmol 6-MMP/g Hb/h.
251
Development and validation of a GC–MS method for the determination of hydroxyzine and its
active metabolite, cetirizine, in whole blood
Maria Katselou, Sotiris Athanaselis, Panagiota Nikolaou, Artemisia Dona, Ioannis Papoutsis
ABSTRACT
A simple, rapid, sensitive and accurate gas chromatography–mass spectrometric method was
developed and validated for the simultaneous determination of hydroxyzine and cetirizine in whole
blood. Solid-phase extraction procedure using Bond Elut LRC Certify II columns was used for the
isolation of hydroxyzine and cetirizine from 1 mL whole blood followed by derivatization with a
mixture of acetic anhydride:n-propanol (1:1, v/v). Limits of detection and quantification were 1.50 and
5.00 ng/mL, respectively. The assay was linear within the concentration range of 5.00–1000.0 ng/mL
and the correlation coefficient was R2 ≥ 0.993 for both analytes. Absolute recovery was determined at
three quality control concentration levels and was found to be at least 87.2% for both substances. Intra-
day and inter-day accuracy values for both hydroxyzine and cetirizine were ranged from −1.2 to 3.8%
and −2.7 to 2.0%, respectively, at the three concentration levels studied, whereas their respective intra-
day and inter-day precision values were less than 9.9 and 6.5%, respectively, in terms of relative
standard deviation (%RSD). The developed method was successfully applied for the quantification of
hydroxyzine and cetirizine concentrations in whole blood, during the investigation of clinical cases
where these two antihistamines were detected.
An on-spot internal standard addition approach for accurately determining colistin A and
colistin B in dried blood spots using ultra high-performance liquid chromatography–tandem
mass spectrometry
I-Lin Tsai, Ching-Hua Kuo, Hsin-Yun Sun, Yu-Chung Chuang, Yun-Jung Tsai
ABSTRACT
Outbreaks of multidrug-resistant Gram-negative bacterial infections have been reported worldwide.
Colistin, an antibiotic with known nephrotoxicity and neurotoxicity, is now being used to treat
multidrug-resistant Gram-negative strains. In this study, we applied an on-spot internal standard
addition approach coupled with an ultra high-performance liquid chromatography-tandem mass
spectrometry (LC–MS/MS) method to quantify colistin A and B from dried blood spots (DBSs). Only
15 μL of whole blood was required for each sample. An internal standard with the same yield of
extraction recoveries as colistin was added to the spot before sample extraction for accurate
quantification. Formic acid in water (0.15%) with an equal volume of acetonitrile (50:50 v/v) was used
as the extraction solution. With the optimized extraction process and LC–MS/MS conditions, colistin
A and B could be quantified from a DBS with respective limits of quantification of 0.13 and 0.27 μg
mL−1, and the retention times were < 2 min. The relative standard deviations of within-run and
between-run precisions for peak area ratios were all < 17.3%. Accuracies were 91.5-111.2% for lower
limit of quantification, low, medium, and high QC samples. The stability of the easily hydrolyzed
prodrug, colistin methanesulfonate, was investigated in DBSs. Less than 4% of the prodrug was found
to be hydrolyzed in DBSs at room temperature after 48 h. The developed method applied an on-spot
internal standard addition approach which benefited the precision and accuracy.
252
Comparative study of single/combination use of Huang-Lian-Jie-Du decoction and berberine on
their protection on sepsis induced acute liver injury by NMR metabolic profiling
Yan Lv, Junsong Wang, Dingqiao Xu, Shanting Liao, Lingyi Kong
ABSTRACT
Sepsis is a serious clinical disease with a high mortality rate all around the world. Liver organ
dysfunction is an important sign for the severity and outcome of sepsis in patients. In this study, 1H
NMR-based metabolomics approach and biochemical assays were applied to investigate the metabolic
profiling for cecal ligation and puncture (CLP) induced acute liver injury, the therapeutical effect of
single/combination use of Huang-Lian-Jie-Du decoction (HLJDD) and berberine, and the interaction
of them. Metabolomics analysis revealed significant perturbations in livers of septic rats, which could
be ameliorated by HLJDD, berberine and their combination treatment. Berberine could better rectified
glycolysis and nucleic acid metabolism in the liver. HLJDD had exceptional better anti-inflammatory,
antibacterial and antioxidative effects than berberine. The interaction of berberine and HLJDD could
further strengthen the anti-inflammation and anti-oxidation, but with poor effect on amino acids
metabolism. These findings highlighted the feasibility of the integrated NMR based metabolomics
approach to understand the pathogenesis of diseases, the action mechanisms of therapy and the herb-
drug interaction.
Screening and confirmation of steroids and nitroimidazoles in urine, blood, and food matrices:
Sample preparation methods and liquid chromatography tandem mass spectrometric
separations
Ádám Tölgyesi, Enikő Barta, Andrea Simon, Thomas J. McDonald, Virender K. Sharma
ABSTRACT
Veterinary drugs containing synthetic anabolic steroid and nitroimidazole active agents are not allowed
for their applications in livestock of the European Union (EU). This paper presents analyses of twelve
selected steroids and six nitroimidazole antibiotics at low levels (1.56 μg/L–4.95 μg/L and 0.17 μg/kg–
2.14 μg/kg, respectively) in body fluids and egg incurred samples. Analyses involved clean-up
procedures, high performance liquid chromatography (HPLC) separation, and tandem mass
spectrometric screening and confirmatory methods. Target steroids and nitroimidazoles in samples
were cleaned by two independent supported liquid extraction and solid phase extraction procedures.
Separation of the selected compounds was conducted on Kinetex XB C-18 HPLC column using
gradient elution. The screening methods utilised supported liquid extraction that enabled fast and cost
effective clean-up. The confirmatory methods were improved by extending the number of matrices and
compounds, and by introducing an isotope dilution mass spectrometry for nitroimidazoles. The new
methods were validated according to the recommendation of the European Union Reference
Laboratories and the performance characteristics evaluated met fully the criteria. The methods were
applied to incurred samples in the proficiency tests. The obtained results of Z-scores demonstrated the
applicability of developed protocols of the methods to real samples. The confirmatory methods were
applied to the national monitoring program and natural contamination of prednisolone could be
detected in urine at low concentration in few samples.
253
Validation of an HPLC-UV method for analysis of Kaempferol-loaded nanoemulsion and its
application to in vitro and in vivo tests
Mariana Colombo, Gabriela de Lima Melchiades, Fabrício Figueiró, Ana Maria Oliveira Battastini,
Letícia Scherer Koester
ABSTRACT
A simple and reliable HPLC-UV method for Kaempferol (KPF) determination in a Kaempferol-loaded
nanoemulsion (KPF-NE), samples from mucosa permeation/retention studies, and murine brain was
developed and validated according to international guidelines. The analyses were performed on a
reversed-phase C18 column at 35 °C and under UV detection at 368 nm. The mobile phase was
composed of methanol:formic acid 0.1% (75:25, v/v) and was eluted at an isocratic flow rate of 1.0
mL/min. The method was selective and sensitive for KPF analysis in matrix extracts, and linear in the
range of 0.25–7.5 μg/mL. The method was also considered precise, accurate, and robust. The recovery
rates of KPF from the porcine nasal mucosa and murine brain were higher than 85%. Low matrix
effect was observed to determine KPF, including biological matrices. The applicability of the method
was confirmed in all different approaches, i.e., quantification of KPF in nanoemulsion, in vitro
permeation/retention of KPF across porcine nasal mucosa, and in vivo quantification of KPF in brain
samples after nasal administration in rats. Thus, the method is effective and reliable to determine KPF
in different real samples. The proposed method, therefore, provides a useful quantification approach to
routine processes, to the development of drug delivery systems, and to KPF quantification in different
biological matrices. Furthermore, the method is applicable in bioavailability studies and the developed
formulation (KPF-NE) is suitable for preclinical trials in different brain disorders.
Development of direct assays for Toxoplasma gondii and its use in genomic DNA sample
Lívia M. Alves, Vinícius R. Rodovalho, Ana C.H. Castro, Márcia A.R. Freitas, Ana G. Brito-Madurro
ABSTRACT
This work describes an approach for the selection and detection of specific DNA probes related to
Toxoplasma gondii, a protozoan parasite responsible for toxoplasmosis. The detection system was
developed on graphite carbon electrode modified with poly(3-hydroxybenzoic acid) sensitized with
ToxG1 probe. The hybridization of the specific genomic DNA related to T. gondii showed good
response by direct detection of guanine residue oxidation using differential pulse voltammetry (DPV).
The biosensor was able to distinguish both the complementary and non-complementary targets and
detect up to 100 ng μL−1 of the T. gondii genomic DNA. The hybridization (ToxG1: T. gondii
genomic DNA) was confirmed by optical measurement. Optical assays using gold
nanoparticles:ToxG1 probe showed a significant change in the absorbance peak in the presence of the
T. gondii genomic DNA according to the electrochemical results. This novel biosensor shows potential
as electrochemical transducer and was successfully applied in the biological sample.
254
Investigation and structural elucidation of a new impurity in bulk drug of cilostazol by
LC/MS/MS, FT-IR and NMR
Zhaoxia Hu, Sanguo Gao, Jue Gao
ABSTRACT
A new impurity was detected in bulk cilostazol (CIL) crude during routine analysis. The impurity
(∼4%, the specification for unknown impurity in crude is not more than 0.20%) has a relative retention
time of 1.46. Based on MS, NMR and IR spectral data, the impurity was identified as 6,6′-bis(4-(1-
cyclohexyl-1H-tetrazol-5-yl) butoxy)-3,3′,4,4′-tetrahydro-[7,7′-biquinoline]-2,2′(1H,1′H)-dione(CIL-
dimer). The precursor of CIL-dimer is an oxidative product of starting material 6-Hydroxy-3,4-
dyhydro-1H-quinolin–2-one(6-HQ), CIL-dimer was formed in the following reaction with 5-(3-
Chloro-propyl)-1-cyclohexyl-1H-tetrazol(CHCBT).
Selective screening of glutaric acid acidurias by capillary electrophoresis-mass spectrometry
Jaime Fernández-Bravo, Fernando de Andrés, Mohammed Zougagh, Ángel Ríos
ABSTRACT
A sensitive and selective method for the separation and quantification of the three organic acids 3-
hydroxy-3-methylglutaric acid, 3-methylglutaric acid, and glutaric acid in human urine samples by CE
with mass spectrometry detection has been developed. This methodology is faster, simpler and less
time-consuming, than other methodologies previously described, and requires of reduced amounts of
reagents as well. Samples are first filtered and then diluted in water. For the electrophoretic separation,
a 20 mM ammonium acetate and 10% methanol solution at pH 9.1 was selected as the running
electrolyte. With 5-s hydrodynamic injection, detection limits ranging from 15.5 to 39.3 μM and linear
responses ranging from the LOQ calculated for each analyte to more than 400 μM were obtained for
the analysis of the different organic acids in less than 13 min. Remarkable selectivity is achieved by
mass spectrometry detection using 0.25% of formic acid in 50% v/v 2-propanol-water solution as
sheath liquid, and enough sensitivity without interferences from the matrices was obtained as well.
This methodology has revealed as an efficient approach to help the 3-hydroxy-3-methylglutaric
aciduria diagnoses in order to discard or confirm the occurrence of the disease as of the presence or
absence of the expected increased levels of these analytes in samples of potential patients.
255
Online turbulent flow extraction coupled with liquid chromatography–tandem mass
spectrometry for high throughput screening of anabolic steroids in horse urine
Hyun Du Shin, Joon Hyuk Suh, Junghyun Kim, Hyun-Deok Cho, Sang Beom Han
ABSTRACT
A high throughput method for simultaneous screening of anabolic steroids and their metabolites (4-
esterendione, trenbolone, boldenone, oxandrolone, nandrolone, methandrostenolone, testosterone, 1-
androstendione, ethisterone, normethandrolone, methyltestosterone, 16β-Hydroxystanozolol,
epitestosterone, bolasterone, norethandrolone, danazol, stanozolol and androstadienone) in equine
urine by online turbulent flow extraction coupled with liquid chromatography-tandem mass
spectrometry was developed. The use of turbulent flow chromatography could simplify pretreatment of
horse urine, which has complex matrices as well as high viscosity. The urine was extracted by mixed-
mode cation exchange solid phase extraction, and hydrolyzed using β-glucuronidase/arylsulfatase.
Then, the sample was automatically loaded on the TurboFlow Cyclone extraction column for removal
of further matrix, followed by separation on a fused core C18 column before MS/MS detection.
Optimization and validation of the method were discussed in detail. All analytes were rapidly detected
within 10 min with high sensitivity (picogram to nanogram per milliliter level), and no interference
was observed. The linearity range was from 0.1–10 ng/mL for nine steroids and 1.0–50 ng/mL for the
others, with correlation of coefficient values over 0.995. Precision and accuracy ranged from 0.1 to
14.5% and 1.7 to 12.4%, respectively. The developed method was successfully applied to the analysis
of anabolic steroids in horse urine after administration of a model drug.
Development of a mixed-mode chromatography with tandem mass spectrometry method for the
quantitative analysis of 23 underivatized amino acids in human serum
Min Sun Choi, Shaheed Ur Rehman, In Sook Kim, Hi-Joon Park, Hye Hyun Yoo
ABSTRACT
In this study, a robust, selective and simplified method was developed and validated for the
simultaneous quantitative analysis of 23 underivatized amino acids in human serum using mixed-mode
chromatography with tandem mass spectrometry (LC–MS/MS). Serum samples were deproteinized
with acetonitrile and subjected to LC–MS/MS analysis. The chromatographic separation of amino
acids was achieved using a mixed-mode column (150 × 3 mm, 3 μm) with a gradient elution system;
the mobile phase consisted of 50 mM ammonium formate and 0.1% formic acid in acetonitrile. The
total run time was 22 min. Eluted compounds were detected in the electrospray ionization-positive
mode with multiple reaction monitoring. The validation study evaluated linearity, repeatability, intra
and inter-day accuracy and precision, and matrix effect. The validation results were satisfactory in all
the tested parameters. This method was successfully applied to the analysis of amino acids in the
clinical sample of human serum.
256
Inhibitory assay for degradation of collagen IV by cathepsin B with a surface plasmon resonance
sensor
Atsushi Shoji, Yumiko Suenaga, Atsushi Hosaka, Yuuki Ishida, Masao Sugawara
ABSTRACT
We describe a simple method for evaluating the inhibition of collagen IV degradation by cathepsin B
with a surface plasmon resonance (SPR) biosensor. The change in the SPR signal decreased with an
increase in the concentration of cathepsin B inhibitors. The order of the inhibitory constant (Ki)
obtained by the SPR method was CA074Me ≈ Z-Phe-Phe-FMK < leupeptin. This order was different
from that obtained by benzyloxycarbonyl-Phe-Phe-Fluoromethylketone (Z-Phe-Phe-FMK) as a peptide
substrate. The comparison of Ki suggested that CA074 and Z-Phe-Phe-FMK inhibited exopeptidase
activity, and leupeptin inhibited the endopeptidase activity of cathepsin B more strongly.
Liquid chromatography-tandem mass spectrometry assay to quantify plitidepsin in human
plasma, whole blood and urine
L. van Andel, H. Rosing, S. Fudio, P. Avilés, J.H. Beijnen
ABSTRACT
Plitidepsin is an anti-cancer drug currently evaluated in phase I/II/III clinical trials. This article
describes the development and validation of a bioanalytical assay to quantify plitidepsin in human
plasma, urine and whole blood using HPLC–MS/MS. The analyte was extracted from the matrix by
liquid–liquid extraction using tert-butyl methyl ether. Final extracts were injected onto a C18 column,
gradient elution was applied for chromatographic separation and detection was performed on a triple
quadrupole mass spectrometer operating in the positive ion mode. The assay was linear over the range
0.1–100 ng/mL, with acceptable accuracy and precision values. This is the first reported bioanalytical
assay quantifying plitidepsin using a stable isotopically labelled standard, achieving a lower limit of
quantification of 0.1 ng/mL in all three matrices, allowing the quantification of trace level of
plitidepsin, and accomplishing this in an analysis time of two minutes only. The presented method was
successfully applied in a mass balance study with plitidepsin in patients with advanced cancer.
257
Rapid analysis of benzalkonium chloride using paper spray mass spectrometry
Jingjing Liu, Wenjie Deng, Muqian Yu, Ruizhi Wen, Bo Chen
ABSTRACT
A paper spray mass spectrometry (PS-MS) method for rapid and reliable analysis of benzalkonium
chloride (BAC) in compound eye drops and body surface disinfectant was developed. The sample was
dropped onto triangular filter paper, and high voltage (3.5 kV) was applied to form an electrospray.
This method can provide the composition of benzalkonium chloride in samples without pretreatment,
solvent or chromatographic separation, and the analysis time is only 10 s. The primary homologues
C12-BAC, C14-BAC and C16-BAC of benzalkonium chloride were quantitatively analyzed using PS-
MS. Samples were subjected to simple dilution and quantified using the internal standard method. Ion
trap mass spectrometry was scanned using SIM mode. The linear ranges of C12-BAC, C14-BAC and
C16-BAC were 1–100 μg mL−1; the linear regression coefficients were 0.998–0.999; the detection
limits (LODs) were 0.1 μg mL−1; the limit of quantifications (LOQs) was <1 μg mL−1, and the
method validation indicated that the method precision and accuracy were good. Compared with HPLC-
UV methods, there was no significant difference in the quantitative determination of the actual
samples, but the analysis time for PS-MS is shorter (2 min). In addition, reagent consumption in PS-
MS is small, and no chromatographic separation is needed, suggesting that PS-MS is especially
suitable for high-throughput analysis.
Rapid screening of non-steroidal anti-inflammatory drugs illegally added in anti-rheumatic
herbal supplements and herbal remedies by portable ion mobility spectrometry
Mengjiao Li, Haiyan Ma, Jinglin Gao, Lina Zhang, Ye Jiang
ABSTRACT
In this work, for the first time, a high-performance ion mobility spectrometry with electrospray
ionization (ESI-HPIMS) method has been employed as a rapid screening tool for the detection of
acetaminophen, ibuprofen, naproxen, diclofenac sodium and indomethacin illegally added in anti-
rheumatic herbal supplements and herbal remedies. Samples were dissolved and filtered through a 0.45
μm microporous membrane, then the filtrate was directly injected into the high-performance ion
mobility spectrometry for analysis. Using this approach, the screening of illegal additions can be
accomplished in as rapid as two to three minutes with no pretreatment required. The proposed method
provided a LOD of 0.06–0.33 μg mL−1, as well as a good seperation of the five NSAIDs. The
precision of the method was 0.1–0.4% (repeatability, n = 6) and 0.9–3.3% (reproducibility, n = 3). The
proposed method appeared to be simple, rapid and highly specific, thus could be effective for the in-
situ screening of NSAIDs in anti-rheumatic herbal supplements and herbal remedies.
258
Simultaneous quantitative analysis of polyethylene glycol (PEG), PEGylated paclitaxel and
paclitaxel in rats by MS/MSALL technique with hybrid quadrupole time-of-flight mass
spectrometry
Heping Sun, Qi Zhang, Zhi Zhang, Jin Tong, Jingkai Gu
ABSTRACT
PEGylation is practically one of most important modifications of drugs including small molecules,
peptides and proteins, which has been proven to dramatically improve physicochemical properties and
pharmacokinetic behavior of the PEGylated drugs. However, it is a challenge currently to
quantitatively analyze PEG and PEGylated drugs by various analytical methods, even mass
spectrometry because of multiple parent ion distribution of PEG caused by its polydispersity of
molecular weight. Here we developed a robust method with MS/MSALL technique using electrospray
ionization (ESI) source coupled high resolution Quadrupole Time-of-Flight (Q-TOF) mass
spectrometry for the quantification of PEG2K-Paclitaxel (PEG-PTX) and its two metabolites, PEG and
Paclitaxel (PTX). The analysis was performed on a 300SB-C18 column with acetonitrile and 0.1%
formic acid as the mobile phase. Samples were simply prepared by protein precipitation in a small
quantity of plasma (50 μL). Calibration curve was linear within the range of 50.0–4000 ng/mL for
PEG and PEG-PTX and 1.0–1000 ng/mL for PTX. The intra- and inter-day precisions were 3.2–6.9%
and 3.1–6.9% for PEG, 4.1–7.8% and 4.0–9.9% for PEG-PTX, and 3.3–4.8% and 3.1–6.9% for PTX,
respectively. The recoveries were greater than 90% with low matrix effects. Afterwards, the newly
developed method was successfully applied to support a preclinical pharmacokinetic study in six rats
after single intravenous injection of PEG-PTX (51.7 mg/kg).
Lyophilic matrix method for dissolution and release studies of nanoscale particles
Jenni Pessi, Sami Svanbäck, Ilkka Lassila, Edward Hæggström, Jouko Yliruusi
ABSTRACT
We introduce a system with a lyophilic matrix to aid dissolution studies of powders and particulate
systems. This lyophilic matrix method (LM method) is based on the ability to discriminate between
non-dissolved particles and the dissolved species. In the LM method the test substance is embedded in
a thin lyophilic core-shell matrix. This permits rapid contact with the dissolution medium while
minimizing dispersion of non-dissolved particles without presenting a substantial diffusion barrier. The
method produces realistic dissolution and release results for particulate systems, especially those
featuring nanoscale particles. By minimizing method-induced effects on the dissolution profile of
nanopowders, the LM method overcomes shortcomings associated with current dissolution tests.
259
Incorporation of 14C-cholesterol in human adrenal corticocarcinoma H295R cell line and
online-radiodetection of produced 14C-steroid hormone metabolites
Jonas Abdel-Khalik, Erland Björklund, Frederik Knud Nielsen, Martin Hansen
ABSTRACT
This study demonstrates the addition of 14C-cholesterol to the human cell line H295R will in-situ form
radiolabeled steroid hormones allowing for new mechanistic and metabolic insights. The aim of the
present study was to in-situ radiolabel steroid hormones from cell line-incorporated 14C-cholesterol
using the OECD guideline 456, H295R steroidogenesis in-vitro assay. Radiodetection of the steroid
metabolites of the steroidogenic pathway allows for an improved understanding of the various
enzymatic mechanisms involved without necessarily being dependent on quantification. Generated
radiolabeled steroids were analyzed using HPLC hyphenated with a Flow Scintillation Analyzer
(FSA). H295R cells were incubated with radiolabeled cholesterol and cell media were collected and
prepared by solid phase extraction and analyzed with HPLC-FSA. For successful radiolabeling of the
steroids in the steroidogenesis of H295R cells, radioactive cholesterol may potentially only need to be
added just before the cells are incubated for 72 h in well plates. Based on the obtained HPLC-FSA
chromatograms, and confirmation of the observations by studies in the literature, a qualitative time
profile for the production of steroid hormones was estimated. Multiple radiolabeled steroid hormones
were identified by means of analytical standards and UV (ultraviolet) co-chromatography, though the
elucidation of multiple metabolites remains unresolved. Although online radiodetection proved to
suffer from suboptimal sensitivity, the concept of radiolabeling the steroidogenesis in H295R cells
with 14C-cholesterol and detecting the radiolabeled steroid hormones online was proved and may
assist in further toxicological studies.
Verification of the effectiveness of the Fourier transform infrared spectroscopy computational
model for colorectal cancer
J. Depciuch, E. Kaznowska, A. Koziorowska, J. Cebulski
ABSTRACT
Colorectal cancer is one of the most common cancers. Its formation is influenced by genetic and
environmental factors. Despite the continuous development of diagnostic tools and cancer therapies,
there are no methods that allow a real-time estimation of treatment efficiency. This method can be a
vibrational spectroscopy. The resulting infrared spectrum (FTIR) of the tissue gives us information
about the chemical composition and the content of the individual components. We have noticed that
tumor tissues, healthy and after chemotherapy tissues, have different vibrational spectra. It was also
shown that spectra acquired from normal (benign) tissues were similar to those derived from tissues
post-chemotherapy. The similarity was greater, when the effectiveness of chemotherapy, confirmed by
medical documentation, was better. Therefore, we decided to use the physical model proposed in our
earlier paper to verify its correctness and to show whether a particular type of chemotherapy was
effective or not. Comparison of the results obtained from the physical model with patients data have
been found as close to the physical condition.
260
Quantification of paracetamol and 5-oxoproline in serum by capillary electrophoresis:
Implication for clinical toxicology
Tomáń Hloņek, Tomáń Kříņek, Petr Tůma, Miroslava Bursová, Radomír Čabala
ABSTRACT
High anion gap metabolic acidosis frequently complicates acute paracetamol overdose and is generally
attributed to lactic acidosis or compromised hepatic function. However, metabolic acidosis can also be
caused by organic acid 5-oxoproline (pyroglutamic acid). Paracetamol‘s toxic intermediate, N-acetyl-
p-benzoquinoneimine irreversibly binds to glutathione and its depletion leads to subsequent disruption
of the gamma glutamyl cycle and an excessive 5-oxoproline generation. This is undoubtedly an
underdiagnosed condition because measurement of serum 5-oxoproline level is not readily available. A
simple, cost effective, and fast capillary electrophoresis method with diode array detection (DAD) for
simultaneous measurement of both paracetamol (acetaminophen) and 5-oxoproline in serum was
developed and validated. This method is highly suitable for clinical toxicology laboratory diagnostic,
allowing rapid quantification of acidosis inducing organic acid 5-oxoproline present in cases of
paracetamol overdose. The calibration dependence of the method was proved to be linear in the range
of 1.3–250 μg mL−1, with adequate accuracy (96.4–107.8%) and precision (12.3%). LOQ equaled 1.3
μg mL−1 for paracetamol and 4.9 μg mL−1 for 5-oxoproline.
Quantification of cyclocreatine in mouse and rat plasma using hydrophilic-interaction ultra-
performance liquid chromatography-tandem mass spectrometry
Amy Q. Wang, Emma Hughes, Wenwei Huang, Edward H. Kerns, Xin Xu
ABSTRACT
An accurate, rapid and selective method was developed to quantify cyclocreatine in mouse and rat
plasma using hydrophilic interaction (HILIC) ultra-performance liquid chromatography-tandem mass
spectrometry (UPLC–MS/MS). The plasma samples were prepared by protein precipitation with
acetonitrile:methanol (70:30). Chromatographic separation was performed on a HILIC BEH amide
column (2.1 mm × 50 mm, 1.7 μm) with a 3 min gradient elution at a flow rate of 0.5 mL/min. For
mass spectrometric detection, selected reaction monitoring (SRM) was used; the SRM transitions were
m/z 144 → 98 and m/z 144 → 56 for cyclocreatine and m/z 148 → 102 for the internal standard (D4-
cyclocreatine) in the positive ionization mode. No endogenous components interfered with the analysis
of cyclocreatine and the internal standard in mouse and rat plasma. Plasma calibration curves were
constructed in the range of 0.01–25 μM. The correlation coefficient of the calibration curves was
greater than 0.99. The mean intraday assay accuracy for all quality control (QC) replicates was
between 93 and 105%. The mean intraday assay precision (CV%) was 1.9-11% for all QC levels. The
HILIC–UPLC–MS/MS method was successfully applied in pharmacokinetic (PK) studies of
cyclocreatine in mice and rats for the first time. After a single 30 mg/kg oral administration in mice
and rats, the AUC0-∞ (area under the curve) was 84.1 μg h/mL and 91.7 ± 18.0 μg h/mL, respectively.
261
Volumetric adsorptive microsampling-liquid chromatography tandem mass spectrometry assay
for the simultaneous quantification of four antibiotics in human blood: Method development,
validation and comparison with dried blood spot
Sebastiano Barco, Elio Castagnola, Andrea Moscatelli, James Rudge, Giuliana Cangemi
ABSTRACT
Summary: In this paper we show the development and validation of a volumetric absorptive
microsampling (VAMS™)-LC–MS/MS method for the simultaneous quantification of four antibiotics:
piperacillin-tazobactam, meropenem, linezolid and ceftazidime in 10 μL human blood. The novel
VAMS-LC–MS/MS method has been compared with a dried blood spot (DBS)-based method in terms
of impact of hematocrit (HCT) on accuracy, reproducibility, recovery and matrix effect. Antibiotics
were extracted from VAMS and DBS by protein precipitation with methanol after a re-hydration step
at 37 °C for 10 min. LC–MS/MS was carried out on a Thermo Scientific™ TSQ Quantum™ Access
MAX triple quadrupole coupled to an Accela ™UHPLC system. The VAMS-LC–MS/MS method is
selective, precise and reproducible. In contrast to DBS, it allows an accurate quantification without any
HCT influence. It has been applied to samples derived from pediatric patients under therapy. VAMS is
a valid alternative sampling strategy for the quantification of antibiotics and is valuable in support of
clinical PK/PD studies and consequently therapeutic drug monitoring (TDM) in pediatrics.
PLGA Ethionamide Nanoparticles for Pulmonary Delivery: Development and in vivo evaluation
of dry powder inhaler
Sujit Kumar Debnath, Srinivasan Saisivam, Abdelwahab Omri
ABSTRACT
PLGA (50:50) nanoparticles were prepared to sustain the release of Ethionamide in order to decrease
the dose and dosing frequency. It further modified in the form of dry powder inhaler to make suitable
for pulmonary administration and increase drug residency in lungs. Ethionamide loaded PLGA
nanoparticles were prepared by solvent evaporation method. Freeze dried nanoparticles and anhydrous
inhalable grade lactose were mixed manually using geometrical dilution process to modify the
nanoparticles in the form of dry powder inhaler. Animal study was conducted to correlate between in-
vivo and in-vitro. PLGA nanoparticles showed initial burst release followed by zero order release up to
95.17 ± 3.59% in 24 h. Aerodynamic particle size of optimized dry powder inhaler was found as 1.79
μm. There was no significant aggregation of dry powder inhaler during 6 months of stability study.
Area under the concentration-time curve from 0 h to infinity (AUC0−∞) signifies the prolong
residency of ETH in body compartment, revealed from animal study. PLGA 50:50 coated
nanoparticles released Ethionamide for the period of 24 h in simulated lungs fluid. Correlation
between in-vitro dissolution and in-vivo study was established after performing animal study. Prepared
dry powder inhaler maintained Ethionamide concentration above minimum inhibitory concentration
for more than 12 h after single dose administration.
262
Simultaneous determination and pharmacokinetics of danshensu, protocatechuic aldehyde, 4-
hydroxy-3-methyloxyphenyl lactic acid and protocatechuic acid in human plasma by LC–
MS/MS after oral administration of Compound Danshen Dripping Pills
Wei Li, Hongjie Zhou, Yang Chu, Xiangyang Wang, He Sun
ABSTRACT
Compound Danshen Dripping Pills (CDDP), a herbal patent medicine, is widely used in China for the
prevention and treatment of cardiovascular diseases. A simple, sensitive and reliable method for
simultaneous determination of danshensu (DSS), protocatechuic aldehyde (PCA), and their related
metabolites, 4-hydroxy-3-methyloxyphenyl lactic acid (HMLA) and protocatechuic acid (PAA) in
human plasma was developed and validated based on liquid chromatography tandem mass
spectrometry (LC–MS/MS). The analytes and internal standard (IS), vanillic acid (VAA), were
extracted from plasma with ethyl acetate and separated on a C18 column by using the mobile phase
consisted of methanol-0.1% formic acid via gradient elution. The electrospray ionization (ESI) source
was applied and operated under the multiple reaction monitoring (MRM) mode. The linear calibration
curves were obtained at the concentration ranges of 0.46–1000 ng/mL for DSS and PAA, and 1.38–
1000 ng/mL for PCA and HMLA, respectively. The inter- and intra-day precisions (RSD%) were less
than 13.5%, and the accuracy (±RE%) was within 13.4%. The described method was successfully
applied for the clinical pharmacokinetics of CDDP in Chinese healthy volunteers.
LC–MS bioanalysis of Trastuzumab and released emtansine using nano-surface and molecular-
orientation limited (nSMOL) proteolysis and liquid–liquid partition in plasma of Trastuzumab
emtansine-treated breast cancer patients
Noriko Iwamoto, Akihiko Shimomura, Kenji Tamura, Akinobu Hamada, Takashi Shimada
ABSTRACT
Antibody-drug conjugates (ADCs) consist of monoclonal antibody and cytotoxic drugs covalently
attached via stable crosslinkers, and are prospective antibody drugs for cancer therapy. To cover the
overall pharmacokinetic understanding of ADCs, both the antibody and the released drugs are
necessary for practical clinical observation. The nano-surface and molecular-orientation limited
(nSMOL) proteolysis is a universal approach for antibody bioanalysis that enable Fab-selective
proteolysis, which maintains antibody sequence specificity while decreasing excess analyte peptides.
In this study, we describe quantitative assays for ADC in human plasma using nSMOL for the
antibody and polarity-selective liquid–liquid partition with a methanol/ethyl acetate mixed solvent for
the cytotoxic drugs. This approach led to the successful development of LC–MS validated bioanalysis
of the antibody and released drugs within 20% for lower limit of quantitation and 15% for another
concentration setting of Trastuzumab emtansine (T-DM1), Trastuzumab antibody and emtansine
conjugated with crosslinker (DM1-MCC). The validated concentration ranges in human plasma were
0.06–250 μg/mL for T-DM1 and 0.39–200 ng/mL for DM1-MCC. These results indicate that LC–MS
method with a two-sided approach, using nSMOL and liquid–liquid partition, show potential for the
precise pharmacokinetic study for ADC development and treatment.
263
Separation of furostanol saponins by supercritical fluid chromatography
Jie Yang, Lingling Zhu, Yang Zhao, Yongwei Xu, Baiping Ma
ABSTRACT
Supercritical fluid chromatography (SFC) has good separation efficiency and is suitable for separating
weakly polar compounds. Furostanol saponins, as an important kind of steroidal saponins, generally
have two sugar chains, which are polar and hydrophilic. The hydroxyl group at the C-22 position of
furostanol saponins is active and easily reacts with lower alcohols under appropriate conditions. The
separation of hydrophilic furostanol saponins was tested by SFC in this study. The effects of
chromatographic conditions on the separation of the mixed furostanol saponins and their hydroxyl
derivatives at the C-22 position were studied. The conditions for SFC, which included different
column polarity, modifier, additive, and column temperature, were tested. After optimization, the
mixed 10 similar structures of furostanol saponins were separated in 22 min on the Diol column at a
temperature of 40 °C. The mobile phase was CO2 (mobile phase A) and methanol (containing 0.2%
NH3∙H2O and 3% H2O) (mobile phase B). The backpressure was maintained isobarically at 11.03
MPa. SFC was found to be effective in separating the furostanol saponins that shared the same
aglycone but varied in sugar chains. SFC was sensitive to the number and type of sugars. The
resolution of furostanol saponin isomers was not ideal. The extract of Dioscorea zingiberensis C. H.
Wright was profiled by SFC–quadrupole time-of-flight mass spectrometry. The main saponins of the
extract were well separated. Therefore, SFC could be used for separating hydrophilic furostanol
saponins and analyzing traditional Chinese medicines that mainly contained steroidal saponins.
Macroporous monoliths for biodegradation study of polymer particles considered as drug
delivery systems
M.V. Volokitina, V.A. Korzhikov-Vlakh, T.B. Tennikova, E.G. Korzhikova-Vlakh
ABSTRACT
Nanostructures based on biodegradable polymers are often considered as drug delivery systems. The
properties of these nanomaterails towards in vitro biodegradation are very important and usually are
studied using the model physiological conditions. In this work the novel approach based on application
of monolithic immobilized enzyme reactors (IMERs) as the systems for biodegradation study of the
nanoobjects of different nature and morphology was suggested. Rigid nanospheres based on
poly(lactic acid) and self-assembled nanoobjects formed from block-copolymer of glutamic acid and
phenylalanine were applied as model nanomaterials. For that, two enzymes, namely, esterase and
papain were chosen for preparation of the monolithic IMERs. The properties of immobilized enzymes
were compared to those obtained for soluble biocatalysts in the reaction of poly(lactic acid) and
poly(glutamic acid) degradation. The monitoring of substrate destruction process was carried out using
different HPLC modes (anion-exchange, cation-exchange or precipitation-redissolution based process)
also based on application of the same modern stationary phase, namely, macroporous monoliths (CIM
disks and lab-made column). Finally, the applicability of monolithic immobilized enzyme reactors for
degradation of polyester and polypetide-based particles was demonstrated and compared to the process
observed in human blood plasma.
264
A novel enantioseparation approach based on liposome electrokinetic capillary chromatography
Xiaoqi Li, Yingxiang Du, Zijie Feng, Xiaodong Sun, Zhifeng Huang
ABSTRACT
As a novel separation mode of capillary electrophoresis (CE), liposome electrokinetic capillary
chromatography (LEKC) has aroused considerable attention in recent years; however, the
enantioseparation based on this new system has not been previously investigated. In this study, we
proposed a brand-new LEKC chiral separation approach using liposomes comprised of
phosphatidylcholine (PC) and cholesterol as pseudo-stationary phase and sulfobutyl ether-β-
cyclodextrin (SBE-β-CD) as chiral selector. Compared with the single CD system and CD-SDS-
MEKC system, this LEKC method presented an obviously preferable enantioseparation of four model
drugs (naproxen, warfarin, ketoprofen and amlodipine). In this new established system, all the
enantiomers represented baseline separations with the resolution and selectivity respectively achieving
1.584/1.067 (for naproxen), 2.226/1.045 (for warfarin), 1.537/1.038 (for ketoprofen) and 2.592/1.097
(for amlodipine), while other two comparative systems demonstrated no separation or a poor
separation. Several important parameters affecting the enantioseparation, such as buffer pH,
concentration of liposomes, phosphate buffer solution (PBS) and chiral selector (SBE-β-CD), and
applied voltage were systematically investigated. Satisfactory repeatability was achieved through intra-
day, inter-day and batch-to-batch investigations with relative standard deviations less than 3.40%.
Furthermore, the established method was successfully applied to test the chiral impurity of naproxen
sample.
Comparative study on the anticancer activities and binding properties of a hetero metal
binuclear complex [Co(dipic)2Ni(OH2)5]·2H2O (dipic = dipicolinate) with two carrier proteins
Somaye Shahraki, Fereshteh Shiri, Mostafa Heidari Majd, Zohreh Razmara
ABSTRACT
Recognizing of binding mechanisms between drugs and carrier proteins is basic for us to understand
the pharmacokinetics and pharmacodynamics of them. In this research, the anticancer activities of a
binuclear complex [Co(dipic)2Ni(OH2)5]·2H2O (dipic = dipicolinate) against MDA-MB-231 cell
lines were studied. Results of MTT assay and flow cytometry analysis revealed that above complex
can induce the cytotoxicity and the apoptosis in breast cancer cell lines. So, this complex was selected
to investigate its binding to human serum albumin (HSA) and bovine β-lactoglobulin (βLG) by
spectroscopic methods (UV–visible, fluorescence and FT-IR) along with molecular docking technique.
The fluorescence data showed Co-Ni complex quench the fluorescence of both proteins by a static
quenching mechanism and HSA has stronger binding affinity toward Co-Ni complex than βLG. The
binding constant (Kb), number of binding sites (n) and thermodynamic parameters were calculated and
showed that the Co-Ni complex binds to protein (HSA and βLG) through hydrogen bonding and van
der Waals forces with one binding site. The results of UV–visible measurements indicated that the
binding of above complex to HSA and βLG may induce conformational and micro-environmental
changes of studied proteins. Protein–ligand docking analysis confirmed that the Co-Ni complex binds
to residues located in the subdomain IIA of HSA and site II of βLG.
265
Characterization and quantitative analysis of phenolic derivatives in Longxuetongluo Capsule by
HPLC-DAD-IT-TOF-MS
Jing Sun, Yuelin Song, Hui Sun, Wenjing Liu, Jun Li
ABSTRACT
Longxuetongluo Capsule (LTC), which is derived from the total phenolic extract of Chinese dragon‘s
blood, has been proved to be safe as well as effective towards ischemic stroke. However, the effective
material basis remains unclear. The present study thereby focused on the clarification of the qualitative
and quantitative properties for the phenolic derivatives in LTC. Regarding homolog-focused chemical
profiling, the mass fragmentation patterns of the primary subtypes of phenolic compounds such as
homoisoflavanones, flavanes, chalcones, and flavonoid oligomers were summarized by assaying
authentic references with hybrid ion trap time-of-flight mass spectrometry, and the chemical structures
of 124 phenolic compounds, in total, were unambiguously or tentatively annotated in LTC by
matching the accurate mass spectral profiles with the proposed mass cracking rules and those reference
substances. Afterwards, simultaneous determination of 12 primary phenolic compounds was carried
out in different batches of LTC using HPLC-DAD, after that the method was proved to be accurate,
precise, and reproducible according to diverse method validation assays. The obtained findings are
expected to be meaningful for clarifying the effective substances and quality assessment of LTC.
Volume 145 October 2017
Two-dimensional liquid chromatography in pharmaceutical analysis Instrumental aspects,
trends and applications
Marion Iguiniz, Sabine Heinisch
ABSTRACT
The interest in two-dimensional liquid chromatography (2D-LC) has been growing up since the last
decades. This promising technique appears as a relevant solution for various analytical challenges
encountered in pharmaceutical analysis. The objective of this review is to give an overview of past,
current and emerging trends in 2D-LC techniques applied to pharmaceutical compounds. The
referenced studies cover the late 1980s to the present. Information regarding the different aspects of
this analytical technique, including chromatographic conditions, instrumental setup and compounds of
interest, was compiled and summarized into a synoptic table. Particular attention is paid to key features
including (i) the interfaces used for coupling the two dimensions, (ii) the application fields, and (iii)
the chromatographic modes that can be combined together. Finally an attempt is made to predict future
advances in two-dimensional separation techniqes for pharmaceutical analysis.
266
Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A
generic method development approach
Balázs Bobály, Valentina D‘Atri, Alain Beck, Davy Guillarme, Szabolcs Fekete
ABSTRACT
Hydrophilic interaction liquid chromatography (HILIC) is a well-established technique for the
separation and analysis of small polar compounds. A recently introduced widepore stationary phase
expanded HILIC applications to larger molecules, such as therapeutic proteins. In this paper, we
present some generic HILIC conditions adapted for a wide range of FDA and EMA approved
recombinant monoclonal antibody (mAb) species and for an antibody-drug conjugate (ADC). Seven
approved mAbs possessing various isoelectric point (pI) and hydrophobicity as well as a cysteine
conjugated ADC were used in this study. Samples were digested by IdeS enzyme and digests were
further fragmented by chemical reduction. The resulting fragments were separated by HILIC. The
main benefit of HILIC was the separation of polar variants (glycovariants) in a reasonable analysis
time at the protein level, which is not feasible with other chromatographic modes. Three samples were
selected and chromatographic conditions were further optimized to maximize resolution. A
commercial software was used to build up retention models. Experimental and predicted
chromatograms showed good agreement and the average error of retention time prediction was less
than 2%. Recovery of various species and sample stability under the applied conditions were also
discussed.
A new platform for serological analysis based on porous 3-dimensional polyethylene sinter
bodies
Mohammed Alasel, Michael Keusgen
ABSTRACT
A new sensitive and selective platform, three-dimensional immunosensor, has been developed for a
rapid serological diagnosis; detection of a Borrelia infection was considered as a model assay. The
immunosensor is based on a 3-dimensional (3D) porous solid surface (sinter body) with dimensions of
2 × 2.5 mm where a recombinant variable lipoprotein surface-exposed protein (VlsE; Borrelia-antigen)
is immobilized by different techniques. The sinter body served as a robust and inexpensive carrier,
which facilitated a successful hydrophobic adsorption as well as covalent immobilization of the
antigen with sufficient amounts of on the surface. Because of sinter body‘s porosity, the detection
could be performed in an immune affinity flow system based on a little disposable plastic column. The
flow of reagents through the column is advantageous in terms of reducing the non-specific interaction
and shortening the test time. Furthermore, three labels were tested for a colorimetric detection: i) a
horseradish peroxidase (HRP) labeled secondary antibody, ii) nanoparticles based on Sudan IV, and
iii) gold nanoparticles modified with protein A. HRP secondary labeled antibody provides the most
sensitive test, 1000 fold dilution of serum sample can be clearly detected in only 20 min. Gold
nanoparticles modified with protein A were used as a direct label or as a catalyst for reduction of silver
ions. Direct detection with gold nanoparticles provides short time of analysis (5 min) while detection
of metallic silver required longer time (12 min) but with improved sensitivity. Nanoparticles based on
Sudan IV showed high background and were less favorable. The assay is distinctive because of the
rapid analysis time with all used labels, longest 20 min. Compared to classical serological methods for
Borrelia diagnosis, the developed method offers a simple, rapid and reliable tool of analysis with
minimal cost and can be easily transferred to other infectious diseases.
267
Designing a calibration set in spectral space for efficient development of an NIR method for
tablet analysis
Md Anik Alam, James Drennen, Carl Anderson
ABSTRACT
Designing a calibration set is the first step in developing a multivariate spectroscopic calibration
method for quantitative analysis of pharmaceutical tablets. This step is critical because successful
model development depends on the suitability of the calibration data. For spectroscopic-based
methods, traditional concentration based techniques for designing calibration sets are prone to have
redundant information while simultaneously lacking necessary information for a successful calibration
model. A method for designing a calibration set in spectral space was developed. The pure component
spectra of a tablet formulation were used to define the spectral space of that formulation. This method
maximizes the information content of measurements and minimizes sample requirements to provide an
efficient means for developing multivariate spectroscopic calibration. A comparative study was
conducted between a commonly employed full factorial approach to calibration development and the
newly developed technique. The comparison was based on a system to quantify a model drug,
acetaminophen, in pharmaceutical compacts using near infrared spectroscopy. A 2-factor full factorial
design (acetaminophen with 5 levels and MCC:Lactose with 3 levels) was used for calibration
development. Three replicates at each design point resulted in a total of 45 tablets for the calibration
set. Using the newly developed spectral based method, 11 tablets were prepared for the calibration set.
Partial least square (PLS) models were developed from respective calibration sets. Model performance
was comprehensively assessed based on the ability to predict acetaminophen concentrations in
multiple prediction sets.
On-line prediction of the glucose concentration of CHO cell cultivations by NIR and Raman
spectroscopy: Comparative scalability test with a shake flask model system
Bence Kozma, Edit Hirsch, Szilveszter Gergely, László Párta, András Salgó
ABSTRACT
In this study, near-infrared (NIR) and Raman spectroscopy were compared in parallel to predict the
glucose concentration of Chinese hamster ovary cell cultivations. A shake flask model system was
used to quickly generate spectra similar to bioreactor cultivations therefore accelerating the
development of a working model prior to actual cultivations. Automated variable selection and several
pre-processing methods were tested iteratively during model development using spectra from six shake
flask cultivations. The target was to achieve the lowest error of prediction for the glucose
concentration in two independent shake flasks. The best model was then used to test the scalability of
the two techniques by predicting spectra of a 10 l and a 100 l scale bioreactor cultivation. The NIR
spectroscopy based model could follow the trend of the glucose concentration but it was not
sufficiently accurate for bioreactor monitoring. On the other hand, the Raman spectroscopy based
model predicted the concentration of glucose in both cultivation scales sufficiently accurately with an
error around 4 mM (0.72 g/l), that is satisfactory for the on-line bioreactor monitoring purposes of the
biopharma industry. Therefore, the shake flask model system was proven to be suitable for scalable
spectroscopic model development.
268
Conventional and accelerated-solvent extractions of green tea (camellia sinensis) for
metabolomics-based chemometrics
Joshua J. Kellogg, Emily D. Wallace, Tyler N. Graf, Nicholas H. Oberlies, Nadja B. Cech
ABSTRACT
Metabolomics has emerged as an important analytical technique for multiple applications. The value of
information obtained from metabolomics analysis depends on the degree to which the entire
metabolome is present and the reliability of sample treatment to ensure reproducibility across the
study. The purpose of this study was to compare methods of preparing complex botanical extract
samples prior to metabolomics profiling. Two extraction methodologies, accelerated solvent extraction
and a conventional solvent maceration, were compared using commercial green tea [Camellia sinensis
(L.) Kuntze (Theaceae)] products as a test case. The accelerated solvent protocol was first evaluated to
ascertain critical factors influencing extraction using a D-optimal experimental design study. The
accelerated solvent and conventional extraction methods yielded similar metabolite profiles for the
green tea samples studied. The accelerated solvent extraction yielded higher total amounts of extracted
catechins, was more reproducible, and required less active bench time to prepare the samples. This
study demonstrates the effectiveness of accelerated solvent as an efficient methodology for
metabolomics studies.
Phase separation of in situ forming poly (lactide-co-glycolide acid) implants investigated using a
hydrogel-based subcutaneous tissue surrogate and UV–vis imaging
Yu Sun, Henrik Jensen, Nickolaj J. Petersen, Susan W. Larsen, Jesper Østergaard
ABSTRACT
Phase separation of in situ forming poly (lactide-co-glycolide acid) (PLGA) implants with agarose
hydrogels as the provider of nonsolvent (water) mimicking subcutaneous tissue was investigated using
a novel UV–vis imaging-based analytical platform. In situ forming implants of PLGA-1-methyl-2-
pyrrolidinone and PLGA-triacetin representing fast and slow phase separating systems, respectively,
were evaluated using this platform. Upon contact with the agarose hydrogel, the phase separation of
the systems was followed by the study of changes in light transmission and absorbance as a function of
time and position. For the PLGA-1-methyl-2-pyrrolidinone system, the rate of spatial phase separation
was determined and found to decrease with increasing the PLGA concentration from 20% to 40%
(w/w). Hydrogels with different agarose concentrations (1% and 10% (w/v)) were prepared for
providing the nonsolvent, water, to the in situ forming PLGA implants simulating the injection site
environment. The resulting implant morphology depended on the stiffness of hydrogel matrix,
indicating that the matrix in which implants are formed is of importance. Overall, the work showed
that the UV–vis imaging-based platform with an agarose hydrogel mimicking the subcutaneous tissue
holds potential in providing bio-relevant and mechanistic information on the phase separation
processes of in situ forming implants.
269
Mid-infrared and near-infrared spectroscopy for rapid detection of Gardeniae Fructus by a
liquid-liquid extraction process
Lingyan Tao, Zhonglin Lin, Jiashan Chen, Yongjiang Wu, Xuesong Liu
ABSTRACT
Gardeniae Fructus is widely used in the pharmaceutical industry, and many studies have confirmed its
medical and economic value. In this study, samples collected from different liquid-liquid extraction
batches of Gardeniae Fructus were detected by mid-infrared (MIR) and near-infrared (NIR)
spectroscopy. Seven analytes, neochlorogenic acid (5-CQA), cryptochlorogenic acid (4-CQA),
chlorogenic acid (3-CQA), geniposidic acid (GEA), deacetyl-asperulosidic acid methyl ester
(DAAME), genipin-gentiobioside (GGB), and gardenoside (GA), were chosen as quality property
indexes of Gardeniae Fructus. The two kinds of spectra were each used to build models by single
partial least squares (PLS). Additionally, both spectral data were combined and modeled by multiblock
PLS. For single spectroscopy modeling results, NIR had a better prediction for high-concentration
analytes (3-CQA, DAAME, GGB, and GA) whereas MIR performed better for low-concentration
analytes (5-CQA, 4-CQA, and GEA). The multiblock methodology was found to be better compared
to single spectroscopy models for all seven analytes. Specifically, the coefficients of determination
(R2) of the NIR, MIR, and multiblock PLS calibration models of all seven components were higher
than 0.95. Relative standard errors of prediction (RSEP) were all less than 7%, except for models of
GGB, which were 10.36%, 13.24%, and 8.15% for the NIR-PLS, MIR-PLS, and multiblock models,
respectively. These results indicate that MIR and NIR spectrographic techniques could provide a new
choice for quality control in industrial production of Gardeniae Fructus.
Quantitative analysis of a biopharmaceutical protein in cell culture samples using automated
capillary electrophoresis (CE) western blot
Dong Xu, Kentaro Marchionni, Yunli Hu, Wei Zhang, Zoran Sosic
ABSTRACT
An effective control strategy is critical to ensure the safety, purity and potency of biopharmaceuticals.
Appropriate analytical tools are needed to realize such goals by providing information on product
quality at an early stage to help understanding and control of the manufacturing process. In this work,
a fully automated, multi-capillary instrument is utilized for size-based separation and western blot
analysis to provide an early readout on product quality in order to enable a more consistent
manufacturing process. This approach aims at measuring two important qualities of a
biopharmaceutical protein, titer and isoform distribution, in cell culture harvest samples. The acquired
data for isoform distribution can then be used to predict the corresponding values of the final drug
substance, and potentially provide information for remedy through timely adjustment of the
downstream purification process, should the expected values fall out of the accepted range.
270
Synchronous characterization of carbohydrates and ginsenosides yields deeper insights into the
processing chemistry of ginseng
Shan-Shan Zhou, Jun Xu, Ming Kong, Ka-Man Yip, Hu-Biao Chen
ABSTRACT
Carbohydrates and ginsenosides in ginseng are biologically interrelated. Their synchronous analysis is
therefore essential in chemical research on ginseng to characterize its ―holistic‖ quality. Here we
investigated the processing chemistry of red ginseng (RG), a ginseng product processed by water-
steaming, for which both carbohydrates and ginsenosides were qualitatively and quantitatively
determined through multiple analytical techniques. Results revealed that the steam-processing not only
qualitatively and quantitatively altered the ginsenosides but also affected the polymeric carbohydrates
via changing their physiochemical parameters, i.e. water-solubility, molecular size, types and ratios of
constituent monosaccharides. Potential mechanisms involved in the transformation of ginseng
chemicals are proposed and discussed, including hydrolysis (deglycosylation, demalonylation,
deacetylation), dehydration, polymerization, volatilization, reduction and the Maillard reaction. The
study strengthens the research on the processing chemistry of RG, and therefore should be helpful for
elucidating the scientific basis of RG preparation and application.
Development and validation of an ICP-MS method for the determination of elemental impurities
in TP-6076 active pharmaceutical ingredient (API) according to USP 〈232〉/〈233〉
Osama Chahrour, John Malone, Mark Collins, Vrushali Salmon, Nick Dunwoody
ABSTRACT
The new guidelines of the United States pharmacopeia (USP), European pharmacopeia (EP) and
international conference on harmonization (ICH) regulating elemental impurities limits in
pharmaceuticals signify the end of unspecific analysis of metals as outlined in USP 〈231〉. The new
guidelines specify both daily doses and concentration/limits of elemental impurities in pharmaceutical
final products, active pharmaceutical ingredients (API) and excipients. In chapter USP 〈233〉
method implementation, validation and quality control during the analytical process are described. We
herein report the use of a stabilising matrix that overcomes low spike recovery problem encountered
with Os and allows the determination of all USP required elemental impurities (As, Cd, Hg, Pb, V, Cr,
Ni, Mo, Cu, Pt, Pd, Ru, Rh, Os and Ir) in a single analysis. The matrix was used in the validation of a
method to determine elemental impurities in TP-6076 active pharmaceutical ingredient (API) by ICP-
MS according to the procedures defined in USP〈233〉 and to GMP requirements. This validation
will support the regulatory submission of TP-6076 which is a novel tetracycline analogue effective
against the most urgent multidrug-resistant gram-negative bacteria. Evaluation of TP-6076 in IND-
enabling toxicology studies has led to the initiation of a phase 1 clinical trial.
271
Comparison of SEC and CE-SDS methods for monitoring hinge fragmentation in IgG1
monoclonal antibodies
Oluwatosin O. Dada, Romesh Rao, Natalie Jones, Nomalie Jaya, Oscar Salas-Solano
ABSTRACT
Fragmentation of monoclonal antibodies is a critical quality attribute routinely monitored to assess the
purity and integrity of the product from development to commercialization. Cleavage in the upper
hinge region of IgG1 monoclonal antibodies is a common fragmentation pattern widely studied by size
exclusion chromatography (SEC). Capillary electrophoresis with sodium dodecylsulfate (CE-SDS) is a
well-established technique commonly used for monitoring antibody fragments as well, but its
comparability to SEC in monitoring hinge fragments has not been established until now. We report a
characterization strategy that establishes the correlation between hinge region fragments analyzed by
SEC and CE-SDS. Monoclonal antibodies with elevated hinge fragments were generated under low pH
stress conditions and analyzed by SEC and CE-SDS. The masses of the fragments generated were
determined by LC-MS. Electrophoretic migration of the hinge fragmentation products in CE-SDS
were determined based on their mass values. Comparative assessment of fragments by SEC, and CE-
SDS showed similar correlation with incubation time. This study demonstrates that CE-SDS can be
employed as a surrogate technique to SEC for monitoring hinge region fragments. Most importantly,
combination of these techniques can be used to obtain comprehensive understanding of fragment
related characteristics of therapeutic protein products.
Revisiting blood-brain barrier: A chromatographic approach
Xavier Subirats, Laura Muñoz-Pascual, Michael H. Abraham, Martí Rosés
ABSTRACT
Drugs designed to reach a pharmacological CNS target must be effectively transported across the
blood-brain barrier (BBB), a thin monolayer of endothelial cells tightly attached together between the
blood and the brain parenchyma. Because of the lipidic nature of the BBB, several physicochemical
partition models have been studied as surrogates for the passive permeation of potential drug
candidates across the BBB (octanol-water, alkane-water, PAMPA...). In the last years, biopartition
chromatography is gaining importance as a noncellular system for the estimation of biological
properties in early stages of drug development. Microemulsions (ME) are suitable mobile phases,
because of their ease of formulation, stability and adjustability to a large number of compositions
mimicking biological structures. In the present work, several microemulsion liquid chromatographic
(MELC) systems have been characterized by means of the Abraham‘s solvation parameter model, in
order to assess their suitability as BBB distribution or permeability surrogates. In terms of similarity
between BBB and MELC systems (dispersion forces arising from solute non-bonded electrons,
dipolarity/polarizability, hydrogen-bond acidity and basicity, and molecular volume), the passive
permeability surface area product (log PS) for neutral (including zwitterions), fully and partially
ionized drugs was found to be well correlated with the ME made of 3.3% SDS (w/v; surfactant) 0.8%
heptane (w/v; oil phase) and 6.6% 1-butanol (w/v; co-surfactant) in 50 mM aqueous phosphate buffer,
pH 7.4.
272
Liquid chromatographic enantioseparation of carbocyclic β-amino acids possessing limonene
skeleton on macrocyclic glycopeptide-based chiral stationary phases
Tímea Orosz, Nóra Grecsó, Gyula Lajkó, Zsolt Szakonyi, Antal Péter
ABSTRACT
Polar-ionic and reversed-phase high-performance liquid chromatographic separations of limonene-
based cyclic β-amino acid enantiomers were carried out by using macrocyclic glycopeptide-based
chiral selectors applying Chirobiotic T, TAG and R columns. The effects of additives, concentration of
the co- and counter-ions and the temperature in polar-ionic mobile phase systems were studied. The
influence of pH, MeOH content and alcohol additives were investigated in the reversed-phase mode.
The difference in the change in standard enthalpy Δ(ΔH°), entropy Δ(ΔS°), and free energy Δ(ΔG°)
was calculated from the linear van't Hoff plots derived from the ln α vs 1/T curves in the temperature
range 5–40 °C. Unusual temperature behavior was observed on Chirobiotic TAG for most of the
analytes: decreased retention times were accompanied with increased separation factors with
increasing temperature, and separation was entropically-driven. For two of the studied analytes
enthalpically-driven enantioseparations were observed. The elution sequence was determined in all
cases, but no general rule could be established.
On-line coupling of molecularly imprinted solid phase extraction with liquid chromatography
for the fast determination of coumarins from complex samples
Andrea Machyňáková, Ivona Lhotská, Katarína Hroboňová, Dalibor Ńatínský
ABSTRACT
In this work, an on-line SPE-HPLC method with spectrophotometric detection was developed for the
determination of coumarins in complex samples. For the on-line cleanup of samples, a molecularly
imprinted polymer was packed into the column cartridge and coupled directly with HPLC (MISPE-
HPLC) using a column switching system. The separation of coumarins was performed on a C18 core-
shell column (100 × 4.6 mm, 5 μm) with a mobile phase consisting of 0.3% acetic acid/acetonitrile
with gradient elution at a flow-rate of 1 mL min−1. The total time of the whole analytical run,
including the extraction step, was 13.25 min. The on-line MISPE-HPLC method was optimized and
validated. The results showed good linearity (0.10–100 μg mL−1) with correlation coefficients higher
than 0.99. The LOD values were from 0.03 to 0.15 μg mL−1. The proposed method was successfully
applied for analysis of real samples (Cassia cinnamon, chamomile tea, and Tokaj specialty wines) and
obtained recoveries varied from 78.7% to 112.2% with an RSD less than 9%.
273
Metabolic profiling analysis of Siraitia grosvenorii revealed different characteristics of green
fruit and saccharified yellow fruit
Chengnan Fang, Qingqing Wang, Xinyu Liu, Guowang Xu
ABSTRACT
Siraitia grosvenorii is an economic and medicinal plant, its fruit is considered to be good to health for
its diverse bioactive ingredients. However, the clarification of chemical composition and their changes
after saccharification procedure are not well performed. In present study, a metabolomics method
based on ultra-high-performance liquid chromatography tandem quadrupole time-of-flight mass
spectrometry was developed for metabolic profiling acquisition of Siraitia grosvenorii extract.
Furthermore, information dependent analysis (IDA) combined with self-constructed LC–MS/MS
identification system for metabolites were employed to identify primary and secondary metabolites in
Siraitia grosvenorii. A total of 126 metabolites were identified or tentatively identified. The obvious
differences of metabolic profiling between green fruit and saccharified yellow fruit were observed, and
metabolites showed their own distribution characteristics in peel, flesh and seed. The majority of the
nutrients and effective components were more distributed in flesh and peel, and saccharification was
conducive to accumulation of sweet glycosides. This study not only expanded metabolite composition
information of Siraitia grosvenorii, but also specified distribution characteristics of identified
metabolites.
Comparison of α-glucosidase inhibitory effect and bioactive constituents of Anemarrhenae
Rhizoma and Fibrous Roots
Si-Hui Nian, Hui-Jun Li, E-Hu Liu, Ping Li
ABSTRACT
Comprehensive utilization of medicinal plant resources is of great significance for sustainable
development of traditional Chinese medicines. In the present study, the α-glucosidase inhibitory
activities of the rhizome and fibrous root of Anemarrhena Asphodeloides Bunge, were compared
detailedly, and a high performance liquid chromatography coupled with electrospray ionization tandem
triple quadrupole mass spectrometry (HPLC-QQQ/MS) method was developed for simultaneous
quantification of seven bioactive constituents including neomangiferin, mangiferin, isomangiferin,
timosaponin BII, timosaponin B, timosaponin AIII, and timosaponin N in 40 batches of samples. The
results demonstrated that fibrous root extracts had more potent α-glucosidase inhibitory activity than
rhizome extracts. Mangiferin and isomangiferin were abundant in fibrous root, while the analyzed
saponins were rich in rhizome. Based on the chemometrics methods including principal component
analysis (PCA), orthogonal partial least square discriminant analysis (OPLS-DA), and partial least
square (PLS), mangiferin and isomangiferin might be mainly responsible for α-glucosidase inhibitory
activity of the genus. These findings indicate that the established HPLC-QQQ/MS method was proven
to be useful and efficient for quality control of Anemarrhena materials, and fibrous root had the
potential to be utilized as anti-diabetic medicinal resource.
274
Characterization of forced degradation products of torasemide through MS tools and
explanation of unusual losses observed during mass fragmentation of drug and degradation
products through density functional theory
Moolchand Kurmi, Neha Patel, Shalu Jhajra, Prasad V. Bharatam, Saranjit Singh
ABSTRACT
Mass spectrometry tools (HRMS/LC-HRMS, MSn, and/or on-line H/D exchange) were employed to
establish mass fragmentation pattern of torasemide and to characterize its degradation products.
During collision-induced dissociation, multiple rearrangement processes and unusual losses of sulfur
(S), sulfanyl (HS), sulfur dioxide (SO2), sulphinic acid radical (HSO2), sulfur monoxide (SO), carbon
monoxide (CO), formyl radical (CHO) and C5H3NOS were observed. The same were successfully
explained by study of energy profiles, established by application of density functional theory (DFT).
Metabolic profiling of the traditional Chinese medicine formulation Yu Ping Feng San for the
identification of constituents relevant for effects on expression of TNF-α, IFN-γ, IL-1β and IL-4
in U937 cells
Stefanie Nikles, Marlene Monschein, Huiqin Zou, Yong Liu, Rudolf Bauer
ABSTRACT
Yu Ping Feng San (YPFS) is a classical TCM formulation which has been traditionally used for
treatment of immune system related diseases such as chronic bronchitis, allergic rhinitis and asthma.
The formula is a mixture of Radix Saposhnikoviae (Fangfeng), Radix Astragali (Huangqi), and
Rhizoma Atractylodis macrocephalae (Baizhu). TLC- and LC-DAD-ESI-MS/MS methods have been
developed for the analysis of the metabolic profiles of the single herbs and of the formula. Decoctions
and ASE extracts were analyzed in order to trace components of the individual herbs in YPFS. Nine
constituents of Radix Saposhnikoviae, ten constituents of Radix Astragali and five constituents of
Rhizoma Atractylodis macrocephalae have been assigned in the chemical profiles of the formula,
which now allow the standardisation of YPFS. The pharmacological testing showed that all extracts
significantly inhibited expression of TNF-α, IFN-γ, and IL-1β in U937 cells, while the inhibition of IL-
4 was consistently low. Compared to conventional analyses which are focused on a limited set of
compounds, metabolomics approaches, together with novel data processing tools, enable a more
holistic comparison of the herbal extracts. In order to identify the constituents which are relevant for
the immunomodulatory effects of the formula, metabolomics studies (PCA, OPLS-DA) have been
performed using UPLC/QTOF MS data.
275
A comprehensive stability-indicating HPLC method for determination of chloroquine in active
pharmaceutical ingredient and tablets: Identification of oxidation impurities
Ana Silva Coelho, Clara Elisa Pontes Chagas, Rodrigo Maia de Pádua, Gerson Antônio Pianetti,
Christian Fernandes
ABSTRACT
Malaria is the most common parasitic disease in humans. It is estimated that 3 billion people live under
the risk of contracting this disease in the world. Chloroquine (CQ) is the drug of choice to treat cases
of non-complicated malaria. Forced degradation studies are important to know the drug‘s potentials
degradation products and to develop a stability indicating method. Thus, chloroquine active
pharmaceutical ingredient (API), chloroquine tablets and placebo were submitted to a detailed forced
degradation study, using several stressing agents. The results were used on the development of a
stability indicating method, using high performance liquid chromatography. The method was validated
showing selectivity, precision, accuracy, robustness and linearity in the range of 30–360 μg/mL of
chloroquine. Chloroquine API and tablets were susceptible to alkaline hydrolysis with NaOH 1 mol/L,
and to oxidation with H2O2 3.0%. Two degradation products were formed in oxidative test. Kinetics
of chloroquine degradation in alkaline hydrolysis was performed for both API and tablets. The
calculated decay constant (k1) was 0.223 days−1 for API and 0.182 days−1 for tablets, while the half-
life (t1/2) was 3.1 days for API and 3.8 days for tablets. Chemical structures have been proposed for
the two degradation products formed in the presence of H2O2, using an UHPLC-UV-MS/MS
approach.
Identification of impurities in macrolides by liquid chromatography–mass spectrometric
detection and prediction of retention times of impurities by constructing quantitative structure–
retention relationship (QSRR)
Xia Zhang, Jin Li, Chen Wang, Danqing Song, Changqin Hu
ABSTRACT
Macrolides are multicomponent drugs whose impurity control is always a challenge demanding
analysis method with good sensitivity and selectivity. Three separate, sensitive, accurate liquid
chromatography tandem mass spectrometry methods (LC–MS) were developed for the measurement
of three 16-membered ring macrolides (josamycin, josamycin propionate and midecamycin acetate)
and related substances in commercial samples. The characteristics of impurities in macrolides were
summarized as useful guidance for the impurity analysis of this class of drugs. For each drug, a large
number of unknown components have been detected with the high-sensitive MS detector and possible
structures of the majority of them were postulated based on the summarized fragmentation rules of 16-
membered ring macrolides. A QSRR model was constructed by multilinear regression to predict the
retention times of identified impurities which were not detected by the LC–MS methods, without
obtaining their reference standards. Satisfactory performance was obtained during leave-one-out cross-
validation with a predictive ability (Q2) of 0.95. The generalisation ability of the model was further
confirmed by an average error of 2.3% in external prediction. The best QSRR model, based on eight
molecular descriptors, exhibited a promising predictive performance and robustness.
276
The impact of ZnO and TiO2 on the stability of clotrimazole under UVA irradiation:
Identification of photocatalytic degradation products and in vitro cytotoxicity assessment
Agata Kryczyk, Paweł Żmudzki, Paulina Koczurkiewicz, Joanna Piotrowska, Urszula Hubicka
ABSTRACT
In order to ensure the safe and effective use of pharmaceutical products especially for topical
administration photostability testing is necessary. The current paper presents an in-depth analysis of
the stability of one of the most common antifungal agents, namely clotrimazole. Clotrimazole has
proven to be stable under UVA irradiation in applied experimental conditions, but the presence of
catalysts such as ZnO and TiO2 has contributed significantly to the degradation of this compound. The
findings indicate that its photocatalytic degradation reactions followed the pseudo first-order kinetics
with rate constant depending on the pH and the used solvent. Using LC–MS/MS, 14 presumable
degradation products of clotrimazole were identified and the plausible transformation pathways were
proposed. The in vitro cytotoxicity risk evaluation based on photostability of clotrimazole was also
performed using the Human skin fibroblast cell line (BJ) ATCC™ CRL-2522. There was no
statistically significant difference between cells viability in all analyzed combinations of clotrimazole,
TiO2/ZnO, and UVA irradiation (p < 0.05).
Chemometrically assisted development and validation of LC–MS/MS method for the analysis of
potential genotoxic impurities in meropenem active pharmaceutical ingredient
Katerina Grigori, Yannis L. Loukas, AnĎelija Malenović, Vicky Samara, Yannis Dotsikas
ABSTRACT
A sensitive Liquid Chromatography tandem mass spectrometry (LC–MS/MS) method was developed
and validated for the quantitative analysis of three potential genotoxic impurities (318BP, M9, S5) in
meropenem Active Pharmaceutical Ingredient (API). Due to the requirement for LOD values in ppb
range, a high concentration of meropenem API (30 mg/mL) had to be injected. Therefore, efficient
determination of meropenem from its impurities became a critical aim of this study, in order to divert
meropenem to waste, via a switching valve. After the selection of the important factors affecting
analytes‘ elution, a Box-Behnken design was utilized to set the plan of experiments conducted with
UV detector. As responses, the separation factor s between the last eluting impurity and meropenem,
as well as meropenem retention factor k were used. Grid point search methodology was implemented
aiming to obtain the optimal conditions that simultaneously comply to the conflicted criteria. Optimal
mobile phase consisted of ACN, methanol and 0.09% HCOOH at a ratio 71/3.5/15.5 v/v. All
impurities and internal standard omeprazole were eluted before 7.5 min and at 8.0 min the eluents were
directed to waste. The protocol was transferred to LC–MS/MS and validated according to ICH
guidelines.
277
Identification and interconversion of isomeric 4,5-functionalized 1,2,3-thiadiazoles and 1,2,3-
triazoles in conditions of electrospray ionization
D.M. Mazur, M.E. Zimens, V.A. Bakulev, A.T. Lebedev
ABSTRACT
1,2,3-Triazoles and 1,2,3-thiadiazoles have been receiving permanent interest due to their exciting
chemical reactivity and interesting biological properties including antibacterial, anticancer and
antiviral activities. There are four compounds bearing 1H-1,2,3-triazole core in clinical studies which
may appear in the market of drugs in nearest future. Definitely reliable methods of their identification
and quantification should be developed by that time. Mass spectrometry showed itself as the most
reliable method of analysis when dealing with trace levels of organic compounds in the mixtures and
in the most complex matrices, including biological ones. In the present study tandem mass
spectrometry was used to study fragmentation pathways of protonated and deprotonated molecules of
isomeric 4,5-functionalized 1,2,3-thiadiazoles and 1,2,3-triazoles in conditions of electrospray
ionization (ESI). A group of marker ions allowing differentiation between the targeted isomeric
compounds was established. Besides, interconversion of these isomers into one another was studied in
the gas phase in conditions mimicking these processes in solution.
Dissolution assessment of allopurinol immediate release tablets by near infrared spectroscopy
Jelena Smetińko, Sneņana Miljanić
ABSTRACT
The purpose of this study was to develop a NIR spectroscopic method for assessment of drug
dissolution from allopurinol immediate release tablets. Thirty three different batches of allopurinol
immediate release tablets containing constant amount of the active ingredient, but varying in excipients
content and physical properties were introduced in a PLS calibration model. Correlating allopurinol
dissolution reference values measured by the routinely used UV/Vis method, with the data extracted
from the NIR spectra, values of correlation coefficient, bias, slope, residual prediction determination
and root mean square error of prediction (0.9632, 0.328%, 1.001, 3.58, 3.75%) were evaluated. The
obtained values implied that the NIR diffuse reflectance spectroscopy could serve as a faster and
simpler alternative to the conventional dissolution procedure, even for the tablets with a very fast
dissolution rate (>85% in 15 minutes). Apart from the possibility of prediction of the allopurinol
dissolution rate, the other multivariate technique, PCA, provided additional data on the non-chemical
characteristics of the product, which could not be obtained from the reference dissolution values.
Analysis on an independent set of samples confirmed that a difference between the UV/Vis reference
method and the proposed NIR method was not significant. According to the presented results, the
proposed NIR method may be suitable for practical application in routine analysis and for continuously
monitoring the product's chemical and physical properties responsible for expected quality.
278
Enhanced and green extraction polyphenols and furanocoumarins from Fig (Ficus carica L.)
leave using deep eutectic solvents
Tong Wang, Jiao Jiao, Qing-Yan Gai, Peng Wang, Yu-Jie Fu
ABSTRACT
Nowadays, green extraction of bioactive compounds from medicinal plants has gained increasing
attention. As green solvent, deep eutectic solvent (DES) have been highly rated to replace toxic
organic solvents in extraction process. In present study, to simultaneous extraction five main bioactive
compounds from fig leaves, DES was tailor-made. The tailor-made DES composed of a 3:3:3 molar
ratio of glycerol, xylitol and D-(−)-Fructose showed enhanced extraction yields for five target
compounds simultaneously compared with traditional methanol and non-tailor DESs. Then, the tailor-
made DES based extraction methods have compared and microwave-assisted extraction was selected
and optimized due to its high extraction yields with lower time consumption. The influencing
parameters including extraction temperature, liquid-solid ratio, and extraction time were optimized
using response surface methodology (RSM). Under optimal conditions the extraction yield of
caffeoylmalic acid, psoralic acid-glucoside, rutin, psoralen and bergapten was 6.482 mg/g, 16.34 mg/g,
5.207 mg/g, 15.22 mg/g and 2.475 mg/g, respectively. Macroporous resin D101 has been used to
recovery target compounds with recovery yields of 79.2%, 83.4%, 85.5%, 81.2% and 75.3% for
caffeoylmalic acid, psoralic acid-glucoside, rutin, psoralen and bergapten, respectively. The present
study suggests that DESs are truly designer and efficient solvents and the method we developed was
efficient and sustainable for extraction main compounds from Fig leaves.mg/g
Site- and species-specific hydrolysis rates of cocaine
Levente Szöcs, Gergely Völgyi, Ákos Urai, Sándor Hosztafi, Béla Noszál
ABSTRACT
The hydroxide-catalyzed non-enzymatic hydrolysis of cocaine is quantified in terms of ten site- and
species-specific rate constants in connection with also ten site- and species-specific acid-base
equilibrium constants, comprising all the twelve coexisting species in solution. This characterization
involves the major and minor decomposition pathways via benzoylecgonine and ecgonine methyl
ester, respectively, leading to ecgonine, the final product. Hydrolysis has been found to be 10–330
times faster at site 2 than at site 3, depending on the ionization status of the amino moiety and the rest
of the molecule. Nitrogen protonation accelerates the hydrolyses approximately ten times both at site 2
and site 3.
279
Comparative stability-indicating chromatographic methods for determination of 4-
hexylresorcinol in pharmaceutical formulation and shrimps
Rafeek F. Shokry, Lories I. Bebawy, Mohamed R. Elghobashy, Samah S. Abbas
ABSTRACT
Three chromatographic stability-indicating methods were developed for determination of 4-
hexylresorcinol in pure form and in a pharmaceutical formulation. Method A was based on a gradient
elution liquid chromatographic HPLC determination of 4-hexylresorcinol, its related impurities and in
presence of its degradation products. UPLC–MS/MS (Method B) was described for determination of
the cited drug in presence of its degradation products. Method C was a thin- layer chromatography
(TLC)-densitometry method for the separation and determination of the active ingredient, one of its
related impurities and in presence of its degradation products. The mechanism of alkali, oxidative and
photodegradation of 4-hexylresorcinol was studied according to ICH guidelines. The degradation
products were characterized by the LC–MS/MS method. Methods A and B were applicable for
determination of 4-hexylresorcinol residues in shrimp meat. The studied drug was easily degraded in
alkali medium giving toxic compounds. The results obtained by the proposed methods were
statistically analyzed and compared with those obtained by applying a reported method.
Enantiomeric separation of seven β-agonists by NACE—Study of chiral selectivity with
diacetone-d-mannitol–boric acid complex
Lili Lv, Lijuan Wang, Jun Li, Yajun Jiao, Hongyuan Yan
ABSTRACT
A rapid and effective nonaqueous capillary electrophoresis (NACE)–ultraviolet (UV) method was
developed for the enantiomeric separation of seven β-agonists. Diacetone-d-mannitol–boric acid
complex was used as a new chiral selector. It was in situ synthesized by the reaction of diacetone-d-
mannitol and boric acid in methanol medium containing triethylamine. The effects of diacetone-d-
mannitol, boric acid, and triethylamine concentrations on the enantioseparation were carefully
investigated. Under the optimized conditions, baseline enantioseparation could be obtained for six of
the tested β-agonists within 12 min. These results were better than that obtained with d-mannitol–boric
acid complex in previous work. 11B nuclear magnetic resonance (11B NMR) was applied to determine
the fraction of boron species and confirm the formation of diacetone-d-mannitol–boric acid complex.
Validation of the established NACE method was also carried out according to ICH guidelines.
Calibration curves showed good linearity with correlation coefficients (r) ≥ 0.9992 over a certain
concentration range for each enantiomer of the tested five β-agonists. The relative standard deviations
(RSDs) of intra-day precisions and inter-day precisions of migration times were ≤1.4% (n = 6), and
≤6.3% (n = 10), respectively. That of peak areas were ≤3.7% (n = 6), and ≤5.6% (n = 10), respectively.
The limits of detection (LODs) and the limits of quantitation (LOQs) based on the signal-to-noise
ratios of 3 and 10 were found below 1.25 μg mL−1 and 5.00 μg mL−1, respectively. The proposed
method was successfully applied to the determination of clenbuterol enantiomers in a multi-component
pharmaceutical dosage form called ―Ambroxol Hydrochloride and Clenbuterol Hydrochloride Oral
Solution‖.
280
An optimized HPLC-UV method for quantitatively determining sesquiterpenes in
Nardostachyos Radix et Rhizoma
Vu Ngoc Han Le, Trong Quan Khong, Min Kyun Na, Kyung Tae Kim, Jong Seong Kang
ABSTRACT
Nardostachyos Radix et Rhizoma (NR), the root and rhizome from either Nardostachys jatamansi
Batal or Nardostachys jatamansi DC, is known to have biological functions including neuro-protective
and anti-pancreatitis activity. The main bioactive compounds within NR are all classified as
sesquiterpenes, and include desoxo-narchinol A, nardosinonediol, and nardosinone. Although NR is a
valuable herb that is widely used in many Asian countries, robust quality control protocols for NR are
still in question, especially those that can analyze the three main active compounds. Current
quantitative methods within the Chinese Pharmacopoeic use nardosinone as a marker compounds. One
compound cannot represent a complicated matrix, and is thus insufficient to control the quality of this
herbal medicine. Moreover, there are no high-performance liquid chromatography (HPLC) methods
that can simultaneously analyze desoxo-narchinol A (DA), nardosinonediol (NE), and nardosinone
(ND) within NR. This study aimed to establish an efficient quality control protocol by developing an
analytical method that simultaneously detects the three sesquiterpenes with HPLC using response
surface methodology (RSM) to optimize sample preparation. Optimized HPLC conditions included a
mobile phase of 0.1% formic acid in water (A), and a 0.1% formic acid in acetonitrile (B) under an
elution program of 20% B–80% B for 30 min at 254 nm. The method was well validated,
demonstrating satisfactory linearity, accuracy, precision, recovery, repeatability, and stability.
Optimized conditions for creating the analytical sample were predicted by RSM using a Box-Behnken
design. These conditions included reflux at 70 °C for 3 h using 24.98% ethanol as the extraction
solvent (solvent: solid ratio = 78.81 mL/g).
Photostability testing using online reactor HPLC hyphenation and mass spectrometric
compound identification illustrated by ketoprofen as model compound
Jaber Assaf, Diego Zulkiewicz Gomes, Bernhard Wuest, Maria Kristina Parr
ABSTRACT
Investigations on the photochemical stability of pharmaceutical substances are mandatory in drug
development and licensing as photo-induced degradation of an active pharmaceutical ingredient (API)
may not only lead to decreased API concentrations but also to toxic or reactive products. Thus, the US
Food and Drug Administration (FDA) and the International Council for Harmonisation of Technical
Requirements for Pharmaceuticals for Human Use (ICH) issued Guidance for Industry Q1B
―Photostability Testing of New Drug Substances and Products‖ for testing of pure but also packed
drugs. However, photoproducts are also known to be generated in vivo under sunlight exposure of the
skin and lead to considerable amounts of adverse drug effects. Herein we present an alternative system
that may be used for photostability testing mimicking both situations. It combines a tailored
photoreactor with an exchangeable pen light source and a modified HPLC system with online-SPE.
Identification of photoproducts may be performed using mass spectrometry. The potential of accurate
mass spectrometry as a tool for identification of photoproducts was demonstrated as well. A
comparison of the online photoreactor system and the traditional photochamber irradiation was
performed using ketoprofen for proof of concept. In both designs acetylbenzophenone and
ethylbenzophenone were detected as main photoproducts.
281
Optoelectronic iron detectors for pharmaceutical flow analysis
Natalia Rybkowska, Robert Koncki, Kamil Strzelak
ABSTRACT
Compact flow-through optoelectronic detectors fabricated by pairing of light emitting diodes have
been applied for development of economic flow analysis systems dedicated for iron ions
determination. Three analytical methods with different chromogens selectively recognizing iron ions
have been compared. Ferrozine and ferene S based methods offer higher sensitivity and slightly lower
detection limits than method with 1,10‐phenantroline, but narrower ranges of linear response. Each
system allows detection of iron in micromolar range of concentration with comparable sample
throughput (20 injections per hour). The developed flow analysis systems have been successfully
applied for determination of iron in diet supplements. The utility of developed analytical systems for
iron release studies from drug formulations has also been demonstrated.
Isoconversional approach for non-isothermal decomposition of un-irradiated and photon-
irradiated 5-fluorouracil
Hala Sh. Mohamed, AbdelRahman A. Dahy, Refaat M. Mahfouz
ABSTRACT
Kinetic analysis for the non-isothermal decomposition of un-irradiated and photon-beam-irradiated 5-
fluorouracil (5-FU) as anti-cancer drug, was carried out in static air. Thermal decomposition of 5-FU
proceeds in two steps. One minor step in the temperature range of (270–283 °C) followed by the major
step in the temperature range of (285–360 °C). The non-isothermal data for un-irradiated and photon-
irradiated 5-FU were analyzed using linear (Tang) and non-linear (Vyazovkin) isoconversional
methods. The results of the application of these free models on the present kinetic data showed quite a
dependence of the activation energy on the extent of conversion. For un-irradiated 5-FU, the non-
isothermal data analysis indicates that the decomposition is generally described by A3 and A4 modeles
for the minor and major decomposition steps, respectively. For a photon-irradiated sample of 5-FU
with total absorbed dose of 10 Gy, the decomposition is controlled by A2 model throughout the
coversion range. The activation energies calculated in case of photon-irradiated 5-FU were found to be
lower compared to the values obtained from the thermal decomposition of the un-irradiated sample
probably due to the formation of additional nucleation sites created by a photon-irradiation.
282
Characterization of impurities in sodium cromoglycate drug substance and eye drops using LC-
ESI-ion trap MS and LC-ESI-QTOF MS
Peixi Zhu, Jingxian Lu, Zhijian Wang, Weike Su, Erwin Adams
ABSTRACT
As requested by regulatory authorities, impurity profiling is an important issue of quality control. In
this work, a simple and sensitive liquid chromatographic (LC) method compatible with mass
spectrometry (MS) was developed to study related substances and degradation products in sodium
cromoglycate drug substance and eye drops. The method used a Sunfire column (4.6 mm × 150 mm,
3.5 μm). Mobile phase A consisted of 10 mM ammonium formate and mobile phase B were
acetonitrile. Linear gradient elution with a post-run time of 8 min was performed as follows: 0–30 min,
3% B to 50% B; 30–35 min, 50% B. The flow rate was set at 1.0 mL/min. Degradation experiments
were performed to check the stability indicating properties of the developed method. Based on MSn
spectral data and exact mass measurements, the chemical structures of 2 unknown impurities and 6
unknown degradation products were characterized, including impurity C listed in the European
Pharmacopoeia as unknown structure.
Method validation and nanoparticle characterization assays for an innovative amphothericin B
formulation to reach increased stability and safety in infectious diseases
Maraine Catarina Tadini, Ana Maria de Freitas Pinheiro, Daniel Blascke Carrão, Ana Luiza Scarano
Aguillera Forte, Franciane Marquele-Oliveira
ABSTRACT
Drug Delivery Systems (DDS) of known drugs are prominent candidates towards new and more-
effective treatments of various infectious diseases as they may increase drug bioavailability, control
drug delivery and target the site of action. In this sense, the encapsulation of Amphotericin B (AmB) in
Nanostructured Lipid Carriers (NLCs) designed with pH-sensible phospholipids to target infectious
tissues was proposed and suitable analytical methods were validated, as well as, proper nanoparticle
characterization were conducted. Characterization assays by Dinamic Light Scattering (DLS) and
Atomic Force Microscopy demonstrated spherical particles with nanometric size 268.0 ± 11.8 nm and
Zeta Potential −42.5 ± 1.5 mV suggestive of important stability. DSC/TGA and FT-IR assessments
suggested mechanical encapsulation of AmB. The AmB aggregation study indicated that the
encapsulation provided AmB at the lowest cytotoxic form, polyaggregate. Analytical methods were
developed and validated according to regulatory agencies in order to fast and assertively determine
AmB in nanoparticle suspension and, in Drug Encapsulation Efficiency (EE%), release and stability
studies. The quantification method for AmB in NLC suspension presented linearity in 5.05–60.60 μg
mL−1 range (y = 0.07659x + 0.05344) and for AmB in receptor solution presented linearity in 0.15–
10.00 μg mL−1 range (y = 54609x + 263.1), both with r ≥ 0.999. EE% was approximately 100% and
according to the release results, at pH 7.4, a sustained controlled profile was observed for up 46 h. In
the meantime, a micellar AmB solution demonstrated an instability pattern after 7 h of contact with the
medium. Degradation and release studies under acid conditions (infectious condition) firstly depicted a
prominent degradation of AmB (raw-material), with 20.3 ± 3.5% at the first hour, reaching 43.3 ±
7.0% after 7 h of study.
283
Surfactants enhance recovery of poorly soluble drugs during microdialysis sampling:
Implications for in vitro dissolution-/permeation-studies
Sebastian Koplin, Mont Kumpugdee-Vollrath, Annette Bauer-Brandl, Martin Brandl
ABSTRACT
Aim of this project was to investigate the applicability of a recently developed in vitro microdialysis-
sampling approach in connection with a dissolution-/permeation (D/P) system, especially the impact of
surfactants within the perfusion fluid. The D/P-system is based on side-by-side chambers, separated by
a barrier that simulates the intestinal barrier. Here, in contrast to conventional D/P-systems, the
dissolution of the drug (donor chamber concentration) is followed by microdialysis sampling. This
approach appears promising, because it is expected not to disturb the dynamic interplay between drug-
dissolution (-release) and drug permeation. Furthermore, it should allow quantification of the unbound
(free) drug concentration. In the first step, it was assessed, if the addition of the anionic surfactant
sodium dodecyl sulphate (SDS) to the perfusate of the microdialysis system affects the recovery of the
(slightly) water-soluble model drug acyclovir and the poorly water soluble model drug celecoxib
(CXB). SDS had no influence on acyclovir-recovery, but substantially enhanced CXB-recovery, partly
due to improved extraction efficiency, partly due to inhibition of loss of CXB due to non-specific
binding to surfaces and the probe. The fraction of CXB recovered from aqueous CXB-solutions by
microdialysis sampling using SDS-containing perfusates correlated well with the celecoxib
concentration in the samples, but was found independent of the SDS-concentrations (above critical
micelle concentration). In the next step microdialysis sampling with SDS-containing perfusates was
assessed for celecoxib solutions in fasted state simulated intestinal fluid (FaSSIF) and compared to that
in buffer. In FaSSIF, the measured CXB-concentrations were far below the overall CXB concentration,
likely representing the free celecoxib, i.e. the fraction of drug, which is not associated with
taurocholate surfactant micelles. In buffer, the measured concentrations matched the overall CXB
concentrations.
Simultaneous identification and quantification of polymethoxyflavones, coumarin and phenolic
acids in Ageratum conyzoides by UPLC-ESI-QToF-MS and UPLC-PDA
Larissa G. Faqueti, Louis P. Sandjo, Maique W. Biavatti
ABSTRACT
Ageratum conyzoides L. is a plant widely used in traditional medicine of tropical and subtropical
regions for its anti-inflammatory and antinociceptive properties. Nevertheless, the chemical
composition of its medicinal preparation has not been yet accurately established. In this study,
chromatographic methods were developed for the simultaneous identification and quantification of the
main compounds in A. conyzoides aqueous extract, using UPLC-PDA-ESI-QToF-MS. The qualitative
analyses defined by MS/MS analysis allowed the identification of 27 compounds in the aqueous
extract, including the toxic pyrrolizidine alkaloids, phenolic acids, coumarin and
polymethoxyflavones. Among the metabolites, twelve were detected for the first time in the species.
The quantitative method was validated according to the official guidelines, demonstrating to be
specific, linear, precise, accurate and sensitive for the quantification of chlorogenic acid, coumaric
acid, coumarin (1,2-benzopyranone), 5,6,7,3′,4′,5′-hexamethoxyflavone, nobiletin, 5′-methoxynobiletin
and eupalestin.
284
Degradation and metabolite formation of estrogen conjugates in an agricultural soil
Li Ma, Scott R. Yates
ABSTRACT
Estrogen conjugates are precursors of free estrogens such as 17ß-estradiol (E2) and estrone (E1),
which cause potent endocrine disrupting effects on aquatic organisms. In this study, microcosm
laboratory experiments were conducted at 25 °C in an agricultural soil to investigate the aerobic
degradation and metabolite formation kinetics of 17ß-estradiol-3-glucuronide (E2-3G) and 17ß-
estradiol-3-sulfate (E2-3S). The aerobic degradation of E2-3G and E2-3S followed first-order kinetics
and the degradation rates were inversely related to their initial concentrations. The degradation of E2-
3G and E2-3S was extraordinarily rapid with half of mass lost within hours. Considerable quantities of
E2-3G (7.68 ng/g) and E2-3S (4.84 ng/g) were detected at the end of the 20-d experiment, particularly
for high initial concentrations. The major degradation pathway of E2-3G and E2-3S was oxidation,
yielding the primary metabolites 17ß-estrone-3-glucuronide and 17ß-estrone-3-sulfate, respectively.
Common metabolites were E2, the second primary metabolite, and E1, the secondary metabolite.
Additionally, ring B unsaturated estrogens and their sulfate conjugates were tentatively proposed as
minor metabolites. The persistence of E2-3G and E2-3S (up to 20 d) suggests that the high rate of
application of conjugated estrogen-containing substances could be responsible for the frequent
detection of free estrogens in surface and subsurface water.
Chemical analysis and potential endocrine activities of aluminium coatings intended to be in
contact with cosmetic water
Elias Bou-Maroun, Laurence Dahbi, Marie-Pierre Gomez-Berrada, Philippine Pierre, Marie-Christine
Chagnon
ABSTRACT
The objective of the work was to check the presence of Non-Intended Added Substances (NIAS) with
hormonal activities in aluminium coatings extracts coded: AA, BBF, MC and RR, furnished by four
different suppliers. Water samples were prepared at room temperature or at 40 °C for three months to
verify the storage effect on the coatings. Solid phase extraction was used to concentrate and to extract
coating substances. Hormonal activities were checked in vitro using reporter gene bioassays. Except
BBF, all extracts induced a weak but significant estrogenic agonist activity in the human cell line.
Using an estrogenic antagonist (ICI-182, 780), the answer was demonstrated specific in the bioassay.
RR was the only extract to induce a concentration dependent anti-androgenic response in the MDA-
KB2 cell line. Analysis performed using GC–MS and HPLC–MS detected 12 substances in most of the
extracts. 8 NIAS were present. Among them, 4 were identified with certainty: HMBT, BGA, DCU and
BPA. Estrogenic potency was BPA > DCU > BGA > HMBT. HMBT was also anti-androgenic at high
concentration. Combining chemical analysis and bioassays data, we demonstrated that in the RR and
the RR40 extracts, the observed estrogenic response was mainly due to BPA, the anti-androgenic
activity of RR could be due to a synergism between HMBT and BPA. For MC and AA, estrogenic
responses appear to be due to the presence of DCU. Except BBF, storage conditions tended to increase
estrogenic activities in all extracts. However, in term of risk assessment, activities observed were
negligible.
285
Identification and determination of related substances of ceftaroline fosamil in medicinal
product by high performance liquid chromatography with diode array detection and tandem
mass spectrometry
Izabela Karpiuk, Katarzyna Michalska, Katarzyna Bus, Monika Kiljan, Stefan Tyski
ABSTRACT
Ceftaroline fosamil, the prodrug of ceftaroline, is an advanced-generation cephalosporin antibacterial
agent approved for treatment in the European Union in 2012. The drug is dedicated to curing
complicated skin and soft tissue infections and community-acquired pneumonia. The developed
analytical method of high performance liquid chromatography (HPLC) followed by diode array
detector (DAD) set at 243 nm was used to identify and determine degradation products of ceftaroline
fosamil. The elaborated method demonstrated good selectivity and linearity [r > 0.998 for the range
0.8–1.2 mg/mL (80–120%) and r > 0.997 for the range 0.005–0.015 mg/mL (0.5–1.5%)]. Limit of
detection (LOD) and limit of quantification (LOQ) values were equal to 0.15 μg/mL and 0.5 μg/mL,
respectively. The forced decomposition of ceftaroline fosamil carried out under acidic, basic,
oxidative, photolytic and thermal conditions revealed the high sensitivity of ceftaroline on
photodegradation and thermolysis processes. Representative ceftaroline samples were selected and
analysed by LC coupled with electrospray ionisation time-of-flight mass spectrometry (LC-ESI-Q-
TOF-MS) to identify the related substances that appeared under stress conditions. The LC-DAD
method was transferred without any modification to LC–MS/MS system, allowing it to correlate
results back to the LC-DAD method. Eight unknown signals were detected in the photolytic and
thermal stress solutions for ceftaroline fosamil. The evaluated method was applied to the analysis of a
medicinal product containing ceftaroline fosamil − Zinforo™, 600 mg, powder for concentrate for
solution for infusion. Moreover, the optimised conditions allowed for the successful separation of eight
cephalosporin antibiotics possessing a molecular structure similar to ceftaroline (cefepime, cefapirin,
ceftazidime, cefpirome, cefalonium, cefotaxime, cefquinome, cefalotin).
Quantification of concentrated Chinese medicine granules by quantitative polymerase chain
reaction
Yat-Tung Lo, Pang-Chui Shaw
ABSTRACT
Determination of the amount of constituent in a multi-herb product is important for quality control. In
concentrated Chinese medicine granules (CCMG), no dregs are left after dissolution of the CCMG.
This study is the first to examine the feasibility of using quantitative polymerase chain reaction
(qPCR) to find the amount of CCMG in solution form. DNA was extracted from Hirudo and Zaocys
CCMG mixed at different ratios and amplified in qPCR using species-specific primers. The threshold
cycle (CT) obtained was compared with the respective standard curves. Results showed that
reproducible quantification results could be obtained (1) for 5–50 mg CCMG using a modified DNA
extraction protocol, (2) amongst DNA extracted from the same batch of CCMG and (3) amongst
different batches of CCMG from the same company. This study demonstrated the constitute amount of
CCMG in a mixture could be determined using qPCR. This work has extended the application of DNA
techniques for the quantification of herbal products and this approach may be developed for quality
assurance in the CCMG industry.
286
Chiral separation of terbutaline and non-steroidal anti-inflammatory drugs by using a new
lysine–bridged hemispherodextrin in capillary electrophoresis
V. Cucinotta, M. Messina, A. Contino, G. Maccarrone, A. Giuffrida
ABSTRACT
A method for the separation of a mixture of terbutaline and non-steroidal anti-inflammatory drugs was
developed using capillary electrophoresis with a new hemispherodextrin, ad hoc designed, the lysine −
bridged hemispherodextrin (THLYSH). The use of lysine residues to bridge the trehalose capping unit
moiety to the cyclodextrin cavity gives rise to a receptor with two long chains with amine nitrogen
atoms, whose charge can be easily tuned as a function of the solution pH. The new hemispherodextrin
was accurately characterised by ESI–MS and NMR spectroscopy, also highlighting its protonation
behaviour. Circular dichroism and ESR spectroscopy measurements were also carried out to test its
inclusion ability towards anthraquinone-3-sulfonate and its metal coordination ability towards
copper(II) ion, respectively. Analogously to the other hemispherodextrins, the main skill of this new
derivative lies in its chiral selector properties, as shown by the separation of the enantiomeric pairs of
terbutaline and ibuprofen, flurbiprofen, suprofen and tiaprofenic acid by capillary electrophoresis. The
focused use of the solution equilibria involved in the separations made it possible to understand the
phenomena occurring in solution, and to finely tune the charge status of the receptor. In this way the
chiral separation of the racemic mixture was successfully obtained, even if the receptor was
individually used, differently by the other hemispherodextrins previously studied whose chiral
separation capabilities are present only if used as binary mixtures.
Separation and characterization of allergic polymerized impurities in cephalosporins by 2D-
HPSEC × LC-IT-TOF MS
Yu Xu, DanDan Wang, Lan Tang, Jian Wang
ABSTRACT
Eleven unknown allergic impurities in cefodizime, cefmenoxime and cefonicid were separated and
characterized by a trap-free two-dimensional high performance size exclusion chromatography
(HPSEC) and reversed phase liquid chromatography (RP-HPLC) coupled to high resolution ion
trap/time-of-flight mass spectrometry (2D-HPSEC × LC-IT-TOF MS) with positive and negative
modes of electrospray ionization method. Separation and characterization the allergic polymerized
impurities in β-lactam antibiotics were on the basis of column-switching technique which effectively
combined the advantages of HPSEC and the ability of RP-HPLC to identify the special impurities. In
the first dimension HPSEC, the column was Xtimate SEC-120 analytical column (7.8 mm × 30 cm, 5
μm), and the gradient elution used pH 7.0 buffer-acetonitrile as mobile phase And the second
dimension analytical column was ZORBAX SB-C18 (4.6 × 150 mm, 3.5 μm) with ammonium formate
solution (10 mM) and ammonium formate (8 mM) in [acetonitrile-water (4:1, v/v)] solution as mobile
phase. Structures of eleven unknown impurities were deduced based on the high resolution MSn data
with both positive and negative modes, in which nine impurities were polymerized impurities. The
forming mechanism of β-lactam antibiotic polymerization in cephalosporins was also studied. The
question on incompatibility between non-volatile salt mobile phase and mass spectrometry was solved
completely by multidimensional heart-cutting approaches and online demineralization technique,
which was worthy of widespread use and application for the advantages of stability and repeatability.
287
Phytochemical analysis and anti-inflammatory evaluation of compounds from an aqueous
extract of Croton cajucara Benth
Adamara M. Nascimento, Daniele Maria-Ferreira, Fernando T. Dal Lin, Alexandre Kimura, Lauro M.
de Souza
ABSTRACT
Croton cajucara Benth is a medicinal plant popularly used in the Brazilian Amazonia, where it is
known as sacaca, being consumed as tea, decoction or infusion of the leaves and stem bark. From a
decoction of the leaves, a comprehensive phytochemical analysis was developed by liquid
chromatography-mass spectrometry. Many compounds were identified for the first time in C. cajucara,
such as O-glycosides of kaempferol and quercetin, flavonoid-C-glycosides, tannins and cinnamic acid
derivatives. These compounds were fractionated by polarity and assayed for their anti-inflammatory
activity, using a model of mice edema, induced by an intraplantar injection of carrageenan. All
fractions exhibited anti-inflammatory properties.
Supercritical fluid chromatography approach for a sustainable manufacture of new
stereoisomeric anticancer agent
Alina Ghinet, Yasmine Zehani, Emmanuelle Lipka
ABSTRACT
Two routes aimed at the manufacture of unprecedented stereoisomeric combretastatin A-4 analogue
were described: flash chromatography vs supercritical fluid chromatography. The latter has many
advantages over liquid chromatography and was therefore chosen for the small scale separation of
methyl 1-[(3-hydroxy-4-methoxyphenyl) (3,4,5-trimethoxyphenyl)methyl]-5-oxo-l-prolinate 5, with
potential antitumoral activity. After a screening of six different polysaccharide based chiral stationary
phases and four co-solvents, the percentage of co-solvent, the flow-rate and the outlet pressure were
optimized through a design of experiments (DoE). The preparation of 50 mg of each stereoisomer was
achieved successfully on a Chiralpak AD-H with isopropanol as a co-solvent. Productivity (kkd),
solvent usage and environmental factor (E Factor) were calculated. Flash chromatography and
supercritical fluid chromatography approaches were compared in terms of yield and purity of each
stereoisomer manufactured.
288
Use of ionic liquids as headspace gas chromatography diluents for the analysis of residual
solvents in pharmaceuticals
Omprakash Nacham, Tien D. Ho, Jared L. Anderson, Gregory K. Webster
ABSTRACT
In this study, two ionic liquids (ILs), 1-butyl-3-methylimidazolium bis[(trifluoromethyl)
sulfonyl]imide ([BMIM][NTf2]) and trihexyltetradecylphosphonium bis[(trifluoromethyl)sulfonyl]
imide ([P66614][NTf2]) were examined as contemporary diluents for residual solvent analysis using
static headspace gas chromatography (SHS-GC) coupled with flame ionization detection (FID). ILs are
a class of non-molecular solvents featuring negligible vapor pressure and high thermal stabilities.
Owing to these favorable properties, ILs have potential to enable superior sensitivity and reduced
interference, compared to conventional organic diluents, at high headspace incubation temperatures.
By employing the [BMIM][NTf2] IL as a diluent, a 25-fold improvement in limit of detection (LOD)
was observed with respect to traditional HS-GC diluents, such as N-methylpyrrolidone (NMP). The
established IL-based method demonstrated LODs ranging from 5.8 parts-per-million (ppm) to 20 ppm
of residual solvents in drug substances. The optimization of headspace extraction conditions was
performed prior to method validation. An incubation temperature of 140 °C and a 15 min incubation
time provided the best sensitivity for the analysis. Under optimized experimental conditions, the mass
of residual solvents partitioned in the headspace was higher when using [BMIM][NTf2] than NMP as
a diluent. The analytical performance was demonstrated by determining the repeatability, accuracy,
and linearity of the method.
Adduct ion-targeted qualitative and quantitative analysis of polyoxypregnanes by ultra-high
pressure liquid chromatography coupled with triple quadrupole mass spectrometry
Xu Wu, Lin Zhu, Jiang Ma, Yang Ye, Ge Lin
ABSTRACT
Polyoxypregnane and its glycosides (POPs) are frequently present in plants of Asclepiadaceae family,
and have a variety of biological activities. There is a great need to comprehensively profile these
phytochemicals and to quantify them for monitoring their contents in the herbs and the biological
samples. However, POPs undergo extensive adduct ion formation in ESI-MS, which has posed a
challenge for qualitative and quantitative analysis of POPs. In the present study, we took the advantage
of such extensive adduct ion formation to investigate the suitability of adduct ion-targeted analysis of
POPs. For the qualitative analysis, we firstly demonstrated that the sodium and ammonium adduct ion-
targeted product ion scans (PIS) provided adequate MS/MS fragmentations for structural
characterization of POPs. Aided with precursor ion (PI) scans, which showed high selectivity and
sensitivity and improved peak assignment confidence in conjunction with full scan (FS), the
informative adduct ion-targeted PIS enabled rapid POPs profiling. For the quantification, we used
formic acid rather than ammonium acetate as an additive in the mobile phase to avoid simultaneous
formation of sodium and ammonium adduct ions, and greatly improved reproducibility of MS response
of POPs. By monitoring the solely formed sodium adduct ions [M+Na]+, a method for simultaneous
quantification of 25 POPs in the dynamic multiple reaction monitoring mode was then developed and
validated. Finally, the aforementioned methods were applied to qualitative and quantitative analysis of
POPs in the extract of a traditional Chinses medicinal herb, Marsdenia tenacissima (Roxb.) Wight et
Arn., and in the plasma obtained from the rats treated with this herb.
289
Quantitative determination of dobutamine in newborn pig plasma samples by HPLC–MS/MS
O.E. Albóniga, M.L. Alonso, M.E. Blanco, O. González, R.M. Alonso
ABSTRACT
A novel gradient reverse phase high performance liquid chromatography tandem mass spectrometry
(HPLC/MS-MS) was performed as a method for the determination of dobutamine hydrochloride
(DOB) in newborn pig plasma samples. It was developed and validated after optimization of sample
treatment and various chromatographic and mass spectrometric conditions. Trimethoxydobutamine
(TMD) was used as internal standard. Heptafluorobutyric acid (HFBA) and ethyl acetate were used for
the treatment of plasma samples. The separation of dobutamine and internal standard was done using a
Kinetex F5 (50 × 2.1 mm, 2.6 μm, 100 Å) analytical column. The mobile phase was a mixture of
acetonitrile and HCOOH 0.01%. The column oven temperature was optimized at 40° C and the flow
rate was 0.25 mL/min. DOB and TMD were detected by multiple reaction monitoring (MRM) mode in
ESI+, using a cone voltage (CV) of 25 V and a collision energy (CE) of 25 eV. The weighted
calibration curve (1/x2) was found to be linear over the concentration range of 1–100 ng/mL (r2 >
0.999). The limit of quantification (LLOQ) of the method was 1 ng/mL. The values of selectivity,
carryover, LLOQ, linearity, accuracy, precision, matrix effect, stability and recovery obtained meet the
acceptable range according to European Medicines Agency (EMA) and Food and Drug Administration
(FDA) guidelines. The method was efficiently applied to quantify DOB in plasma samples from a
pharmacokinetic/pharmacodynamic study in a disease model of newborn piglet.
An integrative investigation of the toxicity of Aconiti kusnezoffii radix and the attenuation effect
of its processed drug using a UHPLC-Q-TOF based rat serum and urine metabolomics strategy
Zhenyu Sui, Qing Li, Lin Zhu, Zhenru Wang, Kaishun Bi
ABSTRACT
Aconiti kusnezoffii radix (AKR), the root of Aconitum kusnezoffii Reichb, is commonly used in the
treatment of the rheumatoid arthritis. However, the clinical application is limited due to its potential
toxicity. Therefore, to investigate the mechanism of its potential neurotoxicity and nephrotoxicity, a
comprehensive metabolomics study combined with serum biochemistry and histopathology
measurements was carried out. A UHPLC-Q-TOF mass spectrometry based metabolomics approach
was applied to characterize the AKR toxicity, while the toxicity attenuation effects of Aconiti
kusnezoffii radix cocta (AKRC) on Wistar rats were also investigated. Two chromatographic
techniques involving reversed-phase chromatography and hydrophilic interaction chromatography
were combined for the serum and urine detection, which balanced the integrity and selectivity of the
two matrices. Principal component analysis was used to determine the groups, and principal
component analysis discriminant analysis was carried out to confirm the important variables. Then, the
developed integrative toxicity evaluation method was applied to assess the toxicity of AKR and the
attenuation effect of AKRC. The highly sensitive and specific toxic biomarkers, which can provide
practical bases were identified for the diagnosis of the neurotoxicity and nephrotoxicity induced by
AKR. In all, a total of 19 putative biomarkers were characterized, and related metabolic pathways were
identified.
290
Calibration and validation of a MCC/IMS prototype for exhaled propofol online measurement
Felix Maurer, Larissa Walter, Martin Geiger, Jörg Ingo Baumbach, Sascha Kreuer
ABSTRACT
Propofol is a commonly used intravenous general anesthetic. Multi-capillary column (MCC) coupled
Ion-mobility spectrometry (IMS) can be used to quantify exhaled propofol, and thus estimate plasma
drug concentration. Here, we present results of the calibration and analytical validation of a MCC/IMS
pre-market prototype for propofol quantification in exhaled air. Calibration with a reference gas
generator yielded an R2 ≥ 0.99 with a linear array for the calibration curve from 0 to 20 ppbv. The
limit of quantification was 0.3 ppbv and the limit of detection was 0.1 ppbv. The device is able to
distinguish concentration differences > 0.5 ppbv for the concentration range between 2 and 4 ppbv and
> 0.9 ppbv for the range between 28 and 30 ppbv. The imprecision at 20 ppbv is 11.3% whereas it is
3.5% at a concentration of 40 ppbv. The carry-over duration is 3 min. The MCC/IMS we tested
provided online quantification of gaseous propofol over the clinically relevant range at measurement
frequencies of one measurement each minute.
Biochemical and functional analysis of corticotropin releasing factor purified from an aqueous
extract of human placenta used as wound healer
Namrata Singh, Debasish Bhattacharyya
ABSTRACT
Human placental extract constitutes of innumerable therapeutically important components mostly used
in wound healing arising from the skin and burn injuries. However, there is still some bioactive present
in the placental extracts yet to be characterized to better under the complex process of wound healing
mediated by the placental extract. In this study, the presence of corticotropin releasing factor (CRF) in
an aqueous extract of human placenta was detected and quantified by dot blot and CRF-ELISA
immunoassay kit respectively. Subsequently, it was purified by immuno-affinity chromatography and
quantified as 0.45 ± 0.05 μg of CRF per ml of placental extract where its molecular weight found to be
4.78 kDa by MALDI-TOF. To study functional analysis of CRF, an in vitro WI-38 lung fibroblast cell
scratch wound model was used which indicated proliferation, motility of cells after treatment with
purified CRF. Moreover, reduction in apoptosis rate of cells during closure of wound was observed
from microscopy studies and FACS analysis. Also, Antalarmin, an antagonist of CRF type 1 receptor
inhibited the wound closure potency of the purified component. Faster healing of wound with an
elevation of IL-6 and TGF-β during early stages of repair by placental CRF was observed on excision
rat model. The process of healing was accompanied by the decrease in the level of TNF-α and IFN-γ.
291
Toxic compounds from tobacco in placenta samples analyzed by UPLC-QTOF-MS
Somayeh Mohammadi, Celia Domeno, Isabel Nerin, Margarita Aznar, Cristina Nerin
ABSTRACT
Polycyclic aromatic hydrocarbons (PAHs), tobacco-specific nitrosamines (TSNAs) and aromatic
amines are carcinogens present in cigarette smoke. These compounds are distributed in the human
body and they could be transferred to the foetus during the pregnancy. Placenta is the main barrier to
these toxic compounds and its study is the objective of this work. A method based on solid-phase
extraction (SPE) with ultra-performance liquid chromatography−tandem quadrupole-time-of-flight
mass spectrometry (UPLC-QTOF-MS) has been examined and optimized for the analysis of 9 target
analytes (4 tobacco-specific nitrosamines and some of their metabolites, 3 aromatic amines, nicotine
and cotinine) in 26 placenta samples from smoking and non-smoking women. Limits of detection
(LODs) were in the range of 3–27 ng/g of placenta. Nicotine, cotinine, N-nitrosoanatabine (NAT) and
4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone (NNK) metabolite, 4-(methylnitrosamino)-1-(3-
pyridyl)-1-butanol (NNAL) were detected in the placenta samples of smoking woman. Nicotine was
detected in 3 out of 8 placentas from smoking women, always below the limit of quantification (88
ng/g). This could be expected, as the half-life of nicotine in the body is limited to about 0.5–3 h.
Cotinine, the main metabolite from nicotine, was detected in all placentas from smoking women at
concentrations between 17.2 and 61.8 ng/g, reaching the highest values for those women that smoked
the highest number of cigarettes. NAT and NNAL were detected in all placentas from smoking
women, always below the limit of quantification (40 ng/g and 33 ng/g respectively).
HPLC-HG-AFS determination of arsenic species in acute promyelocytic leukemia (APL) plasma
and blood cells
Meihua Guo, Wenjing Wang, Xin Hai, Jin Zhou
ABSTRACT
Arsenic trioxide (ATO) has been successfully used in the treatment of acute promyelocytic leukemia
(APL). To clarify the arsenic species in APL patients, high performance liquid chromatography-
hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) and HG-AFS methods were
developed and validated to quantify the plasma concentrations of inorganic arsenic (As(III) and As(V))
and methylated metabolites (MMA and DMA), and the total amounts of arsenic in blood cells and
plasma. Blood cells and plasma were digested with mixtures of HNO3H2O2 and analyzed by HG-
AFS. For arsenic speciation, plasma samples were prepared with perchloric acid to precipitate protein.
The supernatant was separated on an anion-exchange column within 6 min with isocratic elution using
13 mM CH3COONa, 3 mM NaH2PO4, 4 mM KNO3 and 0.2 mM EDTA-2Na. The methods provided
linearity range of 0.2–20 ng/mL for total arsenic and 2.0–50 ng/mL for four arsenic species. The
developed methods for total arsenic and arsenic species determination were precise and accurate. The
spiked recoveries ranged from 81.2%-108.6% and the coefficients of variation for intra- and inter-
batch precision were less than 9.3% and 12.5%, respectively. The developed methods were applied
successfully for the assay of total arsenic and arsenic species in 5 APL patients. The HPLC-HG-AFS
may be a good alternative for arsenic species determination in APL patients with its simplicity and
low-cost in comparison with HPLC-ICP-MS.
292
Quantification of IDP-73152, a novel antibiotic, in plasma from mice, rats and humans using an
ultra-high performance liquid chromatography/tandem mass spectrometry method for use in
pharmacokinetic studies
Myongjae Lee, Dohee Kim, Jeongcheol Shin, Hee-Yeol Lee, Suk-Jae Chung
ABSTRACT
IDP-73152, a novel inhibitor of a bacterial peptide deformylase, was recently approved as a new,
investigational drug in Korea for the clinical management of infections caused by Gram positive
bacteria. The objective of this study was to develop/validate a simple and robust analytical method for
the determination of IDP-73152 in plasma samples from rodents and humans, and to assess the
feasibility of the assay for use in pharmacokinetic studies using animal models. Plasma samples were
processed using a standard method for protein precipitation and an aliquot of the extract then injected
onto an UHPLC–MS/MS system. The drug and IDP-117293, an internal standard, were analyzed in
the positive ion-mode by electrospray ionization and quantified by monitoring the transition at m/z
555.2 → 245.2 for IDP-73152 and 563.3 → 253.1 for the internal standard, respectively. The lower
and upper limit of the assay was determined to be 5 and 10000 ng/ml, respectively, with an acceptable
linearity (R > 0.999) in the response-concentration relationship. Validation parameters, including
accuracy, precision, dilution, recovery, matrix effect and stability were found to be within the
acceptable ranges recommended by the assay validation guidelines of the United States FDA. The
method was successfully applied to the quantification of IDP-73152 in plasma from mice/rats that had
received a single oral administration of 80 mg/kg IDP-73152, in the form of the mesylate salt.
New approach for the diagnosis of histamine intolerance based on the determination of
histamine and methylhistamine in urine
Oriol Comas-Basté, M.Luz Latorre-Moratalla, Roberta Bernacchia, M.Teresa Veciana-Nogués,
M.Carmen Vidal-Carou
ABSTRACT
Histamine intolerance is a disorder in the homeostasis of histamine due to a reduced intestinal
degradation of this amine, mainly caused by diamine oxidase (DAO) enzyme deficiency, which
provokes its accumulation in plasma and the appearance of adverse health affects. A new approach for
the diagnosis of this intolerance could be the determination of histamine and its metabolites in urine.
The aim of this work was to develop and validate a rapid method to determine histamine and
methylhistamine in human urine by Ultra High Performance Liquid Chromatography and Fluorimetric
detection (UHPLC-FL). The proposed method is a consistent procedure to determine histamine and
methylhistamine in less than 11 min with adequate linearity and sensitivity. Relative standard
deviation was always lower than 5.5%, ensuring method precision; and mean recovery was greater
than 99% for both analytes. The structure of histamine and methylhistamine conjugated with OPA
were confirmed by UHPLC-ITD-FTMS which enabled to unequivocally identify both analytes in
standards and also in urine samples. The analysis of histamine and methylhistamine in urine samples
could be a potential new approach for the routine diagnosis of histamine intolerance, more patient-
friendly and with clear advantages in terms of equipment and personnel demand for sample collection
in comparison with current plasmatic DAO activity determination.
293
Development and validation of sensitive LC–MS/MS method for the quantification of SUVN-502
and its metabolite and its application for first in human pharmacokinetic study
Ramakrishna Nirogi, Devender Reddy Ajjala, Raghupathi Aleti, Lakshmiprasanna Rayapati, Naga
Surya Prakash Padala
ABSTRACT
A sensitive and rapid LC–MS/MS method was developed and validated for the quantification of
SUVN-502 and M1 of SUVN-502, a 5-HT6 receptor antagonist for the treatment of dementia
associated with Alzheimer‘s disease. Following solid-phase extraction, SUVN-502 and M1 of SUVN-
502 and IS were eluted with 10 mM ammonium acetate (pH 4.0) and acetonitrile using a rapid gradient
program on reverse phase column. Multiple reaction monitoring mode was used to monitor the
respective transitions of m/z 478.2 → 377.7 for SUVN-502 and m/z 464.1 → 377.7 for M1 of SUVN-
502. The assay exhibited a linear dynamic range of 10–10000 pg/mL for SUVN-502 and 20–20000
pg/mL for M1 of SUVN-502 in human plasma. Acceptable precision and accuracy were obtained for
concentrations over the standard curve range. The within batch accuracy and precision were within
acceptable limits. All the other validation parameters were within the acceptable limits. The validated
method was applied to analyze human plasma samples obtained from a human pharmacokinetic study
consisting single and multiple ascending doses.
Glycosylation patterns of selected proteins in individual serum and cerebrospinal fluid samples
Isabella Karlsson, Lorena Ndreu, Alessandro Quaranta, Gunnar Thorsén
ABSTRACT
A method we previously developed has been applied to the determination of the glycosylation pattern
of specific proteins in biological samples. Six proteins (alpha-1-antitrypsin, transferrin, haptoglobin,
C1 inhibitor, alpha-1 acid glycoprotein, and immunoglobulin G) were studied in serum samples from
five individuals and cerebrospinal fluid (CSF) samples from three individuals, to investigate the
expected normal distribution of glycosylation patterns and to assess whether this methodology can be
used to discriminate between samples from different individuals. For serum samples, the differences
were shown to be small, while much larger differences were found for the CSF samples, with a greater
number of glycoforms present. This can be linked to the occurrence of differential glycosylation in
proteins expressed in the brain compared with proteins expressed elsewhere in the body. The
developed method could distinguish differences in the glycosylation pattern of specific proteins in the
individual samples, which was not reflected in the glycan content of total CSF. This is the first time
that the glycoforms of several of these proteins have been investigated in CSF.
294
MIL-101(Cr)@GO for dispersive micro-solid-phase extraction of pharmaceutical residue in
chicken breast used in microwave-assisted coupling with HPLC–MS/MS detection
Yudan Wang, Xinpeng Dai, Xi He, Lin Chen, Xiaohong Hou
ABSTRACT
In this work, MIL-101(Cr)@GO (Graphite Oxide) was synthesized using a hydrothermal synthesis
method and was applied as a dispersive micro-solid-phase extraction (D-μ-SPE) sorbent for the
efficient concentration of four residual drugs (metronidazole, MNZ; tinidazole, TNZ;
chloramphenicol, CAP; sulfamethoxazole, SMX). Meanwhile, the extraction process was optimized by
combining it with microwave-assisted extraction. Factors affecting the D-μ-SPE efficiency, such as
selection of sorbent materials, pH of the sample solution, salting-out effect, amount of used material,
extraction time, desorption solvent and desorption time, were studied. Under the optimal extraction
conditions, the linearity ranged from 10 to 1000 ng kg−1 and 1–100 ng kg−1 (r2 ≥ 0.9928) for the
target analytes. The limits of detection were between 0.08 and 1.02 ng kg−1, and the limits of
quantitation were between 0.26 and 3.40 ng kg−1. Additionally, the developed method also exhibited
good precision (RSD ≤ 2.5%), repeatability (RSD ≤ 4.3%), high recoveries (88.9%–102.3%) and low
matrix effects (78.2%–95.1%). The proposed method proved to be an efficient and reliable approach
for the determination of the analytes. Finally, we successfully detected the four drugs in chicken
breast.
Development of a robust reporter gene assay to measure the bioactivity of anti-PD-1/anti-PD-L1
therapeutic antibodies
Lan Wang, Chuanfei Yu, Yalan Yang, Kai Gao, Junzhi Wang
ABSTRACT
Being regarded as the ‗cancer panacea‘, the anti-PD-1/anti-PD-L1 monoclonal antibodies (mAbs) have
become the R&D focus of biopharmaceutical industries. Several marketed such mAbs have been
proved particularly effective in treating various cancers. However, the cell-based bioassay to measure
the biological activities of the anti-PD-1/anti-PD-L1 mAbs as the lot release or stability test has been a
great challenge to quality control laboratories due to the immunomodulating nature of the mAbs. Here,
we describe the development and validation of a reporter gene assay consisting of two-cell systems to
measure the bioactivity of the anti-PD-1/anti-PD-L1 mAbs. We have generated two cell lines, the
CHO-PD-L1-CD3L cell line that stably expresses PD-L1 and the membrane-anchored anti-CD3 single
chain antibody fragment (scFv) named CD3L and the Jurkat-PD-1-NFAT cell line that stably
expresses PD-1 and the luciferase gene under the control of the NFAT response elements from the IL-
2 promoter. The results show good dose-dependent responsiveness to the mAbs and excellent
performance characteristics including specificity, accuracy and precision. The biological relevance of
the assay, the passage stability of the two cell lines, and the capability of measuring various anti-PD-
1/anti-PD-L1 mAbs render this assay applicable not only in lot release and stability test but also in
characterization and development of new anti-PD1/anti-PD-L1 mAbs.
295
LC–MS/MS-ESI method for simultaneous quantification of darolutamide and its active
metabolite, ORM-15341 in mice plasma and its application to a pharmacokinetic study
Sreekanth Dittakavi, Pavan Kumar V.S.P. Nagasuri, Suresh P. Sulochana, Syed Mohd Saim, Ramesh
Mullangi
ABSTRACT
A sensitive and rapid LC–MS/MS method was developed and validated for the simultaneous
quantitation of darolutamide and its active metabolite i.e. ORM-15341 in 50 μL mice plasma using
bicalutamide as an internal standard (I.S.) as per regulatory guidelines. Sample processing was
accomplished through liquid-liquid extraction. Chromatographic separation was achieved using an
Atlantis C18 column with an isocratic mobile phase comprising 0.2% formic acid:acetonitrile (35:65,
v/v) at a flow rate of 0.8 mL/min within 2.5 min. Detection and quantitation were done by multiple
reaction monitoring on a triple quadrupole mass spectrometer following the transitions: m/z 397 →
202, 395 → 202 and 429 → 255 for darolutamide, ORM-15341 and I.S, respectively in the negative
ionization mode. The calibration curve was linear from 0.61–1097 ng/mL for both darolutamide and
ORM-15341. The intra- and inter-day precisions were in the range of 1.34–13.8 and 4.85–12.9 and
3.91–13.7 and 6.54–14.2%, for darolutamide and ORM-15341, respectively. Darolutamide and ORM-
15341 were found to be stable under different stability conditions. The validated method was applied
to a pharmacokinetic study in mice.
Metabolite identification of AZD8055 in Sprague-Dawley rats after a single oral administration
using ultra-performance liquid chromatography and mass spectrometry
Md Mamunur Rashid, Hyun-A Oh, Hyunbeom Lee, Byung Hwa Jung
ABSTRACT
AZD8055 is an ATP-competitive specific dual mTOR inhibitor and exhibited potent antitumor activity
on several types of solid tumors. However, the metabolism of AZD8055 in the body still remains
unknown. In this study, metabolite identification of AZD8055 was performed using ultra high-
performance liquid chromatography-ion trap mass spectrometry (UHPLC-IT-MS) through both in
vitro and in vivo approaches using rat liver microsomes (RLMs) and rat plasma, urine and feces,
respectively. A total of eight putative metabolites (five phase I and three phase II) were identified, and
a tentative metabolic pathway was suggested for the first time. Considering the accurate mass and
mass fragmentations of the detected metabolites, their plausible structures were suggested.
Demethylation, hydroxylation, oxidation and morpholine ring opening were the major
biotransformation processes for the phase-I metabolism, while phase-II metabolites were merely
generated by the glucuronide conjugation reaction. The cumulative excretion of AZD8055 in urine and
feces was 0.13% and 1.11% of the dose, respectively. When the semi-quantitative analysis of the
metabolites was performed using UHPLC–MS/MS (ultra-performance liquid chromatography tandem
mass spectrometry) to evaluate the overall trend of metabolites formation and excretion, AZD8055
was excreted more in the form of the metabolites than itself and their formation was very fast.
Therefore it was presumed that biotransformation was playing a crucial role in its elimination.
Ultimately, this study provides novel insights regarding the in vitro and in vivo biotransformations of
AZD8055. Further investigations of metabolites of this potent anti-cancer compound could be
beneficial for the antitumor drug design and development process.
296
Confirmation of metabolites of the neuroleptic drug prothipendyl using human liver
microsomes, specific CYP enzymes and authentic forensic samples—Benefits for routine drug
testing
M. Krämer, S. Broecker, B. Madea, C. Hess
ABSTRACT
Metabolism of the tricyclic azaphenothiazine neuroleptic drug prothipendyl was investigated with in
vitro studies using human liver microsomes but also specific isoforms of cytochrome P450 (CYP)
enzymes. Identification and analysis of metabolites was done by liquid chromatography (LC) coupled
with quadrupole time of flight mass spectrometry (LC-QTOF-MS) as well as triple quadrupole mass
spectrometry (LC-QQQ-MS). Results of the herein presented study revealed the proof of various
demethylated and oxidized metabolites (-CH2, -C2H4, four derivatives of prothipendyl +O and three
derivatives of prothipendyl -CH2 + O). Metabolic reactions of prothipendyl were mainly catalyzed by
CYP enzymes CYP1A2, CYP2D6, CYP2C19 and CYP3A4. N-demethyl-prothipendyl was
predominantly formed by isoforms CYP2C19 and CYP1A2, while particularly the CYP isoenzyme
3A4 was responsible for the formation of prothipendyl sulfoxide. To confirm the formation of
previously identified metabolites in vivo, cardiac blood samples that were tested positive for
prothipendyl during routine drug testing and serum and urine samples, collected after a voluntary
intake of prothipendyl, were analyzed by LC-QQQ-MS.
Quantification of 16 β-lactams in chicken muscle by QuEChERS extraction and UPLC-Q-
Orbitrap-MS with parallel reaction monitoring
Qing Chen, Xiao-Dong Pan, Bai-Fen Huang, Jian-Long Han
ABSTRACT
A method is described for the analysis of 16 β-lactams in chicken muscle by UPLC-quadrupole(Q)-
Orbitrap-MS with parallel reaction monitoring (PRM). QuEChERS approach includes clean-up step by
sorbent of primary-secondary amine (PSA) and C18 was adopted for sample preparation. Q-Orbitrap
with PRM showed high sensitivity with limits of detection (LODs) ranged from 0.01 μg kg−1 to 0.35
μg kg−1. The method was further validated by intra- and inter-day test with spiking levels less than
MRLs (maximum residue limits, the European Union). Recovery (83–112%) and precision values
(RSDs <15%) for all studied analytes were obtained. The result indicates that UPLC-Q-Orbitrap
coupled with QuEChERS preparation can serve as a routine quantification method for β-lactam
residues in chicken muscles.
297
Characterization of the phase I and phase II metabolic profile of tolvaptan by in vitro studies
and liquid chromatography–mass spectrometry profiling: Relevance to doping control analysis
Monica Mazzarino, Valeria Buccilli, Xavier de la Torre, Ilaria Fiacco, Francesco Botrè
ABSTRACT
Phase I and phase II biochemical reactions involved in the biotransformation pathways of tolvaptan
were characterized by LC–MS-based techniques and in vitro models to identify the most appropriate
marker(s) of intake. The effects of physiological and non-physiological factors on the metabolic profile
of tolvaptan were also evaluated. In vitro approaches were based on the use of pooled human liver
microsomes and recombinant isoforms of cytochrome P450 and uridine diphospho glucuronosyl-
transferase. Sample preparation included liquid/liquid extraction at neutral pH with tert-butyl methyl-
ether. In the case of the study of phase II metabolism an additional enzymatic hydrolysis step was
performed. The chromatographic separation was carried out using reversed-phase chromatography,
whereas detection was performed by either triple-quadrupole or time-of-flight analyzers in positive
electrospray ionization and different acquisition modes. Our data show that tolvaptan is metabolized to
at least 20 phase I metabolites, the biotransformation reactions being catalyzed mainly by CYP3A4
and CYP3A5 isoforms. The phase-I reactions include hydroxylation (in different positions),
carboxylation, oxidation, hydrogenation, dealkylation, isomerization and a combination of the above.
Most of the phase I metabolites undergo glucuronidation, carried out mostly by UGT2B7 and
UGT2B17 isoforms. Dealkylated, mono-hydroxylated and carboxylated metabolites both in the free
and in the glucuronidated form appear to be the most suitable urinary diagnostic markers for the
detection of tolvaptan intake in doping control.
An LC–MS/MS method for simultaneous determination of nine steroidal saponins from Paris
polyphylla var. in rat plasma and its application to pharmacokinetic study
Guangyi Yang, Wei Lu, Meng Pan, Chenning Zhang, Gao Song
ABSTRACT
Paris polyphylla var is an herbal plant herb widely used in Traditional Chinese Medicine. The purpose
of this study is to develop an Ultra Performance Liquid Chromatography-tandem mass spectrometer
(UPLC–MS) method to quantify the major components (i.e., nine saponins) from P. polyphylla in
plasma samples. A UItra BiPh column (100 × 2.1 mm, 5 μm) was used with acetonitrile/0.1% formic
acid in water as mobile phases. The analytes were quantified using a Waters XEVO TQ mass
spectrometer via multiple reaction monitoring (MRM) with positive scan mode. A protein precipitation
method was used to extract the analytes from rat plasma. The inter/intra-day precision, accuracy,
recovery, matrix effect, and stability were evaluated per the FDA guidance. The method showed
linearity in the concentration ranges of 2.4–1250 ng/mL. The intra-day and inter-day precisions (RSD)
of these analytes at three different levels were less than 15.0%. The extraction recoveries of these
analytes were from 83.8% to 109.4% and the matrix effects ranged from 87.4% to 105.4%. The
stabilities of these compounds in plasma were evaluated by analyzing three different concentrations
following storage at 25 °C for 6 h, and −80 °C for 30 days. All the samples displayed less than 15.0%
variations.
298
N-glucuronidation catalyzed by UGT1A4 and UGT2B10 in human liver microsomes: Assay
optimization and substrate identification
Danyi Lu, Qian Xie, Baojian Wu
ABSTRACT
N-glucuronidation is an important pathway for metabolism and disposition of tertiary amines in
humans. This reaction is mainly catalyzed by the enzymes UGT1A4 and UGT2B10. However, the
metabolic patterns of UGT1A4- and UGT2B10-mediated N-glucuronidation are not fully clear. In this
study, we first optimized in vitro reaction conditions for N-glucuronidation by using specific substrates
(i.e., trifluoperazine for UGT1A4, cotinine and amitriptyline for UGT2B10). Furthermore, we found
that hepatic N-glucuronidation showed significant species differences. In addition, UGT1A4 and
UGT2B10 were primarily responsible for N-glucuronidation of many tertiary amines, including
asenapine, loxapine, clozapine, chlorpromazine, dothiepin, doxepin, mirtazapine, mianserin,
chlorcyclizine, cyclizine, promethazine, cyclobenzaprine, imatinib, retrorsine, strychnine and brucine.
In conclusion, this study provides an in vitro assay system for evaluating N-glucuronidation of amines.
Also, UGT1A4- and UGT2B10-mediated N-glucuronidation might play significant roles in
metabolism and detoxification of tertiary amines in humans.
A simple high performance liquid chromatography–mass spectrometry method for Therapeutic
Drug Monitoring of isavuconazole and four other antifungal drugs in human plasma samples
Giovanna Fatiguso, Fabio Favata, Ilaria Zedda, Amedeo De Nicolò, Antonio D‘Avolio
ABSTRACT
Triazoles chanced the prevention and treatment of invasive fungal infections, but their
pharmacokinetic properties are still unclear. In particular, isavuconazole (ISC) is a new broad-
spectrum antifungal triazole approved in 2015 as first-line treatment for intravenous and oral use
against invasive aspergillosis and for mucormycosis. Nowadays, the optimal management of the
treatments with triazoles requires the use of Therapeutic Drug Monitoring (TDM), in order to prevent
sub-therapeutic or toxic concentrations. In turn, the routine use of TDM requires reliable quantification
methods The aim of this work was the development and full validation of a HPLC-mass spectrometry
assay for the simultaneous quantification of fluconazole, itraconazole, isavuconazole, posaconazole
and voriconazole in human samples. Both standards and quality controls were prepared in human
plasma. After the addition of internal standard (6,7-dimethyl-2,3-di(2-pyridyl)quinaxoline for
voriconazole, posaconazole and itraconazole; stable isotope labeled compounds for fluconazole and
isavuconazole), protein precipitation with acetonitrile and dilution with water were performed.
Chromatographic separation was performed on Atlantis® T3 5 μm 4.6 × 150 mm column, with a
gradient of water and acetonitrile, both added with 0.05% formic acid. Accuracy intra-day and inter-
day imprecision fitted FDA and EMA guidelines, while matrix effects and recoveries resulted stable
between samples for each analyte. Stability results were in accordance with previously published data.
Finally, we tested this method by monitoring plasma concentrations in real patients and using external
quality controls with good results. This method resulted very simple, fast, cheap and very useful for
TDM application, to improve clinical management of antifungal therapy in critically ill patients.
299
Metabolic profiling of dehydrodiisoeugenol using xenobiotic metabolomics
Qian-Qian Lv, Xiao-Nan Yang, Dong-Mei Yan, Wei-Qing Liang, Fei Li
ABSTRACT
Dehydrodiisoeugenol (DDIE), a representative and major benzofuran-type neolignan in Myristica
fragrans Houtt., shows anti-inflammatory and anti-bacterial actions. In order to better understand its
pharmacological properties, xenobiotic metabolomics was used to determine the metabolic map of
DDIE and its influence on endogenous metabolites. Total thirteen metabolites of DDIE were identified
through in vivo and in vitro metabolism, and seven of them were reported for the first time in the
present study. The identity of DDIE metabolites was achieved by comparison of the MS/MS
fragmentation pattern with DDIE using ultra-performance chromatography electrospray ionization
quadrupole time-of-flight mass spectrometry (UPLC-ESI- QTOFMS). Demethylation and ring-
opening reaction were the major metabolic pathways for in vivo metabolism of DDIE. Recombinant
cytochrome P450 s (CYPs) screening revealed that CYP1A1 is a primary enzyme contributing to the
formation of metabolites D1-D4. More importantly, the levels of two endogenous metabolites 2,8-
dihydroxyquinoline and its glucuronide were significantly elevated in mouse urine after DDIE
exposure, which explains in part its modulatory effects on gut microbiota.
Development and validation of a reliable method for thiopurine methyltransferase (TPMT)
enzyme activity in human whole blood by LC–MS/MS: An application for phenotypic and
genotypic correlations
Supaporn Wiwattanakul, Santirhat Prommas, Nuttawut Jenjirattithigarn, Siwalee Santon, Chonlaphat
Sukasem
ABSTRACT
A liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed for the
determination of thiopurine methyltransferase (TPMT) activity in human whole blood lysate, based on
conversion of 6-mercaptopurine (6-MP) by TPMT to 6-methylmercaptopurine (6-MMP) using S-
adenosyl-l-methionine (SAM) as the methyl donor. This method was improved from the previous
laborious method for washing of red cell lysate preparation to develop whole blood EDTA lysate. In
addition, the TPMT incubation was optimized and the chromatography was performed in a short
runtime of 7 min on a C18-column by detection via triple quadrupole mass spectrometry. The MS/MS
was optimally tuned to monitor mass to charge a ratio (m/z) for 6-MMP 167.2 → 151.9 and the isotope
6-MMP-d3 with m/z of 170.5 → 152.2 were applied as an internal standard. The calibration curve
covered the range of 2.5–360 ng/ml and the correlation coefficient was greater than 0.999. The
accuracy of this method was determined in four concentrations of control of quality that ranged
between 99.33 and 106.33%. The intra-assay coefficient of variation (CV) was less than 4.41% and the
inter-assay was less than 5.43%. This method developed for measuring TPMT by LC–MS/MS is a
reliable, safe, and simple with a small volume requirement (100 μl of whole blood EDTA). The assay
was used to study TPMT activity in 132 Thai children with a range from 29.0 to 89.1 nmol 6-MMP/g
Hb/h with means and median values of TPMT activity 55.9 ± 12.47 nmol 6-MMP/g Hb/h and 54.2
nmol 6-MMP/g Hb/h. The genotype-phenotype association of TPMT was evaluated for common
ethnic Thai single nucleotide polymorphisms (SNP) in 30 samples and demonstrated good
concordance.
300
Development and validation of a GC–MS method for the determination of hydroxyzine and its
active metabolite, cetirizine, in whole blood
Maria Katselou, Sotiris Athanaselis, Panagiota Nikolaou, Artemisia Dona, Ioannis Papoutsis
ABSTRACT
A simple, rapid, sensitive and accurate gas chromatography–mass spectrometric method was
developed and validated for the simultaneous determination of hydroxyzine and cetirizine in whole
blood. Solid-phase extraction procedure using Bond Elut LRC Certify II columns was used for the
isolation of hydroxyzine and cetirizine from 1 mL whole blood followed by derivatization with a
mixture of acetic anhydride:n-propanol (1:1, v/v). Limits of detection and quantification were 1.50 and
5.00 ng/mL, respectively. The assay was linear within the concentration range of 5.00–1000.0 ng/mL
and the correlation coefficient was R2 ≥ 0.993 for both analytes. Absolute recovery was determined at
three quality control concentration levels and was found to be at least 87.2% for both substances. Intra-
day and inter-day accuracy values for both hydroxyzine and cetirizine were ranged from −1.2 to 3.8%
and −2.7 to 2.0%, respectively, at the three concentration levels studied, whereas their respective intra-
day and inter-day precision values were less than 9.9 and 6.5%, respectively, in terms of relative
standard deviation (%RSD). The developed method was successfully applied for the quantification of
hydroxyzine and cetirizine concentrations in whole blood, during the investigation of clinical cases
where these two antihistamines were detected.
An on-spot internal standard addition approach for accurately determining colistin A and
colistin B in dried blood spots using ultra high-performance liquid chromatography–tandem
mass spectrometry
I-Lin Tsai, Ching-Hua Kuo, Hsin-Yun Sun, Yu-Chung Chuang, Yun-Jung Tsai
ABSTRACT
Outbreaks of multidrug-resistant Gram-negative bacterial infections have been reported worldwide.
Colistin, an antibiotic with known nephrotoxicity and neurotoxicity, is now being used to treat
multidrug-resistant Gram-negative strains. In this study, we applied an on-spot internal standard
addition approach coupled with an ultra high-performance liquid chromatography-tandem mass
spectrometry (LC–MS/MS) method to quantify colistin A and B from dried blood spots (DBSs). Only
15 μL of whole blood was required for each sample. An internal standard with the same yield of
extraction recoveries as colistin was added to the spot before sample extraction for accurate
quantification. Formic acid in water (0.15%) with an equal volume of acetonitrile (50:50 v/v) was used
as the extraction solution. With the optimized extraction process and LC–MS/MS conditions, colistin
A and B could be quantified from a DBS with respective limits of quantification of 0.13 and 0.27 μg
mL−1, and the retention times were < 2 min. The relative standard deviations of within-run and
between-run precisions for peak area ratios were all < 17.3%. Accuracies were 91.5-111.2% for lower
limit of quantification, low, medium, and high QC samples. The stability of the easily hydrolyzed
prodrug, colistin methanesulfonate, was investigated in DBSs. Less than 4% of the prodrug was found
to be hydrolyzed in DBSs at room temperature after 48 h. The developed method applied an on-spot
internal standard addition approach which benefited the precision and accuracy. Results showed that
DBS sampling coupled with the sensitive LC–MS/MS method has the potential to be an alternative
approach for colistin quantification, where the bias of prodrug hydrolysis in liquid samples is
decreased.
301
Comparative study of single/combination use of Huang-Lian-Jie-Du decoction and berberine on
their protection on sepsis induced acute liver injury by NMR metabolic profiling
Yan Lv, Junsong Wang, Dingqiao Xu, Shanting Liao, Lingyi Kong
ABSTRACT
Sepsis is a serious clinical disease with a high mortality rate all around the world. Liver organ
dysfunction is an important sign for the severity and outcome of sepsis in patients. In this study, 1H
NMR-based metabolomics approach and biochemical assays were applied to investigate the metabolic
profiling for cecal ligation and puncture (CLP) induced acute liver injury, the therapeutical effect of
single/combination use of Huang-Lian-Jie-Du decoction (HLJDD) and berberine, and the interaction
of them. Metabolomics analysis revealed significant perturbations in livers of septic rats, which could
be ameliorated by HLJDD, berberine and their combination treatment. Berberine could better rectified
glycolysis and nucleic acid metabolism in the liver. HLJDD had exceptional better anti-inflammatory,
antibacterial and antioxidative effects than berberine. The interaction of berberine and HLJDD could
further strengthen the anti-inflammation and anti-oxidation, but with poor effect on amino acids
metabolism. These findings highlighted the feasibility of the integrated NMR based metabolomics
approach to understand the pathogenesis of diseases, the action mechanisms of therapy and the herb-
drug interaction.
Screening and confirmation of steroids and nitroimidazoles in urine, blood, and food matrices:
Sample preparation methods and liquid chromatography tandem mass spectrometric
separations
Ádám Tölgyesi, Enikő Barta, Andrea Simon, Thomas J. McDonald, Virender K. Sharma
ABSTRACT
Veterinary drugs containing synthetic anabolic steroid and nitroimidazole active agents are not allowed
for their applications in livestock of the European Union (EU). This paper presents analyses of twelve
selected steroids and six nitroimidazole antibiotics at low levels (1.56 μg/L–4.95 μg/L and 0.17 μg/kg–
2.14 μg/kg, respectively) in body fluids and egg incurred samples. Analyses involved clean-up
procedures, high performance liquid chromatography (HPLC) separation, and tandem mass
spectrometric screening and confirmatory methods. Target steroids and nitroimidazoles in samples
were cleaned by two independent supported liquid extraction and solid phase extraction procedures.
Separation of the selected compounds was conducted on Kinetex XB C-18 HPLC column using
gradient elution. The screening methods utilised supported liquid extraction that enabled fast and cost
effective clean-up. The confirmatory methods were improved by extending the number of matrices and
compounds, and by introducing an isotope dilution mass spectrometry for nitroimidazoles. The new
methods were validated according to the recommendation of the European Union Reference
Laboratories and the performance characteristics evaluated met fully the criteria. The methods were
applied to incurred samples in the proficiency tests. The obtained results of Z-scores demonstrated the
applicability of developed protocols of the methods to real samples. The confirmatory methods were
applied to the national monitoring program and natural contamination of prednisolone could be
detected in urine at low concentration in few samples.
302
Validation of an HPLC-UV method for analysis of Kaempferol-loaded nanoemulsion and its
application to in vitro and in vivo tests
Mariana Colombo, Gabriela de Lima Melchiades, Fabrício Figueiró, Ana Maria Oliveira Battastini,
Letícia Scherer Koester
ABSTRACT
A simple and reliable HPLC-UV method for Kaempferol (KPF) determination in a Kaempferol-loaded
nanoemulsion (KPF-NE), samples from mucosa permeation/retention studies, and murine brain was
developed and validated according to international guidelines. The analyses were performed on a
reversed-phase C18 column at 35 °C and under UV detection at 368 nm. The mobile phase was
composed of methanol:formic acid 0.1% (75:25, v/v) and was eluted at an isocratic flow rate of 1.0
mL/min. The method was selective and sensitive for KPF analysis in matrix extracts, and linear in the
range of 0.25–7.5 μg/mL. The method was also considered precise, accurate, and robust. The recovery
rates of KPF from the porcine nasal mucosa and murine brain were higher than 85%. Low matrix
effect was observed to determine KPF, including biological matrices. The applicability of the method
was confirmed in all different approaches, i.e., quantification of KPF in nanoemulsion, in vitro
permeation/retention of KPF across porcine nasal mucosa, and in vivo quantification of KPF in brain
samples after nasal administration in rats. Thus, the method is effective and reliable to determine KPF
in different real samples. The proposed method, therefore, provides a useful quantification approach to
routine processes, to the development of drug delivery systems, and to KPF quantification in different
biological matrices. Furthermore, the method is applicable in bioavailability studies and the developed
formulation (KPF-NE) is suitable for preclinical trials in different brain disorders.
Development of direct assays for Toxoplasma gondii and its use in genomic DNA sample
Lívia M. Alves, Vinícius R. Rodovalho, Ana C.H. Castro, Márcia A.R. Freitas, Ana G. Brito-Madurro
ABSTRACT
This work describes an approach for the selection and detection of specific DNA probes related to
Toxoplasma gondii, a protozoan parasite responsible for toxoplasmosis. The detection system was
developed on graphite carbon electrode modified with poly(3-hydroxybenzoic acid) sensitized with
ToxG1 probe. The hybridization of the specific genomic DNA related to T. gondii showed good
response by direct detection of guanine residue oxidation using differential pulse voltammetry (DPV).
The biosensor was able to distinguish both the complementary and non-complementary targets and
detect up to 100 ng μL−1 of the T. gondii genomic DNA. The hybridization (ToxG1: T. gondii
genomic DNA) was confirmed by optical measurement. Optical assays using gold
nanoparticles:ToxG1 probe showed a significant change in the absorbance peak in the presence of the
T. gondii genomic DNA according to the electrochemical results. This novel biosensor shows potential
as electrochemical transducer and was successfully applied in the biological sample.
303
Investigation and structural elucidation of a new impurity in bulk drug of cilostazol by
LC/MS/MS, FT-IR and NMR
Zhaoxia Hu, Sanguo Gao, Jue Gao
ABSTRACT
A new impurity was detected in bulk cilostazol (CIL) crude during routine analysis. The impurity
(∼4%, the specification for unknown impurity in crude is not more than 0.20%) has a relative retention
time of 1.46. Based on MS, NMR and IR spectral data, the impurity was identified as 6,6′-bis(4-(1-
cyclohexyl-1H-tetrazol-5-yl) butoxy)-3,3′,4,4′-tetrahydro-[7,7′-biquinoline]-2,2′(1H,1′H)-dione(CIL-
dimer). The precursor of CIL-dimer is an oxidative product of starting material 6-Hydroxy-3,4-
dyhydro-1H-quinolin–2-one(6-HQ), CIL-dimer was formed in the following reaction with 5-(3-
Chloro-propyl)-1-cyclohexyl-1H-tetrazol(CHCBT).
Selective screening of glutaric acid acidurias by capillary electrophoresis-mass spectrometry
Jaime Fernández-Bravo, Fernando de Andrés, Mohammed Zougagh, Ángel Ríos
ABSTRACT
A sensitive and selective method for the separation and quantification of the three organic acids 3-
hydroxy-3-methylglutaric acid, 3-methylglutaric acid, and glutaric acid in human urine samples by CE
with mass spectrometry detection has been developed. This methodology is faster, simpler and less
time-consuming, than other methodologies previously described, and requires of reduced amounts of
reagents as well. Samples are first filtered and then diluted in water. For the electrophoretic separation,
a 20 mM ammonium acetate and 10% methanol solution at pH 9.1 was selected as the running
electrolyte. With 5-s hydrodynamic injection, detection limits ranging from 15.5 to 39.3 μM and linear
responses ranging from the LOQ calculated for each analyte to more than 400 μM were obtained for
the analysis of the different organic acids in less than 13 min. Remarkable selectivity is achieved by
mass spectrometry detection using 0.25% of formic acid in 50% v/v 2-propanol-water solution as
sheath liquid, and enough sensitivity without interferences from the matrices was obtained as well.
This methodology has revealed as an efficient approach to help the 3-hydroxy-3-methylglutaric
aciduria diagnoses in order to discard or confirm the occurrence of the disease as of the presence or
absence of the expected increased levels of these analytes in samples of potential patients.
304
Online turbulent flow extraction coupled with liquid chromatography–tandem mass
spectrometry for high throughput screening of anabolic steroids in horse urine
Hyun Du Shin, Joon Hyuk Suh, Junghyun Kim, Hyun-Deok Cho, Sang Beom Han
ABSTRACT
A high throughput method for simultaneous screening of anabolic steroids and their metabolites (4-
esterendione, trenbolone, boldenone, oxandrolone, nandrolone, methandrostenolone, testosterone, 1-
androstendione, ethisterone, normethandrolone, methyltestosterone, 16β-Hydroxystanozolol,
epitestosterone, bolasterone, norethandrolone, danazol, stanozolol and androstadienone) in equine
urine by online turbulent flow extraction coupled with liquid chromatography-tandem mass
spectrometry was developed. The use of turbulent flow chromatography could simplify pretreatment of
horse urine, which has complex matrices as well as high viscosity. The urine was extracted by mixed-
mode cation exchange solid phase extraction, and hydrolyzed using β-glucuronidase/arylsulfatase.
Then, the sample was automatically loaded on the TurboFlow Cyclone extraction column for removal
of further matrix, followed by separation on a fused core C18 column before MS/MS detection.
Optimization and validation of the method were discussed in detail. All analytes were rapidly detected
within 10 min with high sensitivity (picogram to nanogram per milliliter level), and no interference
was observed. The linearity range was from 0.1–10 ng/mL for nine steroids and 1.0–50 ng/mL for the
others, with correlation of coefficient values over 0.995. Precision and accuracy ranged from 0.1 to
14.5% and 1.7 to 12.4%, respectively. The developed method was successfully applied to the analysis
of anabolic steroids in horse urine after administration of a model drug.
Development of a mixed-mode chromatography with tandem mass spectrometry method for the
quantitative analysis of 23 underivatized amino acids in human serum
Min Sun Choi, Shaheed Ur Rehman, In Sook Kim, Hi-Joon Park, Hye Hyun Yoo
ABSTRACT
In this study, a robust, selective and simplified method was developed and validated for the
simultaneous quantitative analysis of 23 underivatized amino acids in human serum using mixed-mode
chromatography with tandem mass spectrometry (LC–MS/MS). Serum samples were deproteinized
with acetonitrile and subjected to LC–MS/MS analysis. The chromatographic separation of amino
acids was achieved using a mixed-mode column (150 × 3 mm, 3 μm) with a gradient elution system;
the mobile phase consisted of 50 mM ammonium formate and 0.1% formic acid in acetonitrile. The
total run time was 22 min. Eluted compounds were detected in the electrospray ionization-positive
mode with multiple reaction monitoring. The validation study evaluated linearity, repeatability, intra
and inter-day accuracy and precision, and matrix effect. The validation results were satisfactory in all
the tested parameters. This method was successfully applied to the analysis of amino acids in the
clinical sample of human serum.
305
Inhibitory assay for degradation of collagen IV by cathepsin B with a surface plasmon resonance
sensor
Atsushi Shoji, Yumiko Suenaga, Atsushi Hosaka, Yuuki Ishida, Masao Sugawara
ABSTRACT
We describe a simple method for evaluating the inhibition of collagen IV degradation by cathepsin B
with a surface plasmon resonance (SPR) biosensor. The change in the SPR signal decreased with an
increase in the concentration of cathepsin B inhibitors. The order of the inhibitory constant (Ki)
obtained by the SPR method was CA074Me ≈ Z-Phe-Phe-FMK < leupeptin. This order was different
from that obtained by benzyloxycarbonyl-Phe-Phe-Fluoromethylketone (Z-Phe-Phe-FMK) as a peptide
substrate. The comparison of Ki suggested that CA074 and Z-Phe-Phe-FMK inhibited exopeptidase
activity, and leupeptin inhibited the endopeptidase activity of cathepsin B more strongly.
Liquid chromatography-tandem mass spectrometry assay to quantify plitidepsin in human
plasma, whole blood and urine
L. van Andel, H. Rosing, S. Fudio, P. Avilés, J.H. Beijnen
ABSTRACT
Plitidepsin is an anti-cancer drug currently evaluated in phase I/II/III clinical trials. This article
describes the development and validation of a bioanalytical assay to quantify plitidepsin in human
plasma, urine and whole blood using HPLC–MS/MS. The analyte was extracted from the matrix by
liquid–liquid extraction using tert-butyl methyl ether. Final extracts were injected onto a C18 column,
gradient elution was applied for chromatographic separation and detection was performed on a triple
quadrupole mass spectrometer operating in the positive ion mode. The assay was linear over the range
0.1–100 ng/mL, with acceptable accuracy and precision values. This is the first reported bioanalytical
assay quantifying plitidepsin using a stable isotopically labelled standard, achieving a lower limit of
quantification of 0.1 ng/mL in all three matrices, allowing the quantification of trace levels of
plitidepsin and accomplishing this in an analysis time of two minutes only. The presented method was
successfully applied in a mass balance study with plitidepsin in patients with advanced cancer.
306
Rapid analysis of benzalkonium chloride using paper spray mass spectrometry
Jingjing Liu, Wenjie Deng, Muqian Yu, Ruizhi Wen, Bo Chen
ABSTRACT
A paper spray mass spectrometry (PS-MS) method for rapid and reliable analysis of benzalkonium
chloride (BAC) in compound eye drops and body surface disinfectant was developed. The sample was
dropped onto triangular filter paper, and high voltage (3.5 kV) was applied to form an electrospray.
This method can provide the composition of benzalkonium chloride in samples without pretreatment,
solvent or chromatographic separation, and the analysis time is only 10 s. The primary homologues
C12-BAC, C14-BAC and C16-BAC of benzalkonium chloride were quantitatively analyzed using PS-
MS. Samples were subjected to simple dilution and quantified using the internal standard method. Ion
trap mass spectrometry was scanned using SIM mode. The linear ranges of C12-BAC, C14-BAC and
C16-BAC were 1–100 μg mL−1; the linear regression coefficients were 0.998–0.999; the detection
limits (LODs) were 0.1 μg mL−1; the limit of quantifications (LOQs) was <1 μg mL−1, and the
method validation indicated that the method precision and accuracy were good. Compared with HPLC-
UV methods, there was no significant difference in the quantitative determination of the actual
samples, but the analysis time for PS-MS is shorter (2 min). In addition, reagent consumption in PS-
MS is small, and no chromatographic separation is needed, suggesting that PS-MS is especially
suitable for high-throughput analysis.
Rapid screening of non-steroidal anti-inflammatory drugs illegally added in anti-rheumatic
herbal supplements and herbal remedies by portable ion mobility spectrometry
Mengjiao Li, Haiyan Ma, Jinglin Gao, Lina Zhang, Ye Jiang
ABSTRACT
In this work, for the first time, a high-performance ion mobility spectrometry with electrospray
ionization (ESI-HPIMS) method has been employed as a rapid screening tool for the detection of
acetaminophen, ibuprofen, naproxen, diclofenac sodium and indomethacin illegally added in anti-
rheumatic herbal supplements and herbal remedies. Samples were dissolved and filtered through a 0.45
μm microporous membrane, then the filtrate was directly injected into the high-performance ion
mobility spectrometry for analysis. Using this approach, the screening of illegal additions can be
accomplished in as rapid as two to three minutes with no pretreatment required. The proposed method
provided a LOD of 0.06–0.33 μg mL−1, as well as a good seperation of the five NSAIDs. The
precision of the method was 0.1–0.4% (repeatability, n = 6) and 0.9–3.3% (reproducibility, n = 3). The
proposed method appeared to be simple, rapid and highly specific, thus could be effective for the in-
situ screening of NSAIDs in anti-rheumatic herbal supplements and herbal remedies.
307
Simultaneous quantitative analysis of polyethylene glycol (PEG), PEGylated paclitaxel and
paclitaxel in rats by MS/MSALL technique with hybrid quadrupole time-of-flight mass
spectrometry
Heping Sun, Qi Zhang, Zhi Zhang, Jin Tong, Jingkai Gu
ABSTRACT
PEGylation is practically one of most important modifications of drugs including small molecules,
peptides and proteins, which has been proven to dramatically improve physicochemical properties and
pharmacokinetic behavior of the PEGylated drugs. However, it is a challenge currently to
quantitatively analyze PEG and PEGylated drugs by various analytical methods, even mass
spectrometry because of multiple parent ion distribution of PEG caused by its polydispersity of
molecular weight. Here we developed a robust method with MS/MSALL technique using electrospray
ionization (ESI) source coupled high resolution Quadrupole Time-of-Flight (Q-TOF) mass
spectrometry for the quantification of PEG2K-Paclitaxel (PEG-PTX) and its two metabolites, PEG and
Paclitaxel (PTX). The analysis was performed on a 300SB-C18 column with acetonitrile and 0.1%
formic acid as the mobile phase. Samples were simply prepared by protein precipitation in a small
quantity of plasma (50 μL). Calibration curve was linear within the range of 50.0–4000 ng/mL for
PEG and PEG-PTX and 1.0–1000 ng/mL for PTX. The intra- and inter-day precisions were 3.2–6.9%
and 3.1–6.9% for PEG, 4.1–7.8% and 4.0–9.9% for PEG-PTX, and 3.3–4.8% and 3.1–6.9% for PTX,
respectively. The recoveries were greater than 90% with low matrix effects. Afterwards, the newly
developed method was successfully applied to support a preclinical pharmacokinetic study in six rats
after single intravenous injection of PEG-PTX (51.7 mg/kg).
Lyophilic matrix method for dissolution and release studies of nanoscale particles
Jenni Pessi, Sami Svanbäck, Ilkka Lassila, Edward Hæggström, Jouko Yliruusi
ABSTRACT
We introduce a system with a lyophilic matrix to aid dissolution studies of powders and particulate
systems. This lyophilic matrix method (LM method) is based on the ability to discriminate between
non-dissolved particles and the dissolved species. In the LM method the test substance is embedded in
a thin lyophilic core-shell matrix. This permits rapid contact with the dissolution medium while
minimizing dispersion of non-dissolved particles without presenting a substantial diffusion barrier. The
method produces realistic dissolution and release results for particulate systems, especially those
featuring nanoscale particles. By minimizing method-induced effects on the dissolution profile of
nanopowders, the LM method overcomes shortcomings associated with current dissolution tests.
308
Incorporation of 14C-cholesterol in human adrenal corticocarcinoma H295R cell line and
online-radiodetection of produced 14C-steroid hormone metabolites
Jonas Abdel-Khalik, Erland Björklund, Frederik Knud Nielsen, Martin Hansen
ABSTRACT
This study demonstrates the addition of 14C-cholesterol to the human cell line H295R will in-situ form
radiolabeled steroid hormones allowing for new mechanistic and metabolic insights. The aim of the
present study was to in-situ radiolabel steroid hormones from cell line-incorporated 14C-cholesterol
using the OECD guideline 456, H295R steroidogenesis in-vitro assay. Radiodetection of the steroid
metabolites of the steroidogenic pathway allows for an improved understanding of the various
enzymatic mechanisms involved without necessarily being dependent on quantification. Generated
radiolabeled steroids were analyzed using HPLC hyphenated with a Flow Scintillation Analyzer
(FSA). H295R cells were incubated with radiolabeled cholesterol and cell media were collected and
prepared by solid phase extraction and analyzed with HPLC-FSA. For successful radiolabeling of the
steroids in the steroidogenesis of H295R cells, radioactive cholesterol may potentially only need to be
added just before the cells are incubated for 72 h in well plates. Based on the obtained HPLC-FSA
chromatograms, and confirmation of the observations by studies in the literature, a qualitative time
profile for the production of steroid hormones was estimated. Multiple radiolabeled steroid hormones
were identified by means of analytical standards and UV (ultraviolet) co-chromatography, though the
elucidation of multiple metabolites remains unresolved. Although online radiodetection proved to
suffer from suboptimal sensitivity, the concept of radiolabeling the steroidogenesis in H295R cells
with 14C-cholesterol and detecting the radiolabeled steroid hormones online was proved and may
assist in further toxicological studies.
Verification of the effectiveness of the Fourier transform infrared spectroscopy computational
model for colorectal cancer
J. Depciuch, E. Kaznowska, A. Koziorowska, J. Cebulski
ABSTRACT
Colorectal cancer is one of the most common cancers. Its formation is influenced by genetic and
environmental factors. Despite the continuous development of diagnostic tools and cancer therapies,
there are no methods that allow a real-time estimation of treatment efficiency. This method can be a
vibrational spectroscopy. The resulting infrared spectrum (FTIR) of the tissue gives us information
about the chemical composition and the content of the individual components. We have noticed that
tumor tissues, healthy and after chemotherapy tissues, have different vibrational spectra. It was also
shown that spectra acquired from normal (benign) tissues were similar to those derived from tissues
post-chemotherapy. The similarity was greater, when the effectiveness of chemotherapy, confirmed by
medical documentation, was better. Therefore, we decided to use the physical model proposed in our
earlier paper to verify its correctness and to show whether a particular type of chemotherapy was
effective or not. Comparison of the results obtained from the physical model with patients data have
been found as close to the physical condition.
309
Quantification of paracetamol and 5-oxoproline in serum by capillary electrophoresis:
Implication for clinical toxicology
Tomáń Hloņek, Tomáń Kříņek, Petr Tůma, Miroslava Bursová, Radomír Čabala
ABSTRACT
High anion gap metabolic acidosis frequently complicates acute paracetamol overdose and is generally
attributed to lactic acidosis or compromised hepatic function. However, metabolic acidosis can also be
caused by organic acid 5-oxoproline (pyroglutamic acid). Paracetamol‘s toxic intermediate, N-acetyl-
p-benzoquinoneimine irreversibly binds to glutathione and its depletion leads to subsequent disruption
of the gamma glutamyl cycle and an excessive 5-oxoproline generation. This is undoubtedly an
underdiagnosed condition because measurement of serum 5-oxoproline level is not readily available. A
simple, cost effective, and fast capillary electrophoresis method with diode array detection (DAD) for
simultaneous measurement of both paracetamol (acetaminophen) and 5-oxoproline in serum was
developed and validated. This method is highly suitable for clinical toxicology laboratory diagnostic,
allowing rapid quantification of acidosis inducing organic acid 5-oxoproline present in cases of
paracetamol overdose. The calibration dependence of the method was proved to be linear in the range
of 1.3–250 μg mL−1, with adequate accuracy (96.4–107.8%) and precision (12.3%). LOQ equaled 1.3
μg mL−1 for paracetamol and 4.9 μg mL−1 for 5-oxoproline.
Quantification of cyclocreatine in mouse and rat plasma using hydrophilic-interaction ultra-
performance liquid chromatography-tandem mass spectrometry
Amy Q. Wang, Emma Hughes, Wenwei Huang, Edward H. Kerns, Xin Xu
ABSTRACT
An accurate, rapid and selective method was developed to quantify cyclocreatine in mouse and rat
plasma using hydrophilic interaction (HILIC) ultra-performance liquid chromatography-tandem mass
spectrometry (UPLC–MS/MS). The plasma samples were prepared by protein precipitation with
acetonitrile:methanol (70:30). Chromatographic separation was performed on a HILIC BEH amide
column (2.1 mm × 50 mm, 1.7 μm) with a 3 min gradient elution at a flow rate of 0.5 mL/min. For
mass spectrometric detection, selected reaction monitoring (SRM) was used; the SRM transitions were
m/z 144 → 98 and m/z 144 → 56 for cyclocreatine and m/z 148 → 102 for the internal standard (D4-
cyclocreatine) in the positive ionization mode. No endogenous components interfered with the analysis
of cyclocreatine and the internal standard in mouse and rat plasma. Plasma calibration curves were
constructed in the range of 0.01–25 μM. The correlation coefficient of the calibration curves was
greater than 0.99. The mean intraday assay accuracy for all quality control (QC) replicates was
between 93 and 105%. The mean intraday assay precision (CV%) was 1.9-11% for all QC levels. The
HILIC–UPLC–MS/MS method was successfully applied in pharmacokinetic (PK) studies of
cyclocreatine in mice and rats for the first time. After a single 30 mg/kg oral administration in mice
and rats, the AUC0-∞ (area under the curve) was 84.1 μg h/mL and 91.7 ± 18.0 μg h/mL, respectively.
310
Volumetric adsorptive microsampling-liquid chromatography tandem mass spectrometry assay
for the simultaneous quantification of four antibiotics in human blood: Method development,
validation and comparison with dried blood spot
Sebastiano Barco, Elio Castagnola, Andrea Moscatelli, James Rudge, Giuliana Cangemi
ABSTRACT
Summary: In this paper we show the development and validation of a volumetric absorptive
microsampling (VAMS™)-LC–MS/MS method for the simultaneous quantification of four antibiotics:
piperacillin-tazobactam, meropenem, linezolid and ceftazidime in 10 μL human blood. The novel
VAMS-LC–MS/MS method has been compared with a dried blood spot (DBS)-based method in terms
of impact of hematocrit (HCT) on accuracy, reproducibility, recovery and matrix effect. Antibiotics
were extracted from VAMS and DBS by protein precipitation with methanol after a re-hydration step
at 37 °C for 10 min. LC–MS/MS was carried out on a Thermo Scientific™ TSQ Quantum™ Access
MAX triple quadrupole coupled to an Accela ™UHPLC system. The VAMS-LC–MS/MS method is
selective, precise and reproducible. In contrast to DBS, it allows an accurate quantification without any
HCT influence. It has been applied to samples derived from pediatric patients under therapy. VAMS is
a valid alternative sampling strategy for the quantification of antibiotics and is valuable in support of
clinical PK/PD studies and consequently therapeutic drug monitoring (TDM) in pediatrics.
PLGA Ethionamide Nanoparticles for Pulmonary Delivery: Development and in vivo evaluation
of dry powder inhaler
Sujit Kumar Debnath, Srinivasan Saisivam, Abdelwahab Omri
ABSTRACT
PLGA (50:50) nanoparticles were prepared to sustain the release of Ethionamide in order to decrease
the dose and dosing frequency. It further modified in the form of dry powder inhaler to make suitable
for pulmonary administration and increase drug residency in lungs. Ethionamide loaded PLGA
nanoparticles were prepared by solvent evaporation method. Freeze dried nanoparticles and anhydrous
inhalable grade lactose were mixed manually using geometrical dilution process to modify the
nanoparticles in the form of dry powder inhaler. Animal study was conducted to correlate between in-
vivo and in-vitro. PLGA nanoparticles showed initial burst release followed by zero order release up to
95.17 ± 3.59% in 24 h. Aerodynamic particle size of optimized dry powder inhaler was found as 1.79
μm. There was no significant aggregation of dry powder inhaler during 6 months of stability study.
Area under the concentration-time curve from 0 h to infinity (AUC0−∞) signifies the prolong
residency of ETH in body compartment, revealed from animal study. PLGA 50:50 coated
nanoparticles released Ethionamide for the period of 24 h in simulated lungs fluid. Correlation
between in-vitro dissolution and in-vivo study was established after performing animal study. Prepared
dry powder inhaler maintained Ethionamide concentration above minimum inhibitory concentration
for more than 12 h after single dose administration.
311
Simultaneous determination and pharmacokinetics of danshensu, protocatechuic aldehyde, 4-
hydroxy-3-methyloxyphenyl lactic acid and protocatechuic acid in human plasma by LC–
MS/MS after oral administration of Compound Danshen Dripping Pills
Wei Li, Hongjie Zhou, Yang Chu, Xiangyang Wang, He Sun
ABSTRACT
Compound Danshen Dripping Pills (CDDP), a herbal patent medicine, is widely used in China for the
prevention and treatment of cardiovascular diseases. A simple, sensitive and reliable method for
simultaneous determination of danshensu (DSS), protocatechuic aldehyde (PCA), and their related
metabolites, 4-hydroxy-3-methyloxyphenyl lactic acid (HMLA) and protocatechuic acid (PAA) in
human plasma was developed and validated based on liquid chromatography tandem mass
spectrometry (LC–MS/MS). The analytes and internal standard (IS), vanillic acid (VAA), were
extracted from plasma with ethyl acetate and separated on a C18 column by using the mobile phase
consisted of methanol-0.1% formic acid via gradient elution. The electrospray ionization (ESI) source
was applied and operated under the multiple reaction monitoring (MRM) mode. The linear calibration
curves were obtained at the concentration ranges of 0.46–1000 ng/mL for DSS and PAA, and 1.38–
1000 ng/mL for PCA and HMLA, respectively. The inter- and intra-day precisions (RSD%) were less
than 13.5%, and the accuracy (±RE%) was within 13.4%. The described method was successfully
applied for the clinical pharmacokinetics of CDDP in Chinese healthy volunteers.
LC–MS bioanalysis of Trastuzumab and released emtansine using nano-surface and molecular-
orientation limited (nSMOL) proteolysis and liquid–liquid partition in plasma of Trastuzumab
emtansine-treated breast cancer patients
Noriko Iwamoto, Akihiko Shimomura, Kenji Tamura, Akinobu Hamada, Takashi Shimada
ABSTRACT
Antibody-drug conjugates (ADCs) consist of monoclonal antibody and cytotoxic drugs covalently
attached via stable crosslinkers, and are prospective antibody drugs for cancer therapy. To cover the
overall pharmacokinetic understanding of ADCs, both the antibody and the released drugs are
necessary for practical clinical observation. The nano-surface and molecular-orientation limited
(nSMOL) proteolysis is a universal approach for antibody bioanalysis that enable Fab-selective
proteolysis, which maintains antibody sequence specificity while decreasing excess analyte peptides.
In this study, we describe quantitative assays for ADC in human plasma using nSMOL for the
antibody and polarity-selective liquid–liquid partition with a methanol/ethyl acetate mixed solvent for
the cytotoxic drugs. This approach led to the successful development of LC–MS validated bioanalysis
of the antibody and released drugs within 20% for lower limit of quantitation and 15% for another
concentration setting of Trastuzumab emtansine (T-DM1), Trastuzumab antibody and emtansine
conjugated with crosslinker (DM1-MCC). The validated concentration ranges in human plasma were
0.06–250 μg/mL for T-DM1 and 0.39–200 ng/mL for DM1-MCC. These results indicate that LC–MS
method with a two-sided approach, using nSMOL and liquid–liquid partition, show potential for the
precise pharmacokinetic study for ADC development and treatment.
312
Separation of furostanol saponins by supercritical fluid chromatography
Jie Yang, Lingling Zhu, Yang Zhao, Yongwei Xu, Baiping Ma
ABSTRACT
Supercritical fluid chromatography (SFC) has good separation efficiency and is suitable for separating
weakly polar compounds. Furostanol saponins, as an important kind of steroidal saponins, generally
have two sugar chains, which are polar and hydrophilic. The hydroxyl group at the C-22 position of
furostanol saponins is active and easily reacts with lower alcohols under appropriate conditions. The
separation of hydrophilic furostanol saponins was tested by SFC in this study. The effects of
chromatographic conditions on the separation of the mixed furostanol saponins and their hydroxyl
derivatives at the C-22 position were studied. The conditions for SFC, which included different
column polarity, modifier, additive, and column temperature, were tested. After optimization, the
mixed 10 similar structures of furostanol saponins were separated in 22 min on the Diol column at a
temperature of 40 °C. The mobile phase was CO2 (mobile phase A) and methanol (containing 0.2%
NH3∙H2O and 3% H2O) (mobile phase B). The backpressure was maintained isobarically at 11.03
MPa. SFC was found to be effective in separating the furostanol saponins that shared the same
aglycone but varied in sugar chains. SFC was sensitive to the number and type of sugars. The
resolution of furostanol saponin isomers was not ideal. The extract of Dioscorea zingiberensis C. H.
Wright was profiled by SFC–quadrupole time-of-flight mass spectrometry. The main saponins of the
extract were well separated. Therefore, SFC could be used for separating hydrophilic furostanol
saponins and analyzing traditional Chinese medicines that mainly contained steroidal saponins.
Macroporous monoliths for biodegradation study of polymer particles considered as drug
delivery systems
M.V. Volokitina, V.A. Korzhikov-Vlakh, T.B. Tennikova, E.G. Korzhikova-Vlakh
ABSTRACT
Nanostructures based on biodegradable polymers are often considered as drug delivery systems. The
properties of these nanomaterails towards in vitro biodegradation are very important and usually are
studied using the model physiological conditions. In this work the novel approach based on application
of monolithic immobilized enzyme reactors (IMERs) as the systems for biodegradation study of the
nanoobjects of different nature and morphology was suggested. Rigid nanospheres based on
poly(lactic acid) and self-assembled nanoobjects formed from block-copolymer of glutamic acid and
phenylalanine were applied as model nanomaterials. For that, two enzymes, namely, esterase and
papain were chosen for preparation of the monolithic IMERs. The properties of immobilized enzymes
were compared to those obtained for soluble biocatalysts in the reaction of poly(lactic acid) and
poly(glutamic acid) degradation. The monitoring of substrate destruction process was carried out using
different HPLC modes (anion-exchange, cation-exchange or precipitation-redissolution based process)
also based on application of the same modern stationary phase, namely, macroporous monoliths (CIM
disks and lab-made column). Finally, the applicability of monolithic immobilized enzyme reactors for
degradation of polyester and polypetide-based particles was demonstrated and compared to the process
observed in human blood plasma.
313
A novel enantioseparation approach based on liposome electrokinetic capillary chromatography
Xiaoqi Li, Yingxiang Du, Zijie Feng, Xiaodong Sun, Zhifeng Huang
ABSTRACT
As a novel separation mode of capillary electrophoresis (CE), liposome electrokinetic capillary
chromatography (LEKC) has aroused considerable attention in recent years; however, the
enantioseparation based on this new system has not been previously investigated. In this study, we
proposed a brand-new LEKC chiral separation approach using liposomes comprised of
phosphatidylcholine (PC) and cholesterol as pseudo-stationary phase and sulfobutyl ether-β-
cyclodextrin (SBE-β-CD) as chiral selector. Compared with the single CD system and CD-SDS-
MEKC system, this LEKC method presented an obviously preferable enantioseparation of four model
drugs (naproxen, warfarin, ketoprofen and amlodipine). In this new established system, all the
enantiomers represented baseline separations with the resolution and selectivity respectively achieving
1.584/1.067 (for naproxen), 2.226/1.045 (for warfarin), 1.537/1.038 (for ketoprofen) and 2.592/1.097
(for amlodipine), while other two comparative systems demonstrated no separation or a poor
separation. Several important parameters affecting the enantioseparation, such as buffer pH,
concentration of liposomes, phosphate buffer solution (PBS) and chiral selector (SBE-β-CD), and
applied voltage were systematically investigated. Satisfactory repeatability was achieved through intra-
day, inter-day and batch-to-batch investigations with relative standard deviations less than 3.40%.
Furthermore, the established method was successfully applied to test the chiral impurity of naproxen
sample.
Comparative study on the anticancer activities and binding properties of a hetero metal
binuclear complex [Co(dipic)2Ni(OH2)5]·2H2O (dipic = dipicolinate) with two carrier proteins
Somaye Shahraki, Fereshteh Shiri, Mostafa Heidari Majd, Zohreh Razmara
ABSTRACT
Recognizing of binding mechanisms between drugs and carrier proteins is basic for us to understand
the pharmacokinetics and pharmacodynamics of them. In this research, the anticancer activities of a
binuclear complex [Co(dipic)2Ni(OH2)5]·2H2O (dipic = dipicolinate) against MDA-MB-231 cell
lines were studied. Results of MTT assay and flow cytometry analysis revealed that above complex
can induce the cytotoxicity and the apoptosis in breast cancer cell lines. So, this complex was selected
to investigate its binding to human serum albumin (HSA) and bovine β-lactoglobulin (βLG) by
spectroscopic methods (UV–visible, fluorescence and FT-IR) along with molecular docking technique.
The fluorescence data showed Co-Ni complex quench the fluorescence of both proteins by a static
quenching mechanism and HSA has stronger binding affinity toward Co-Ni complex than βLG. The
binding constant (Kb), number of binding sites (n) and thermodynamic parameters were calculated and
showed that the Co-Ni complex binds to protein (HSA and βLG) through hydrogen bonding and van
der Waals forces with one binding site. The results of UV–visible measurements indicated that the
binding of above complex to HSA and βLG may induce conformational and micro-environmental
changes of studied proteins. Protein–ligand docking analysis confirmed that the Co-Ni complex binds
to residues located in the subdomain IIA of HSA and site II of βLG.
314
Characterization and quantitative analysis of phenolic derivatives in Longxuetongluo Capsule by
HPLC-DAD-IT-TOF-MS
Jing Sun, Yuelin Song, Hui Sun, Wenjing Liu, Jun Li
ABSTRACT
Longxuetongluo Capsule (LTC), which is derived from the total phenolic extract of Chinese dragon‘s
blood, has been proved to be safe as well as effective towards ischemic stroke. However, the effective
material basis remains unclear. The present study thereby focused on the clarification of the qualitative
and quantitative properties for the phenolic derivatives in LTC. Regarding homolog-focused chemical
profiling, the mass fragmentation patterns of the primary subtypes of phenolic compounds such as
homoisoflavanones, flavanes, chalcones, and flavonoid oligomers were summarized by assaying
authentic references with hybrid ion trap time-of-flight mass spectrometry, and the chemical structures
of 124 phenolic compounds, in total, were unambiguously or tentatively annotated in LTC by
matching the accurate mass spectral profiles with the proposed mass cracking rules and those reference
substances. Afterwards, simultaneous determination of 12 primary phenolic compounds was carried
out in different batches of LTC using HPLC-DAD, after that the method was proved to be accurate,
precise, and reproducible according to diverse method validation assays. The obtained findings are
expected to be meaningful for clarifying the effective substances and quality assessment of LTC.
Volume 146 November 2017
Analytical characterization of human milk oligosaccharides – potential applications in
pharmaceutical analysis
Márkó Grabarics, Orsolya Csernák, Réka Balogh, Szabolcs Béni
ABSTRACT
Human breast milk is the gold standard for infant feeding and the best possible nourishment a new-
born could have. Breastfeeding is the natural way to provide optimal nutritional, immunological and
emotional nurturing for the healthy growth and development of infants. Human milk is a complex and
dynamic biofluid comprised of many hundreds to thousands of distinct bioactive structures, among
which one of the most abundant substances are the non-conjugated complex carbohydrates referred to
as human milk oligosaccharides (HMOs). Due to their structural diversity and abundance, HMOs
possess many beneficial biological functions. In order to understand human milk composition and
HMO functions, state-of-the-art glycomic methods are inevitable. The industrial, large scale
chemoenzymatic production of the most abundant HMOs became a reality in the last years and it
evokes the need for straightforward and genuine analytical procedures to monitor the synthetic process
and the quality of the products. It is obvious, that HMOs represent the next breakthrough in infant
nutrition, as the addition of HMOs (such as 2′-fucosyllactose or lacto-N-neotetraose) to infant- and
follow-on formulas, processed cereal-based food and baby foods for infants and young children etc.
will revolutionize this field. This review highlights the potential applications of HMOs in the (bio)
pharmaceutical industry, also summarizes the analytical methods available for the characterization of
HMOs. An overview of the structure and function of HMOs along with their determination methods in
complex matrices are provided. Various separation methods including liquid- and gas chromatography
and capillary electrophoresis for the characterization and novel approaches for the quantitation of
HMOs are discussed.
315
Analysis of macrolide antibiotics in water by magnetic solid-phase extraction and liquid
chromatography–tandem mass spectrometry
Rosa Ana Pérez, Beatriz Albero, Macarena Férriz, José Luis Tadeo
ABSTRACT
Macrolides are one of the most commonly used families of antibiotics employed in human and
veterinary treatment. These compounds are considered emerging contaminants with potential
ecological and human health risks that could be present in surface water. This paper describes the
development and application of a simple and efficient extraction procedure for the determination of
tilmicosin; erythromycin, tylosin and erythromycin-H2O from water samples. Sample extraction was
carried out using magnetic solid-phase extraction using oleate functionalized magnetic nanoparticles
followed by LC–MS/MS analysis. The effects of several parameters on the extraction efficiency of
MLs from water were evaluated. The recovery results obtained were >84% for most of the compounds,
except for erytromycin. The LOD and LOQ values ranged from 11.5 to 26 ng L−1 and from 34 to 77
ng L−1, respectively. The selected method was applied to monitor these contaminants in water samples
from different sources. Tilmicosin and tylosin were not detected in any of the samples, but
erythromycin and erythromycin-H2O were found in 50% of the surface water samples at levels from
<LOQ to 264 ng L−1 and 149 ng L−1, respectively.
Second harmonic generation microscopy as a tool for the early detection of crystallization in
spray dried dispersions
Clara Correa-Soto, Niraj S. Trasi, Paul D. Schmitt, Yongchao Su, Lynne S. Taylor
ABSTRACT
Various techniques have been used to detect crystallization in amorphous solid dispersions (ASD).
However, most of these techniques do not enable the detection of very low levels of crystallinity
(<1%). The aim of the current study was to compare the sensitivity of second harmonic generation
(SHG) microscopy with powder X-ray diffraction (XRPD) in detecting the presence of crystals in low
drug loading amorphous solid dispersions. Amorphous solid dispersions of the poorly water soluble
compounds, flutamide (FTM, 15 wt.% drug loading) and ezetimibe (EZT, 30 wt.% drug loading) with
hydroxypropyl methylcellulose acetate succinate (HPMCAS) were prepared by spray drying. To
induce crystallization, samples were subsequently stored at 75% or 82% relative humidity (RH) and 40
°C. Crystallization was monitored by XRPD and by SHG microscopy. Solid state nuclear magnetic
resonance spectroscopy (ssNMR) was used to further investigate crystallinity in selected samples. For
flutamide, crystals were detected by SHG microscopy after 8 days of storage at 40 °C/82% RH,
whereas no evidence of crystallinity could be observed by XRPD until 26 days. Correspondingly, for
FTM samples stored at 40 °C/75% RH, crystals were detected after 11 days by SHG microscopy and
after 53 days by XRPD. The evolution of crystals, that is an increase in the number and size of
crystalline regions, with time could be readily monitored from the SHG images, and revealed the
formation of needle-shaped crystals. Further investigation with scanning electron microscopy indicated
an unexpected mechanism of crystallization, whereby flutamide crystals grew as needle-shaped
projections from the surface of the spray dried particles. Similarly, EZT crystals could be detected at
earlier time points (15 days) with SHG microscopy relative to with XRPD (60 days). Thus, SHG
microscopy was found to be a highly sensitive method for detecting and monitoring the evolution of
crystals formed from spray dried particles, providing much earlier detection of crystallinity than XRPD
under comparable run times.
316
Sensitive analysis of bioactive secondary metabolites in lichen species using liquid
chromatography–mass spectrometry
Syed Ghulam Musharraf, Fareeha Siddiqi, Arslan Ali, Vinitha Moolchand Thadhani
ABSTRACT
Lichens are a large group of valuable lower plants with unique features and diverse applications
worldwide such as in medicine, cosmetics, food, and textile industries. They are also well known for
their potential in observing climate and environmental monitoring. Their successful exploitations
require reliable analytical methods to check and maintain quality and efficacy of the products based on
them. This study focuses on the development of a sensitive and reliable quantification method for the
analysis of important depsides, depsidones, dibenzofuran and monocyclic phenols inseven known and
an unidentified lichen species. Multiple Reaction Monitoring (MRM) approach using UHPLC-QqQ-
MS instrument was employed for the development of the quantitative method. Both LC and MS
parameters were optimized to ensure maximum separation. High sensitivity, and selectivity. LODs and
LOQs were found to be in the range of 2.1–71.5 ng/mL and 6.3–212.9 ng/mL, respectively. The
accuracy (% bias) and precision (% RSD) were found to be <5% in most cases. Metabolites 1–9 were
found in the range of 0.5–41429 μg/g in the analysed lichen extracts. The analysis revealed that
metabolites 1, 2 and 3 are the predominant ones. This method can be used for the identification and
absolute quantification of secondary metabolites in lichen extracts, and herbal or consumer products
based upon them.
Semiautomated determination of neonicotinoids and characteristic metabolite in urine samples
using TurboFlow™ coupled to ultra high performance liquid chromatography coupled to
Orbitrap analyzer
Marina López-García, Roberto Romero-González, Marina Lacasaña, Antonia Garrido Frenich
ABSTRACT
A semiautomated method based on ultra-high performance liquid chromatography (UHPLC) coupled
to Orbitrap high resolution mass spectrometry has been developed for the determination of
neonicotinoids (imidacloprid, acetamiprid, clothianidin, dinotefuran, nitenpyram, thiacloprid and
thiamethoxam) and the metabolite acetamiprid-n-desmethyl in urine samples. Two automated methods
were tested (solid-phase extraction ―SPE‖ and turbulent flow chromatography ―TurboFlow™‖),
obtaining the best results when TurboFlow™ was applied. The total analysis time for the developed
method was 14 min. The optimized method was validated, obtaining suitable results for all validation
parameters. Recoveries ranged from 78% to 116% meanwhile repeatability and reproducibility were
evaluated obtaining values lower than 10% and 20% respectively (except for dinotefuran and
nitenpyram at 0.2 μg L−1). The limit of quantification (LOQ) for all compounds was established at 0.2
μg L−1. The proposed analytical methodology was applied to analyze the target compounds in thirty
six urine samples from pregnant women living in agricultural areas of Almería (Spain). Imidacloprid,
acetamiprid and acetamiprid-n-desmethyl were detected in some of the samples at concentrations
ranging from 0.23 to 1.57 μg L−1. Furthermore, dinotefuran was identified in two samples at trace
levels.
317
A new polymorph of ciprofloxacin saccharinate: Structural characterization and pharmaceutical
profile
Pawanpreet Singh, Renu Chadha
ABSTRACT
In this study, a new polymorph of ciprofloxacin saccharinate has been thoroughly evaluated with
respect to structural as well as biopharmaceutical properties. Preliminary characterization of the new
polymorph was performed by differential scanning calorimetry and thermogravimetric analysis, later
confirmed by Fourier transform infra-red spectroscopy. The crystal structure was determined from the
powder X-ray diffraction pattern using the direct-space algorithm. It lies in the triclinic P-1 space
group. It is having lattice parameters different from previously reported forms. The solid-state 13C
NMR data calculated from the crystal structure by exploiting density functional theory were found to
be in excellent agreement with corresponding experimental 13C NMR data, thus providing a robust
validation of the authenticity of the structure. The prepared polymorph shows enhanced aqueous
solubility and dissolution rate in contrast to previously reported polymorph. The new form
demonstrated improved oral bioavailability and inhibition of bacterial species.
Qualitative and quantitative measurement of cannabinoids in cannabis using modified
HPLC/DAD method
Bhupendra Patel, Daniel Wene, Zhihua (Tina) Fan
ABSTRACT
This study presents an accurate and high throughput method for the quantitative determination of
various cannabinoids in cannabis plant material using high pressure liquid chromatography (HPLC)
with a diode array detector (DAD). Sample extraction and chromatographic analysis conditions for the
measurement of cannabinoids in the complex cannabis plant material matrix were optimized. The
Agilent Poroshell 120 SB-C18 column provided high resolution for all target analytes with a short run
time (10 minutes) given the core shell technology. The aqueous buffer mobile phase was optimized
with ammonium acetate at pH 4.75. The change in the mobile phase and the new column ensured a
separation between cannabidiol (CBD and cannabigerol (CBG) along with cannabigerol and
tetrahydrocannabinolic acid (THCA), which were not well separated by previous publications,
improved buffering capacity, and provided analytical performance stability. Moreover, baseline
drifting was significantly minimized by the use of a low concentration buffer solution (25 mM
ammonium acetate). In addition, evaporation and reconstitution of the sample residue with a methanol-
organic pure (OP) water solution (65:35) significantly reduced the matrix interference. The modified
extraction produced good recoveries (>91%) for each of the eight cannabinoids. The optimized method
was validated for specificity, linearity, sensitivity, precision, accuracy, and stability. The combined
relative standard deviation (%RSD) for intra-day and inter-day precision for all eight analytes varied
from 2.5% to 5.2% and 0.28% to 5.5%, respectively. The %RSD for the repeatability study varied
from 1.1% to 5.5%. The recoveries from spiked cannabis matrix samples were greater than 90% for all
analytes, except delta-8-tetrahydrocannabinol (Δ8-THC), which was 80%. The recoveries varied from
81% to 107% with a precision of 0.7-8.1%RSD. Delta-9-tetrahydrocannabinol (Δ9-THC) in all of the
cannabis samples (n = 635) was less than 10%, which is in compliance with the NJ Medicinal
Marijuana regulation.
318
Quantification of gabapentin polymorphs in gabapentin/excipient mixtures using solid state 13C
NMR spectroscopy and X-ray powder diffraction
Radaduen Tinmanee, Sarah C. Larsen, Kenneth R. Morris, Lee E. Kirsch
ABSTRACT
Gabapentin was used as a model pharmaceutical compound with susceptibility to polymorphic
transformation as a function of environmental and mechanical stress. The utility of 13C CP/MAS
NMR and XRPD as stability-indicating methods to quantify polymorphic transformation kinetics was
investigated. Polymorphic Form II and III were distinguishable based on their chemical shift and
distinct diffraction peak differences. Reproducible and accurate quantification of polymorphic
composition in the presence of selected excipients was demonstrated using both signals from 13C
CP/MAS NMR spectra and XRPD patterns. The effect of excipients on polymorphic transformations
(Form II → III) was determined by measuring the transformation after co-milling. Both 13C CP/MAS
NMR and XRPD were capable of measuring polymorphic composition in co-milled excipient mixtures
without excipient peak interference. The amounts of Form III present in co-milled mixtures containing
colloidal silicon dioxide, starch, hydroxy propyl cellulose and dibasic calcium phosphate were 8.7, 21,
33, and 39 mol%, respectively. A quenching procedure for obtaining 13C CP/MAS NMR spectra and
environmentally-controlled XRPD were devised to determine polymorphic transformation kinetics of
co-milled excipient mixtures during storage.
Accurate recognition and feature qualify for flavonoid extracts from Liang-wai Gan Cao by
liquid chromatography-high resolution-mass spectrometry and computational MS/MS
fragmentation
Min He, Hai Wu, Juan Nie, Pan Yan, Rui Pei
ABSTRACT
In this study, Liquid Chromatography (LC) separation combined with quadrupole-Time-Of-Flight
Mass Spectrometry (qTOF-MS) detection was used to analyze the characteristic ions of the flavonoids
from Liang-wai Gan Cao (Radix Glycyrrhizae uralensis). First, accurate mass measurement and
isotope curve optimization could provide reliable molecular prediction after noise deduction, baseline
calibration and ―ghost peak recognition‖. Thus, some spectral features in the LC–MS data could be
clearly explained. Secondly, the chemical structure of flavonoids was deduced by MS/MS fragment
ions, and the in-silico spectra by MS-FINDER program provided strong support for overcoming the
bottleneck of phytochemical identification. For a predicted formula and experimental MS/MS
spectrum, the MS-FINDER program could sort the candidate compounds in the public database based
on a comprehensive weighted score, and we took the first 20 reliable compounds to seek the target
compound in an in-house database. Certainly, those fragmentation pathways could also be deduced
and described as Retro-Diels-Alder (RDA) fragmentation reaction, losses of C4H8, C5H8, CH3, CO,
CO2 and others. Accordingly, 63 flavonoids were identified, and their in-silico bioactivity were clearly
disclosed by some bioinformatics tools. In this experiment, the flavonoids obtained by the four
extraction processes were tested by LC-qTOF-MS. We looked for possible Q-markers from these data
matrices and then quantified them; their similarities/differences were also described. The results also
indicated that the Macroporous Adsorption Resins (MARs) purification is a low cost, environmentally
friendly and effective approach.
319
Sample preparation composite and replicate strategy case studies for assay of solid oral drug
products
Beverly Nickerson, Brent Harrington, Fasheng Li, Michele Xuemei Guo
ABSTRACT
Drug product assay is one of several tests required for new drug products to ensure the quality of the
product at release and throughout the life cycle of the product. Drug product assay testing is typically
performed by preparing a composite sample of multiple dosage units to obtain an assay value
representative of the batch. In some cases replicate composite samples may be prepared and the
reportable assay value is the average value of all the replicates. In previously published work by
Harrington et al. (2014) [5], a sample preparation composite and replicate strategy for assay was
developed to provide a systematic approach which accounts for variability due to the analytical method
and dosage form with a standard error of the potency assay criteria based on compendia and regulatory
requirements. In this work, this sample preparation composite and replicate strategy for assay is
applied to several case studies to demonstrate the utility of this approach and its application at various
stages of pharmaceutical drug product development.
Separation and characterization of chemical constituents in Ginkgo biloba extract by off-line
hydrophilic interaction × reversed-phase two-dimensional liquid chromatography coupled with
quadrupole-time of flight mass spectrometry
Shuai Ji, Dan-dan He, Tian-yun Wang, Jie Han, Dao-quan Tang
ABSTRACT
Ginkgo biloba extract (GBE), derived from the leaves of Ginkgo biloba L., is one of the most widely
used traditional Chinese medicines worldwide. Due to high structural diversity and low abundance of
chemical constituents in GBE, conventional reversed-phase liquid chromatography has limited power
to meet the needs of its quality control. In this study, an off-line hydrophilic interaction × reversed-
phase two-dimensional liquid chromatography (HILIC × RP 2D-LC) system coupled with diode array
detection (DAD) and quadrupole time-of-flight mass spectrometry (qTOF-MS) was established to
comprehensively analyze the chemical constituents of GBE. After optimizing the chromatographic
columns and mobile phase of 2D-LC, a Waters XBridge Amide column using
acetonitrile/water/formic acid as the mobile phase was selected as the first dimension to fractionate
GBE, and the obtained fractions were further separated on an Agilent Zorbax XDB-C18 column with
methanol/water/formic acid as the mobile phase. As a result, a total of 125 compounds were detected
in GBE. The orthogonality of the 2D-LC system was 69.5%, and the practical peak capacity was 3864
and 2994, respectively, calculated by two different methods. The structures of 104 compounds were
tentatively characterized by qTOF-MS analysis, and 21 of them were further confirmed by comparing
with reference standards. This established HILIC × RP 2D-LC-qTOF/MS system can greatly improve
the separation and characterization of natural products in GBE or other complicated herbal extracts.
320
Double-Track Electrochemical Green Approach for Simultaneous Dissolution Profiling of
Naproxen Sodium and Diphenhydramine Hydrochloride
Mostafa A. Shehata, Esraa M. Fawaz, Mohamed K.Abd El-Rahman, Ezzat M. Abdel-Moety
ABSTRACT
Acquisition of the dissolution profiles of more than single active ingredient in a multi-analyte
pharmaceutical formulation is a mandatory manufacturing practice that is dominated by utilization of
the off-line separation-based chromatographic methods. This contribution adopts a new ―Double-
Track‖ approach with the ultimate goal of advancing the in-line potentiometric sensors to their most
effective applicability for simultaneous acquisition of the dissolution profiles of two active ingredients
in a binary pharmaceutical formulation. The unique abilities of these sensors for real-time
measurements is the key driver for adoption of ―green analytical chemistry‖ (GAC) principles aiming
to expand the application of eco-friendly analytical methods With the aim of performing a side-by-side
comparison, this work investigates the degree of adherence of ISEs to the 12 principles of GAC in
multicomponent dissolution profiling with respect to the HPLC. For the proof of concept, a binary
mixture of naproxen sodium (NAPR) and diphenhydramine hydrochloride (DIPH) marketed as Aleve
pm® tablets was selected as a model for which dissolution profiles were attained by two techniques.
The first ―Double-Track‖ in-line strategy depends on dipping two highly integrated membrane sensors
for continuous monitoring of the dissolution of each active pharmaceutical ingredient (API) by tracing
the e.m.f change over the time scale. For the determination of NAPR, sensor I was developed using
tridodecyl methyl ammonium chloride as an anion exchanger, while sensor II was developed for the
determination of DIPH using potassium tetrakis (4-chlorophenyl) borate as a cation exchanger. The
second off-line strategy utilizes a separation-based HPLC method via off-line tracking the increase of
peak area by UV detection at 220 nm over time using a mobile phase of acetonitrile: water (90:10) pH
3. The advantages of the newly introduced ―Double-Track‖ approach regarding GAC principles are
highlighted, and the merits of these benign real-time analyzers (ISEs) that can deliver equivalent
analytical results as HPLC while significantly reducing solvent consumption/waste generation are
described.
A workflow for column interchangeability in liquid chromatography using modeling software
and quality-by-design principles
Róbert Kormány, Katalin Tamás, Davy Guillarme, Szabolcs Fekete
ABSTRACT
The goal of the present study was to develop a generic workflow to evaluate the chromatographic
resolution in a large design space and easily find some replacement column for the method. To attain
this objective from a limited number of initial experiments, modern LC modeling software (Drylab)
was employed to study the behaviour of the compounds and visually compare the parts of design
spaces obtained with different columns, where a given criterion of critical resolution is fullfilled. A
zone of robust space can then easily be found by overlapping design spaces. By using 50 × 2.1 mm
columns packed with sub–2 μm fully porous particles (UHPLC), the resolution in the entire design
space can be modeled on the basis of only 2–3 h experimental work per column. To demonstrate the
applicability of the developed procedure, amlodipine and its related pharmacopeia impurities were
selected as a case study. It was demonstrated that two columns from different providers (Waters
Acquity HSS C18, Thermo Hypersil Gold C18) can be interchanged, providing a sufficient resolution
at the same working point and a high degree of robustness around this condition.
321
Absolute quantification of poly(dl-lactide-co-glycolide) in microspheres using quantitative 1H
NMR spectroscopy
Qi Zhang, Ningzi Guo, Yue Sun, Xiaodong Li, Huaxin Yang
ABSTRACT
The complex nature and the manufacturing process of poly(dl-lactide-co-glycolide) (PLGA), a key
component of PLGA-based microspheres, have made the quantification of this copolymer difficult.
The main purpose of the current study was to investigate the potential of three different methods for
the quantitative analysis of the PLGA content of clinical products. In this regard, leuprorelin acetate
microspheres from different vendors were chosen as templates to validate quantitative 1H nuclear
magnetic resonance (qHNMR) spectroscopy, size exclusion chromatography (SEC), and high-
performance liquid chromatography (HPLC) methods qHNMR proved to be an excellent and rapid
PLGA quantification method compared to the other two. The recovery value was 99.12% and the
linearity correlation coefficient was 0.9999. The results obtained from the qHNMR method were found
to match the data provided by the vendor, suggesting that qHNMR can be utilized as a reliable quality
control and inspection tool for PLGA-based clinical products.
A novel LC-MS/MS method for the quantitative measurement of the acetate content in
pharmaceutical peptides
Rani J. Qasem, Ibrahim K. Farh, Mohammed A. Al Essa
ABSTRACT
Most pharmaceutical peptides are supplied as acetate salts and the relative amount of acetate to peptide
in the final product is one quality criterion required by regulatory agencies to approve the product for
clinical use. The objective of the present study was to develop a validated LC-MS/MS method that
allows the quantitative determination of the acetate content in pharmaceutical peptide preparations and
simultaneous monitoring and collection of qualitative and quantitative information on the peptide
during manufacture and in the final product. The method uses reversed phase C18-chromatography to
elute the acetate ions under acidic conditions, pH 3, followed by post-column infusion of ammonium
hydroxide 0.6 M, methanolic solution (30:70) at a rate of 0.5 mL/hr. The acetate ions were monitored
in negative polarity mass spectrometry by pseudo multiple reaction monitoring (pseudo MRM) against
1, 2- 13C labelled acetate, the internal standard used in the method. The method was linear for acetate
concentrations between 0.4 and 25 μg/mL with a coefficient of determination (r2) equal to 0.9999. The
minimum level of detection and minimum level of quantification were at 0.06 μg/mL and 0.18 μg/mL
respectively. Accuracy of the method was judged by determining the acetate content in a commercial
product of the peptide pharmaceutical tetracosactide (TCS) and parallel comparison to the amounts
determined by a reversed phase HPLC method with detection at a wavelength of 210 nm. The amounts
determined by the two methods were in agreement with a RSD that was less than 2%. Additional
confirmation of method accuracy was determined by spiking the pharmaceutical peptide with varying
amounts of acetate. The recoveries ranged on average between 101 and 102% for the spiked amounts.
Accuracy was also determined by calculating the percentage relative error of the predicted to actual
acetate concentration in quality controls and was determined to be less than 5%. The LC-MS/MS
method was precise with an intra- and inter-day RSD of less than 5%. The standard solutions were
stable for at least one month when kept frozen at −80 °C with no loss in response and an inter-day
RSD of less than 5%.
322
A quality by design-based approach to a capillary electrokinetic assay for the determination of
dextromepromazine and levomepromazine sulfoxide as impurities of levomepromazine
Stephan Niedermeier, Gerhard K.E. Scriba
ABSTRACT
Using a quality by design approach, a capillary electrophoresis method for the simultaneous
determination of dextromepromazine and the oxidation product levomepromazine sulfoxide in
levomepromazine was developed. The analytical target profile was defined that the method should be
able to quantify 0.1% of both impurities with a precision of ≤10%. Hydroxypropyl-γ-cyclodextrin was
used as chiral selector. The critical process parameters cyclodextrin concentration, buffer pH and
concentration as well as temperature and applied voltage were studied using a fractional factorial
resolution V+ design for defining the knowledge space. A central composite face centered design was
used as response surface methodology for deriving the design space by Monte Carlo simulations. The
selected working point was a 100 mM citric acid buffer, pH 2.85, containing 3.6 mg/mL
hydroxypropyl-γ-cyclodextrin, a temperature of 15 °C and a voltage of 25 kV. Robustness was
estimated using a Plackett-Burman design. The method was subsequently validated in the relative
concentration range of 0.1%–1.0% of the impurities for a solution containing 0.25 mg/mL
levomepromazine. The method was applied to the determination of the purity of the reference
substance of the European Pharmacopoeia and of the drug in a commercial injection solution.
An HPLC–MS/MS method for quantitation of Gly-MCA in mouse plasma: Application to a
pharmacokinetic study
Jia Liu, Guan Lian, Ting Wang, Yuanheng Ma, Yuxin Yin
ABSTRACT
A simple, sensitive and rapid method using high performance liquid chromatography-tandem mass
spectrometry (HPLC–MS/MS) was developed and validated for quantification of glycine-β-muricholic
acid (Gly-MCA) in mouse plasma for the first time. Plasma samples were pretreated with protein
precipitation. The analyte and internal standard were separated on a Shimadzu Shim-pack XR-ODS
column (4.6 × 50 mm, 2.2 μm) using 0.1% formic acid-water-methanol as mobile phase, with a
runtime of 5 min. Detection was performed using negative ion electrospray tandem mass spectrometry
via multiple reaction monitoring (MRM) scan mode. The linear range was 5 ng–2 μg/ml (correlation
coefficient >0.995) for Gly-MCA with a lower limit of quantitation of 5 ng/ml. The intra-day and
inter-day precision were less than 9.2% for the analyte and accuracy was from 0.4% to 7.0%. This
analytical method was then successfully applied to a pharmacokinetic study of Gly-MCA following
oral administration and intraperitoneal injection in mice.
323
Development and validation of a sensitive LC–MS/MS method without derivatization/ion-
pairing agents for etimicin quantification in rat plasma, internal ear and kidney
Lan Yao, Fang Zhou, Mingmin Cai, Ying Peng, Jingwei Zhang
ABSTRACT
Etimicin (ETM), which belongs to the newest generation of aminoglycosides (AGs), has been proven
to not only maintain but also strengthen the advantages of former AGs with relatively less toxicity.
Now, it is widely applied for the treatment of bacterial infections in the clinic. Nevertheless,
nephrotoxicity and ototoxicity are unavoidable issues for AGs, and while ETM is no exception, the
seriousness of these issues is different. To explore the reason why ETM exhibits less toxicity and to
better direct the optimization and development of new AGs, it is of great necessity and importance to
monitor the pharmacokinetic behaviors of ETM in its potential toxicity target organs, the kidney and
internal ear, as well as in plasma. Therefore, a novel, sensitive and efficient LC–MS/MS method
without derivatization or ion-pairing agents had been developed and validated for quantification of
ETM in rat plasma, kidney and internal ear for the first time. This method showed good linearity over
the range of 50–2000 ng/mL for rat plasma/internal ear and 100–5000 ng/mL for rat kidney. The
precision was less than 4.4% and the accuracy was below 4.8%. Recovery and matrix effects were
71.3%–82.8% and 97.6%–108.5%, respectively. After intravenous administration of a single dose of
ETM, plasma drug concentrations fit well with a two-compartmental model, and the AUC0-∞, t1/2α,
t1/2β, MRT and CL were 127.96 ± 5.52 μg *h/mL, 0.53 ± 0.03 h, 3.32 ± 1.11 h, 1.01 ± 0.03 h and
234.80 ± 10.05 mL/h/kg, respectively. Particularly, ETM showed a considerably long half-life in
kidney and internal ear, up to 155.96 ± 19.95 h and 83.11 ± 26.60 h, respectively, which might
contribute greatly to its toxicity.
Bioanalysis of monomethyl fumarate in human plasma by a sensitive and rapid LC–MS/MS
method and its pharmacokinetic application
Imam Pasha S, Murali Balaram Varanasi, Ibrahim Mohammed
ABSTRACT
Dimethyl fumarate (DMF) is the methyl ester of fumaric acid, after oral administration completely
converts to its active metabolite monomethyl fumarate (MMF). A simple, rapid and sensitive LC–
MS/MS method was developed and validated for the quantification of MMF in human plasma.
Monomethyl fumarate d3 was used as an internal standard (IS). The analyte and the IS were extracted
from plasma using a selective solid phase extraction technique. The clean samples were
chromatographed on a C18 column using formic acid and acetonitrile (25:75, v/v) as mobile phase. An
API-4000 LC–MS/MS system equipped with turbo ion spray (TIS) source and operated in multiple
reactions monitoring (MRM) mode was used for the study. The method was validated for linearity in
the range of 5.03-2006.92 ng/mL. Also, a number of stability tests were conducted to evaluate the
stability of analyte, IS in plasma samples and in neat samples, the results comply with recent
bioanalytical guidelines. A shortest run time helped us to analyze more than 300 samples in a day. The
method was applied to a pharmacokinetic study in ten healthy male Indian subjects and the study data
was authenticated by conducting incurred sample reanalysis (ISR).
324
Simultaneous determination of tenofovir alafenamide and its active metabolites tenofovir and
tenofovir diphosphate in HBV-infected hepatocyte with a sensitive LC–MS/MS method
Bingchen Ouyang, Fang Zhou, Le Zhen, Ying Peng, Jingwei Zhang
ABSTRACT
Tenofovir (TFV), a first-line anti-viral agent, has been prepared as various forms of prodrugs for better
bioavailability, lower systemic exposure and higher target cells loading of TFV to enhance efficacy
and reduce toxicity. TFV undergoes intracellular phosphorylation to form TFV diphosphate (TFV-DP)
in target cell to inhibit viral DNA replication. Hence, TFV-DP is the key active metabolite that exhibits
anti-virus activity, its intracellular exposure and half-life determine the final activity. Therefore,
simultaneous monitoring prodrug, TFV and TFV-DP in target cells will comprehensively evaluate
TFV prodrugs, both considering the stability of ester prodrug, and the intracellular exposure of TFV-
DP. Thus we intended to develop a convenient general analytical method, taking tenofovir alafenamide
(TAF) as a representative of TFV prodrugs. A sensitive LC–MS/MS method was developed, and TAF,
TFV and TFV-DP were separated on a XSelect HSS T3 column (4.6 mm × 150 mm, 3.5 μm, Waters)
with gradient elution after protein precipitation. The method provided good linearity for all the
compounds (2–500 nM for TFV and TAF; 20–5000 nM for TFV-DP) with the correlation coefficients
(r) greater than 0.999. Intra- and inter-day accuracies (in terms of relative error, RE < 10.4%) and
precisions (in terms of coefficient of variation, CV < 14.1%) satisfied the standard of validation. The
matrix effect, recovery and stability were also within acceptable criteria. Finally, we investigated the
intracellular pharmacokinetics of TAF and its active metabolites in HepG2.2.15 cells with this method.
LC–MS/MS method for preclinical pharmacokinetic study of QX-OH, a novel long-acting local
anesthetic, in sciatic nerve blockade in rats
YuJun Zhang, DeYing Gong, QingShan Zheng, Jin Liu, WenSheng Zhang
ABSTRACT
QX-OH, a new synthetic local anesthetic, produced concentration-dependent, reversible, and long-
acting local anesthesia in animal models, with moderate local toxicity. As part of preclinical research
for drug development, we developed and validated a method for the determination of QX-OH in the
plasma, muscle, and sciatic nerve using liquid chromatography–mass spectrometry. After a simple
protein precipitation procedure, analysis was performed on an Extend C18 column (100 mm × 3 mm,
3.5 μm) by isocratic elution with 0.05% formic acid/acetonitrile (78:22, v/v) at a flow rate of 0.3
mL/min. A multiple-reaction monitoring mode at the transitions of m/z 279.1 → 102.1 for QX-OH and
m/z 275.2 → 126.1 for an internal standard (ropivacaine hydrochloride) was used for the
quantification, with a positive electrospray ionization interface. The approach was validated as per the
U.S. Food and Drug Administration guidelines and successfully used in a pharmacokinetic study of
QX-OH after a sciatic nerve block with 0.2 mL of 35 mM QX-OH. The results demonstrated that the
new local anesthetic, QX-OH, had a high concentration in tissue, low systemic exposure, and long
duration in the sciatic nerve.
325
Capillary electrophoresis study on the base-catalyzed formation of bioactive oxidized metabolites
of 20-hydroxyecdysone
Halima Meriem Issaadi, Attila Hunyadi, Krisztina Németh
ABSTRACT
A novel capillary electrophoretic method was developed for the analysis and monitoring of the base-
catalyzed autoxidation of 20-hydroxyecdysone, a worldwide used non-hormonal anabolic food
supplement. An effective separation of the starting material and its bioactive oxidized derivatives was
achieved by using sulfobutyl-β-cyclodextrin as selector at pH 11 and by fixing the separation voltage
at +30 kV. Only a dilution step was inserted before injecting the sample, taken from the crude reaction
mixture, to the capillary electrophoresis instrument. The same alkaline pH was used for the analysis as
for the reaction, unlike the previously reported HPLC study where sample neutralization was required
prior to the measurement. Due to the very short analysis time (6 min) in capillary electrophoresis, more
frequent sampling and more detailed time scale analysis could be carried out. Furthermore, in contrast
with the preceding HPLC results, the previously unobserved calonysterone could also be detected by
capillary electrophoresis as a minor primary product. Our novel method demonstrated higher
resolution than the one before. Baseline separation could be achieved and the resolution values were in
the range of 1.9–7.0. The limit of detection was below 71 μg/ml, the relative standard deviation values
of the migration time and peak area for intra- and inter-day precision were less than 10%. The more
precise, direct monitoring of the time dependency of the oxidation process is expected to have a
significant impact on yield optimization initiatives to allow related pharmacological studies in the near
future.
Quantification of the antimalarial drug pyronaridine in whole blood using LC–MS/MS —
Increased sensitivity resulting from reduced non-specific binding
Daniel Blessborn, Karnrawee Kaewkhao, Lijiang Song, Nicholas J. White, Joel Tarning
ABSTRACT
Malaria is one of the most important parasitic diseases of man. The development of drug resistance in
malaria parasites is an inevitable consequence of their widespread and often unregulated use. There is
an urgent need for new and effective drugs. Pyronaridine is a known antimalarial drug that has
received renewed interest as a partner drug in artemisinin-based combination therapy. To study its
pharmacokinetic properties, particularly in field settings, it is necessary to develop and validate a
robust, highly sensitive and accurate bioanalytical method for drug measurements in biological
samples. We have developed a sensitive quantification method that covers a wide range of clinically
relevant concentrations (1.5 ng/mL to 882 ng/mL) using a relatively low volume sample of 100 μL of
whole blood. Total run time is 5 min and precision is within ±15% at all concentration levels.
Pyronaridine was extracted on a weak cation exchange solid-phase column (SPE) and separated on a
HALO RP amide fused-core column using a gradient mobile phase of acetonitrile–ammonium formate
and acetonitrile-methanol. Detection was performed using electrospray ionization and tandem mass
spectrometry (positive ion mode with selected reaction monitoring). The developed method is suitable
for implementation in high-throughput routine drug analysis, and was used to quantify pyronaridine
accurately for up to 42 days after a single oral dose in a drug-drug interaction study in healthy
volunteers.
326
Simultaneous extraction of propofol and propofol glucuronide from hair followed by validated
LC–MS/MS analyses
Alexandra Maas, Christoph Maier, Stefanie Iwersen-Bergmann, Burkhard Madea, Cornelius Hess
ABSTRACT
Besides its clinical application, the anaesthetic agent propofol is being increasingly misused, mostly by
healthcare professionals, and its abuse potential gained worldwide attention after the tragic death of
Michael Jackson in 2009. Due to the short duration of its narcotic effects, propofol abuse is especially
easy to hide compared with the use of other recreational drugs. However, propofol possesses a very
narrow therapeutic window between the desired effect and potentially fatal toxicity, making abuse of
the drug extremely dangerous even in experienced physicians. Consequently, it is important that
forensic laboratories possess a sensitive and specific method for the detection of chronic propofol
abuse. We present a simple, fast and reliable method to simultaneously extract propofol and its main
metabolite propofol glucuronide from hair, followed by sensitive LC–MS/MS analyses, allowing to
determine a chronic propofol abuse. Difficulties regarding the detection of propofol using LC–MS/MS
were solved by using a derivatization reaction with 2-fluoro-1-methylpyridinium-p-toluene-sulfonate
and triethylamine. Reliability of extraction method and subsequent LC–MS/MS analyses was
confirmed under consideration of the validation parameters selectivity, linearity, accuracy and
precision, analytical limits, processed sample stability, matrix effects and recovery. Appropriate
quantification (LLOQ = 10 pg/mg hair) and detection limits (3.6 pg/mg hair for propofol and 7.8
pg/mg hair for propofol glucuronide) could be achieved, enabling to detect even small amounts of both
analytes. Applicability of the method was confirmed by analysis of three human hair samples from
deceased with suspicion of chronic propofol abuse.
11-nor-9-carboxy-Δ9-tetrahydrocannabinol glucuronide exhibits acyl-migration isomers
Stephanie Hanisch, Alexander Paulke, Stefan W. Toennes
ABSTRACT
11-nor-Δ9-Tetrahydrocannabinol-9-carboxylic acid glucuronide (THCCOOH-glucuronide) is an 1-β-
O-acyl glucuronide which degrades not only to 11-nor-9-carboxy-Δ9-THC (THCCOOH) but,
additionally, to an isomer with a currently unknown structure. The present study was carried out to
examine whether acyl glucuronide isomers are formed by acyl migration and if they are involved in
formation of this isomer. THCCOOH-glucuronide was incubated in phosphate buffer (pH 7.4, 37 °C, 7
days) and a variety of glucuronide cleavage procedures were performed. Samples of the incubation
mixture and of different biological specimens from cannabis users were analyzed using liquid
chromatography-mass spectrometry (LC–MS/MS). A total of six chromatographically separated
isomeric acyl glucuronides were detected during incubation of THCCOOH-glucuronide reference
substance. In biological specimens of cannabis users, two additional isomers were found. However, the
main glucuronide present in human specimens was different from that of a commercially available
reference substance. Both, the commercial and the authentic glucuronide were cleaved by β-
glucuronidases, the other formed isomers by alkaline hydrolysis only. Mass spectrometric
investigations (i.e. product ion, precursor ion and neutral loss scans) confirmed identity. The
THCCOOH isomer was detected in all authentic samples, but not in those after buffer incubation. By
analyzing THCCOOH-glucuronide in authentic samples, it has to be taken into account that the
authentic glucuronide is different from that of the commercial reference standard.
327
Simple and rapid LC-MS/MS method for the absolute determination of cetuximab in human
serum using an immobilized trypsin
Kaito Shibata, Takafumi Naito, Jun Okamura, Seiji Hosokawa, Junichi Kawakami
ABSTRACT
Proteomic approaches using liquid chromatography coupled to tandem mass spectrometry (LC-
MS/MS) without an immunopurification technique have not been applied to the determination of
serum cetuximab. This study developed a simple and rapid LC-MS/MS method for the absolute
determination of cetuximab in human serum and applied it to clinical settings. Surrogate peptides
derived from cetuximab digests were selected using a Fourier transform mass spectrometer. Reduced-
alkylated serum cetuximab without immunopurification was digested for 20 minutes using
immobilized trypsin, and the digestion products were purified by solid-phase extraction. The LC-
MS/MS was run in positive ion multiple reaction monitoring mode. This method was applied to the
determination of serum samples in head and neck cancer patients treated with cetuximab. The
chromatographic run time was 10 minutes and no peaks interfering with surrogate peptides in serum
digestion products were observed. The calibration curve of absolute cetuximab in serum was linear
over the concentration range of 4–200 μg/mL. The lower limit of quantification of cetuximab in human
serum was 4 μg/mL. The intra-assay and inter-assay precision and accuracy were less than 13.2% and
88.0–100.7%, respectively. The serum concentration range of cetuximab was 19–140 μg/mL in
patients. The serum cetuximab concentrations in LC-MS/MS were correlated with those in ELISA (r =
0.899, P < 0.01) and the mean bias was 1.5% in cancer patients. In conclusion, the present simple and
rapid method with acceptable analytical performance can be helpful for evaluating the absolute
concentration of serum cetuximab in clinical settings.
Development of quantitative immunochromatographic assay for rapid and sensitive detection of
carbohydrate antigen 19-9 (CA 19-9) in human plasma
Kwaku Baryeh, Sunitha Takalkar, Michelle Lund, Guodong Liu
ABSTRACT
A quantitative immunochromatographic assay (QIA) was developed by using gold nanoparticle
(GNP)-based lateral flow strip biosensor (LFSB) and a portable strip reader for rapid and sensitive
quantitation of Carbohydrate Antigen 19-9 (CA 19-9) in human plasma. CA 19-9 is a biomarker that
has been associated with cancers (such as pancreatic and colorectal cancers) and various non-
cancerous diseases. The principle is based on sandwich-type immunoreactions between gold
nanoparticle (GNP)-labelled detection antibody, anti-CA 19-9 capture antibody and CA 19-9to capture
the GNPs on the test zone of LFSB. The accumulation of GNPs on the test zone gave a red line whose
intensity was read with a portable strip reader to quantify the concentration of CA 19-9. Assay
parameters including the membrane type, antibody concentration, amount of GNP-anti-CA 19-9
conjugates and the components of the running buffer were optimized to obtain the best sensitivity and
reproducibility of the assay. The detection limit of the assay was determined to be 5 U mL−1 (S/N = 3)
with a linear range of 5 U mL−1–100 U mL−1. CA 19-9 concentrations in healthy human and
pancreatic cancer patient plasma samples were successfully evaluated using the developed quantitative
immunochromatographic assay (QIA), and the results were in accordance with that obtained with
enzyme linked immunosorbent assay (ELISA). The developed assay shows great promise for clinical
application and biomedical diagnosis, particularly in limited resource settings.
328
Simultaneous determination of pentoxifylline, metabolites M1 (lisofylline), M4 and M5, and
caffeine in plasma and dried blood spots for pharmacokinetic studies in preterm infants and
neonates
Madhu Page-Sharp, Tobias Strunk, Sam Salman, Julie Hibbert, Kevin T. Batty
ABSTRACT
Advances in bioanalytical methods are facilitating micro-volume and dried blood spot (DBS) analysis
of drugs in biological matrices for pharmacokinetic studies in children and neonates. We sought to
develop a UPLC–MS/MS assay for simultaneous measurement of caffeine, pentoxifylline (PTX) and
three metabolites of PTX in both plasma and DBS. Caffeine, PTX, the metabolites M1 (lisofylline),
M4 and M5, and the internal standards (caffeine-d9 and PTX-d6) were separated using a Waters
Aquity T3 UPLC C18 column and gradient mobile phase (water-methanol-formic acid). Retention
times for caffeine, M5, M4, PTX and M1 were 1.6, 1.7, 1.9, 2.0 and 2.1 min, respectively, with a run
time of 5 min. The precision (≤10%) and accuracy (≤15%) across the concentration range 0.1–50 mg/L
for caffeine, PTX and the three metabolites in plasma and DBS were within accepted limits, as were
the limits of quantification (100 μg/L for caffeine and 10 μg/L for PTX, M1, M4 and M5). Caffeine,
PTX and the metabolites were stable in DBS for >34 days at room and refrigerated temperatures.
Plasma and DBS samples were obtained from 24 preterm infants recruited into a clinical
pharmacokinetic study of PTX.
Pharmacokinetics and tissue distribution of five major triterpenoids after oral administration of
Rhizoma Alismatis extract to rats using ultra high-performance liquid chromatography–tandem
mass spectrometry
Wen Xu, Xiaoyan Li, Na Lin, Xue Zhang, Shuisheng Wu
ABSTRACT
Rhizoma Alismatis (RA) was wildly used for treatment of dysuria, pyelonephritis, hyperlipidemia,
enteritis diarrhea, diabetes, inflammation, and cancer. Triterpenoids are the major active components
of RA, and its extract is mainly composed of alisol A (ALA), alisol B (ALB), alisol C 23-acetate
(ALC-23A), alisol A 24-acetate (ALA-24A), and alisol B 23-acetate (ALB-23A). In this study, a
simple, reliable, and sensitive ultra high-performance liquid chromatography with triple quadrupole
mass spectrometry (UHPLC–MS/MS) method was created and validated for the quantification of the
five major triterpenoids in rat plasma and various tissues biosamples (including intestine, stomach,
liver, kidney, fat, muscle, brain, heart, lung, spleen, and testes). The plasma and tissues biosamples
were pretreated by direct precipitation deproteinization method with acetonitrile. 17α-
Hydroxyprogesterone was used as internal standard (IS). The chromatography was performed on a
Phenomenex C8 column (30 × 2.00 mm, 1.8 μm) at room temperature with gradient elution.
Compounds were quantified by selected multi-reactions monitoring (SRM) scanning with positive
electric spray ionization mode. The linearity of detection for each triterpene was respectively from 1 to
1000 ng/mL for ALC-23A and ALA, from 4 to 4000 ng/mL for ALA-24A, from 10 to 10,000 ng/mL
for ALB, and from 2 to 2000 ng/mL for ALB-23B (r > 0.99) with low quantification limits of 1–10
ng/mL for all analytes. All of the other validation parameters were also in an acceptable range. The
validated UHPLC–MS/MS method subsequently applied for the pharmacokinetic and tissue
distribution studies of RA extract. After orally given 100 mg/kg of RA extract, ALA was the most
exposed component, followed by ALB and ALA-24A.
329
Solid-phase microextraction coupled to gas chromatography–mass spectrometry followed by
multivariate data analysis for the identification of volatile organic compounds as possible
biomarkers in lung cancer tissues
Federica Bianchi, Nicolò Riboni, Paolo Carbognani, Letizia Gnetti, Maria Careri
ABSTRACT
Solid-phase microextraction and gas chromatography–mass spectrometry followed by multivariate
data analysis were used to analyze lung tissues from both healthy and carcinogenic patients. A total of
78 volatile compounds belonging to different chemical classes were identified, seven of which were
able to discriminate between the two groups. Discriminant analysis allowed to correctly classify 98.3%
of the cases. By using the leave-one-method, 100% of the cross-validated samples belonging to the
―tumor‖ group was correctly classified, whereas 2 cross-validated healthy samples out of 48 were
erroneously allocated in the ―tumor‖ group. Achieved results suggest the need of further investigation
to assess the role of the seven identified compounds as lung cancer biomarkers in breath analysis, thus
allowing the development of low-cost diagnostic devices.
UPLC–MS/MS assay of riluzole in human plasma and cerebrospinal fluid (CSF): Application in
samples from spinal cord injured patients
Mahua Sarkar, Robert G. Grossman, Elizabeth G. Toups, Diana S.-L. Chow
ABSTRACT
In the present study, a sensitive and robust LC–MS/MS method has been developed and validated for
the quantification of riluzole in human plasma and cerebrospinal fluid (CSF) in clinical samples from
patients with spinal cord injury (SCI). Riluzole and its labeled internal standard (IS) were isolated from
plasma and CSF by liquid–liquid extraction using ethyl acetate. Riluzole (m/z 235 → 166) and IS (m/z
238 → 169) were detected by electrospray ionization (ESI) using multiple reaction monitoring (MRM)
in a positive mode. The assay was linear in the concentration range of 0.5 (LLOQ, signal/noise ratio >
10)–800 ng/ml in plasma, and 1.0 (LLOQ)–800 ng/ml in CSF samples. The intra- and inter-day
accuracy in plasma were 94.2–110.0% and 97.8–102.0%, respectively, and those in CSF were 87.6–
105.1% and 91.9–98.8%, respectively. The intra- and inter-day precision were 2.2–7.2% and 4.0–
9.1%, respectively, in plasma, and 1.4–14.1% and 2.6–11.5%, respectively in CSF. Matrix effect was
negligible from both matrices with signal percentages of 97.6–100.6% in plasma and 99.4–106.4% in
CSF. The recoveries were >75% in plasma, >84% in CSF with low protein (53.9 mg/dl), and >68% in
CSF with high protein (348.2 mg/dl). This method was successfully applied to quantify riluzole
concentrations in plasma and CSF from patients with SCI.
330
Simultaneous determination and pharmacokinetic study of twelve bioactive compounds in rat
plasma after intravenous administration of Xuebijing injection by UHPLC-Q-Orbitrap HRMS
Lihua Zuo, Qiuyue Zhong, Zhenhui Wang, Zhi Sun, Xiaojian Zhang
ABSTRACT
Xuebijing injection (XBJ) is a traditional Chinese herbal prescription widely used in the treatment of
sepsis. Extensive studies revealed that the major bioactive constituents of XBJ injection, including
phenolic acids, flavonoids, monoterpene glycosides, lactones and organic acids, play an important role
in the treatment. In this study, a rapid, sensitive and accurate ultra high performance liquid
chromatography – Q Exactive hybrid quadrupole – orbitrap high-resolution accurate mass
spectrometry (UHPLC-Q-Orbitrap HRMS) method was developed for simultaneous determination of
twelve bioactive compounds in rat plasma after intravenous administration of XBJ injection. A
gradient elution for separation was achieved on a waters ACQUITY UHPLC® BEH C18 column (2.1
mm × 50 mm, 1.7 μm) column with acetonitrile-water (containing 0.1% formic acid) as mobile phase
at a flow rate of 0.2 mL/min. All compounds and IS were monitored by parallel reaction monitoring
assay both in positive and negative ion mode. The developed method was validated for intra-day and
inter-day accuracy and precision whose values fell in the acceptable limits. Extraction recoveries at
three levels of QC concentrations were all more than 80% for all compounds and IS, and matrix effects
were found in the range of 80.0–120.0%. Stability results showed that all analytes were stable at the
investigated conditions. The validated method was successfully applied to a pharmacokinetic study of
Xuebijing injection following intravenous administration of 2.5, 5.0, 10.0 mL/kg to rats respectively.
And the result indicated that the pharmacokinetic behavior of XBJ injection is positively related to
dosage at the range of 2.5–10 mg/kg. This study will provide a meaningful basis for evaluating the
rationality of XBJ injection for clinical application.
A surrogate analyte-based liquid chromatography-tandem mass spectrometry method for the
determination of endogenous cyclic nucleotides in rat brain
Jie Chen, Ali Tabatabaei, Doug Zook, Yan Wang, Kathe Stauber
ABSTRACT
A robust high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS) assay was
developed and qualified for the measurement of cyclic nucleotides (cNTs) in rat brain tissue. Stable
isotopically labeled 3′,5′-cyclic adenosine-13C5 monophosphate (13C5-cAMP) and 3′,5′-cyclic
guanosine-13C,15N2 monophosphate (13C15N2-cGMP) were used as surrogate analytes to measure
endogenous 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate
(cGMP). Pre-weighed frozen rat brain samples were rapidly homogenized in 0.4 M perchloric acid at a
ratio of 1:4 (w/v). Following internal standard addition and dilution, the resulting extracts were
analyzed using negative ion mode electrospray ionization LC–MS/MS. The calibration curves for both
analytes ranged from 5 to 2000 ng/g and showed excellent linearity (r2 > 0.996). Relative surrogate
analyte-to-analyte LC–MS/MS responses were determined to correct concentrations derived from the
surrogate curves. The intra-run precision (CV%) for 13C5-cAMP and 13C15N2-cGMP was below
6.6% and 7.4%, respectively, while the inter-run precision (CV%) was 8.5% and 5.8%, respectively.
The intra-run accuracy (Dev%) for 13C5-cAMP and 13C15N2-cGMP was <11.9% and 10.3%,
respectively, and the inter-run Dev% was <6.8% and 5.5%, respectively. Qualification experiments
demonstrated high analyte recoveries, minimal matrix effects and low autosampler carryover.
331
Determination of ketamine and its main metabolites by liquid chromatography coupled to
tandem mass spectrometry in pig plasma: Comparison of extraction methods
Cindy Ramiole, Benoit D‘Hayer, Vincent Boudy, Josette Legagneux, Pascal Houzé
ABSTRACT
A rapid, sensitive and specific liquid chromatography coupled to tandem mass spectrometry method
was developed for the simultaneous quantification pig plasma of ketamine and its two principal
metabolites, norketamine and dehydronorketamine. Three extraction procoles were assessed including
acetonitrile precipitation, Oase™ microplate extraction, and liquid–liquid extraction. Oase™
microplate extraction induced no significant matrix effect, important signal/noise ratio and good
recoveries, ranging from 82 to 87% for the considered compounds. Using this extraction procedure,
the assay was linear in the dynamic range 10–3000 ng/mL (R2 > 0.99) regardless of the analytes. Intra-
and inter-day accuracies were less than 12% for all compounds and intra- and inter-day precisions
expressed as RSD were within <9.9%. Samples were stable in different storage conditions. High
ketamine, norketamine and dehydronorketamine concentrations up to 15,000 ng/mL can be determined
with good precision using appropriate sample dilution. The assay was successfully applied to pig
plasma samples to determine the pharmacokinetics of ketamine and the consecutive metabolites after
buccal administration of a 4 mg/kg ketamine base solutions.
Ketamine metabolites with antidepressant effects: Fast, economical, and eco-friendly
enantioselective separation based on supercritical-fluid chromatography (SFC) and single
quadrupole MS detection
Georg M. Fassauer, Robert Hofstetter, Mahmoud Hasan, Stefan Oswald, Andreas Link
ABSTRACT
Increasing evidence accumulates that metabolites of the dissociative anesthetic ketamine contribute
considerably to the biological effects of this drug and could be developed as next generation
antidepressants, especially for acute treatment of patients with therapy-refractory major depression.
Analytical methods for the simultaneous determination of the plethora of hydroxylated,
dehydrogenated and/or demethylated compounds formed after administration of ketamine
hydrochloride are a prerequisite for future clinical investigations and a deeper understanding of the
individual role of the isomers of these metabolites. In this study, we present development and
validation of a method based on supercritical-fluid chromatography (SFC) coupled to single
quadrupole MS detection that allows the separation of ketamine as well as all of its relevant
metabolites detected in urine of healthy volunteers. Inherently to SFC methods, the run times of the
novel protocol are four times shorter than in a comparable HPLC method, the use of organic solvents
is reduced and we were able to demonstrate and validate the successful enantioselective separation and
quantification of R- and S-ketamine, R- and S-norketamine, R- and S-dehydronorketamine and
(2R,6R)- and (2S,6S)-hydroxynorketamine isomers differing in either constitution, stereochemistry, or
both, in one run. The developed method may be useful in investigating the antidepressant efficacy of
ketamine in clinical trials.
332
A new method based on supercritical fluid extraction for polyacetylenes and polyenes from
Echinacea pallida (Nutt.) Nutt. roots
Massimo Tacchini, Antonella Spagnoletti, Virginia Brighenti, Francesco Pio Prencipe, Federica Pellati
ABSTRACT
The genus Echinacea (Asteraceae) includes species traditionally used in phytotherapy. Among them,
Echinacea pallida (Nutt.) Nutt. root extracts are characterized by a representative antiproliferative
activity, due to the presence of acetylenic compounds. In this study, supercritical fluid extraction
(SFE) was applied and compared with conventional Soxhlet extraction (SE) in order to obtain a
bioactive extract highly rich in polyacetylenes and polyenes from E. pallida roots. The composition of
the extracts was monitored by means of HPLC–UV/DAD and HPLC–ESI–MSn by using an Ascentis
Express C18 column (150 mm × 3.0 mm I.D., 2.7 μm, Supelco, Bellefonte, PA, USA) with a mobile
phase composed of (A) water and (B) acetonitrile, under gradient elution. By keeping SFE time at the
threshold of 1 h (15 min static and 45 min dynamic for 1 cycle) with the oven temperature set at 40–45
°C and 90 bar of pressure, an overall extraction yield of 1.18–1.21% (w/w) was obtained, with a high
selectivity for not oxidized lipophilic compounds. The biological activity of the extracts was evaluated
against human non-small lung A549 and breast carcinoma MCF-7 cancer cell lines. The cytotoxic
effect of the SFE extract was more pronounced towards the MCF-7 than the A549 cancer cells, with
IC50 values ranging from 21.01 ± 2.89 to 31.11 ± 2.l4 μg/mL; cell viability was affected mainly
between 24 and 48 h of exposure. The results show the possibility of a new ―green‖ approach to obtain
extracts highly rich in genuine polyacetylenes and polyenes from E. pallida roots. The bioactivity
evaluation confirmed the cytotoxicity of E. pallida extracts against the considered cancer cell lines,
especially against MCF-7 cells, thus suggesting to represent a valuable tool for applicative purposes in
cancer prevention.
Validated LC–MS/MS method for the simultaneous determination of rotigotine and its prodrug
in rat plasma and an application to pharmacokinetics and biological conversion in vitro
Chunjie Sha, Jiangbin Han, Fengjuan Zhao, Xin Shao, Youxin Li
ABSTRACT
Rotigotine behenate (RGTB), a long chain alkyl ester of the prodrug of rotigotine (RGT), has been
synthesized for use in a sustained delivery system. The aim of the present report was to develop and
validate a simple, sensitive and reliable LC–MS/MS method for the simultaneous determination of
RGT and its prodrug RGTB in rat plasma samples. Detection was performed on a 1290 Infinity UPLC
coupled Triple Quad 4500 mass spectrometer operated in positive MRM mode using an Eclipse XDB-
CN chromatography column (2.1 mm × 100 mm, 3.5 μm) by isocratic elution using a 0.2% formic acid
aqueous solution and acetonitrile, with stable isotope labeled RGT as an internal standard. The sample
preparation method employed 50 μL of a plasma sample and liquid-liquid extraction with a mixture of
diethyl ether–dichloromethane (3:2, v/v) as the extraction solvent. The proposed method was fully
validated by assessing its specificity, linearity, precision and accuracy, recovery, matrix effects and
stability. Good linearity was found within the range of 0.1–10.0 ng/mL for both analytes (r > 0.996).
This method was successfully applied to a pharmacokinetic study of a slow release RGTB formulation
in rats following a single intramuscular injection and biological conversion in vitro.
333
Determination of higenamine in dietary supplements by UHPLC/MS/MS method
A. Stajić, M. AnĎelković, N. Dikić, J. Rańić, B. Jančić-Stojanović
ABSTRACT
From 1st January 2017 higenamine was added on the WADA (World Anti-doping Agency) Prohibited
list under S3 group beta-2 agonists as at all times banned substance for the athletes. The main origine
of higenamine (or norcoclaurine) are different plants including Nandina domestica, Aconitum
carmichaelii, Asarum heterotropioides, Galium divaricatum, Annona squamosa, Nelumbo nucifera etc.
Higenamine main use is related to weight loss and it could be found (un)labeled in different dietary
supplements. The objective of this study was development of sensitive and reliable UHPLC/MS/MS
method for determination of higenamine in various dietary supplement samples. In order to obtain high
method sensitivity, hydrophilic interaction liquid chromatography (HILIC) mode was applied.
Separation was carried out on UHPLC Acquity BEH HILIC analytical column (2.1 mm × 100 mm, 1.7
μm particle size). Mobile phase consisted of 0.1% formic acid in water and acetonitrile, respectively,
was mixed in ratio of 30:70, v/v. Flow rate was set at 0.2 mL min−1. Quercetin was used as an internal
standard. ESI (+) source ionization mode using multi reaction monitoring (MRM) mode was utilized
and three ion transitions of higenamine were followed 272.08 → 107.01, 272.08 → 161.07 and 272.08
→ 77.08. Developed method was fully validated and applied for identification and quantification of
higenamine in different dietary supplements. According to the results, the most of investigated
supplements were free of higenamine, and on the other hand, presence of higenamine was confirmed
in some samples while it was not declared on the label.
Determination of genotoxic epoxide at trace level in drug substance by direct injection GC/MS
Liqin Chen, Wei Zhang, Steven Hu
ABSTRACT
A novel direct injection gas chromatography method coupled with selective ion monitoring mass
spectrometry (GC/SIM-MS) was developed for quantitation of trace levels of high boiling point (HBP)
epoxide genotoxic impurity (GTI) in drug substance. The injector temperature was optimized with the
aims to minimize matrix effects and enhance SIM signal response. The final injector temperature 160
°C was selected after balancing between these two factors. The column screening was conducted as
well and MN OPTIMA delta-3 silica capillary column was selected since it showed good peak
symmetry without column bleeding. The good linearity was established for the concentration in the
range from 0.0045 μg/mL to 0.5 μg/mL with a R2 = 0.9999. The limit of detection (LOD) and the limit
of quantitation (LOQ) were 0.0014 μg/mL and 0.0045 μg/mL, respectively. The recovery which
ranged from 95.0% to 112.5% could meet the ICH acceptance criteria. The validation results
demonstrated the good linearity, precision and accuracy of the method which can be further adopted as
an adequate quality control tool for quantitation of epoxide impurity at trace levels in drug substance
and drug product.
334
LC–MS/MS assay for the quantitation of the ribonucleotide reductase inhibitor triapine in
human plasma
Julia Matsumoto, Brian F. Kiesel, Robert A. Parise, Jianxia Guo, Jan H. Beumer
ABSTRACT
The ribonucleotide reductase inhibitor and radiosensitizer triapine (3-aminopyridine-2-carboxaldehyde
thiosemicarbazone (3-AP), NSC 663249) is clinically being evaluated via the intravenous (IV) route
for the treatment of cervical and vulvar cancer in combination with primary cisplatin chemoradiation.
The need for a 2-h infusion and frequent administration of triapine is logistically challenging,
prompting us to pursue oral (PO) administration. In support of the clinical trial investigating oral
triapine in combination with chemoradiation, we developed and validated a novel LC–MS/MS assay
for the quantification of triapine in 50 μL human plasma. After protein precipitation, chromatographic
separation of the supernatant was achieved with a Shodex ODP2 column and an isocratic acetonitrile-
water mobile phase with 10% ammonium acetate. Detection with an ABI 4000 mass spectrometer
utilized electrospray positive mode ionization. The assay was linear from 3 to 3,000 ng/mL and proved
to be accurate (97.1–103.1%) and precise (<7.4% CV), and met the U.S. FDA guidance for
bioanalytical method validation. This LC–MS/MS assay will be an essential tool to further define the
pharmacokinetics and oral bioavailability of triapine.
Development and validation of a liquid chromatography-tandem mass spectrometry method for
pharmacokinetic study of TM-53, a novel transcriptional coactivator with PDZ-binding motif
(TAZ) modulator
Ha-Yeon Lee, Nak Jeong Kim, Yong Moon Lee, Jin Sook Song, Sunjoo Ahn
ABSTRACT
Transcriptional coactivator with PDZ-binding motif (TAZ) is considered an attractive target for
osteoporosis, obesity, and muscle regeneration. TM-53, a promising TAZ modulator, was recently
introduced, and here, we developed a rapid, precise, and reliable analytical method for TM-53 and
characterized its pharmacokinetic properties in rat plasma. The hybrid triple quadrupole/linear ion trap
coupled to liquid chromatography method was developed and validated to quantify TM-53.
Additionally, TM-53 concentrations in plasma were analyzed, and its pharmacokinetic parameters
were calculated by non-compartmental analysis. Multiple reaction monitoring at m/z 569.4 → 207.1
showed the most sensitive signals for TM-53, and the linear scope of the standard curve was between
1.5 ng/mL and 500 ng/mL. The intra- and inter-day precisions of the quality control samples were
<15%, and their accuracies were ranged from 86.2% to 111.0%. Furthermore, the matrix effects,
extraction recoveries, and process efficiencies of this analytical method for evaluating TM-53 in rat
plasma were 99.1%, 99.9%, and 99.1% respectively. In short- and long-term stability studies, TM-53
showed good stability under frozen conditions, but TM-53 hydrolysis in the plasma matrix was
observed following storage at room temperature. This analytical method was successfully applied for
pharmacokinetic analysis of TM-53 in rat plasma and demonstrated excellent sensitivity, selectivity,
precision, and accuracy. These data indicated that this method can be applied for further preclinical
studies of TM-53.
335
Development of a sensitive LC–MS/MS method for quantification of coniferyl ferulate and its
metabolite coniferyl alcohol in rat plasma: Application to a pharmacokinetic study
Xinlun Dai, Li Pang, Zhen Zhang, Chunfeng Yang, Yumei Li
ABSTRACT
A rapid and simple LC–MS/MS method was developed and validated for the simultaneous
determination of coniferyl ferulate (CF) and its metabolite coniferyl alcohol (CA) using bavachromene
as an internal standard (IS). A TSQ Quantum Access mass spectrometer was operated under selected-
reaction monitoring mode using negative electrospray ionization. Extraction with ether was used in
sample preparation. The plasma samples were prepared and then chromatographed on a Phenomenex
Luna C18 column (2.1 mm × 50 mm, 1.7 μm; Torrance, USA) at 35 °C, using acetonitrile: water
(65:35, v/v) in an isocratic mode at a flow rate of 0.3 mL/min. Method validation was performed as per
the FDA guidelines and calibration curves showed good linearity over the concentration range of 2.5–
1000 ng/mL for both CF and CA. The intra- and inter-day precision and accuracy were within the
acceptable limits. The developed assay was successfully applied to a pharmacokinetic study of CA in
rats.
LC–MS/MS assay for the quantitation of the ATR kinase inhibitor VX-970 in human plasma
Brian F. Kiesel, Jonas Scemama, Robert A. Parise, Liza Villaruz, Jan H. Beumer
ABSTRACT
DNA damaging chemotherapy and radiation are widely used standard-of-care modalities for the
treatment of cancer. Nevertheless, the outcome for many patients remains poor and this may be
attributed, at least in part, to highly effective DNA repair mechanisms. Ataxia-telangiectasia mutated
and Rad3-related (ATR) is a key regulator of the DNA-damage response (DDR) that orchestrates the
repair of damaged replication forks. ATR is a serine/threonine protein kinase and ATR kinase
inhibitors potentiate chemotherapy and radiation. The ATR kinase inhibitor VX-970 (NSC 780162) is
in clinical development in combination with primary cytotoxic agents and as a monotherapy for tumors
harboring specific mutations. We have developed and validated an LC–MS/MS assay for the sensitive,
accurate and precise quantitation of VX-970 in human plasma. A dilute-and-shoot method was used to
precipitate proteins followed by chromatographic separation with a Phenomenex Polar-RP 80 Å (4 μm,
50 × 2 mm) column and a gradient acetonitrile-water mobile phase containing 0.1% formic acid from a
50 μL sample volume. Detection was achieved using an API 4000 mass spectrometer using
electrospray positive ionization mode. The assay was linear from 3 to 5,000 ng/mL, proved to be
accurate (94.6–104.2%) and precise (<8.4% CV), and fulfilled criteria from the FDA guidance for
bioanalytical method validation. This LC–MS/MS assay will be a crucial tool in defining the clinical
pharmacokinetics and pharmacology of VX-970 as it progresses through clinical development.
336
Quantitative determination of carfilzomib in mouse plasma by liquid chromatography–tandem
mass spectrometry and its application to a pharmacokinetic study
Jee Sun Min, Jiseon Kim, Jung Ho Kim, Doyun Kim, Soo Kyung Bae
ABSTRACT
A highly sensitive and rapid LC–MS/MS method was developed and validated to determine the levels
of carfilzomib in mice plasma by using chlorpropamide as an internal standard. Carfilzomib and
chlorpropamide were extracted from 5 μL of plasma after protein precipitation with acetonitrile.
Chromatographic separation was performed on Phenomenex Luna C18 column (50 × 2.0 mm id, 3
μm). The mobile phase consisted of 0.1% formic acid in acetonitrile −0.1% formic acid in water (1:1
v/v) and the flow rate was 0.3 mL/min. The total chromatographic run time was 2.5 min. Detection
was performed on a triple quadrupole mass spectrometer equipped with positive-ion electrospray
ionization by selected reaction monitoring of the transitions at m/z 720.20 > 100.15 (for carfilzomib)
and m/z 277.05 > 111.05 (for the internal standard). The lower limit of quantification was 0.075 ng/mL
and the linear range was 0.075–1250 ng/mL (r ≥ 0.9974). All validation data, including selectivity,
precision, accuracy, matrix effect, recovery, dilution integrity, stability, and incurred sample
reanalysis, were well within acceptance limits. This newly developed bioanalytical method was
simple, highly sensitive, required only a small volume of plasma, and was suitable for application in
pharmacokinetic studies in mice that used serial blood sampling.
Development and implementation of a pass/fail field-friendly method for detecting sildenafil in
suspect pharmaceutical tablets using a handheld Raman spectrometer and silver colloids
Adam Lanzarotta, Lisa Lorenz, JaCinta S. Batson, Cheryl Flurer
ABSTRACT
A simple, fast, sensitive and effective pass/fail field-friendly method has been developed for detecting
sildenafil in suspect Viagra and unapproved tablets using handheld Raman spectrometers and silver
colloids. The method involves dissolving a portion of a tablet in water followed by filtration and
addition of silver colloids, resulting in a solution that can be measured directly through a glass vial.
Over one hundred counterfeit Viagra and unapproved tablets were examined on three different devices
during the method development phase of the study. While the pass/fail approach was found to be
92.6% effective on average, the efficacy increased to 97.4% on average when coupled with the
software‘s ―Discover Mode‖ feature that allows the user to compare a suspect spectrum to that of a
stored sildenafil spectrum. The lowest concentration of sildenafil in a water/colloid solution that
yielded a ―Pass‖ was found to be 7.6 μg/mL or 7.6 parts per million (ppm). For the analysis of suspect
tablets, this value was found to be as low as 10 μg/mL and as high as 625 μg/mL. This variability was
likely related to the tablet formulation, e.g., concentration of sildenafil, presence and concentration of
water-soluble and/or water-insoluble ingredients. However, since most counterfeit Viagra and
unapproved tablets contain >50 mg sildenafil per tablet, such low concentrations will not be
encountered often. Limited in-lab and in-field validation studies were conducted in which
analysts/field agents followed the procedure outlined in this study for small sample sets. These
individuals were provided with written instructions, a ∼20 min demonstration regarding how to
perform the procedure and use the instrument, and a kit with field-friendly supplies (purified bottled
water from a local grocery store, disposable plastic pipettes, eye-dropper with a silver colloid solution,
etc.). The method proved to be 98.3% and 91.7% effective for the in-lab and in-field validation studies,
respectively, which demonstrated the ruggedness, simplicity and practicality of the method.
337
Multi-residue determination of 47 organic compounds in water, soil, sediment and fish—Turia
River as case study
Eric Carmona, Vicente Andreu, Yolanda Picó
ABSTRACT
A sensitive and reliable method based on solid-liquid extraction (SLE) using McIlvaine-Na2EDTA
buffer (pH = 4.5)-methanol and solid-phase extraction (SPE) clean up prior to ultra-high-performance
liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS) was applied to
determine 47 organic contaminants in fish, soil and sediments. The SPE procedure to clean-up the
extracts was also used as extraction method to determine these compounds in water. Recoveries ranged
from 38 to 104% for all matrices with RSDs < 30%. Limits of Quantification for the target compounds
were in the range of 10–50 ng/g for soil, 2–40 ng/g for sediment, 5–30 ng/g for fish and 0.3–26 ng/L
for water. Furthermore, the proposed method was compared to QuEChERS (widely used for
environmental matrices) that involves extraction with buffered acetonitrile (pH 5.5) and dispersive
SPE clean-up. The results obtained (recoveries>50% for 36 compounds in front of 9, matrix effect <
20% for 31 compounds against 21, and LOQs <25 ng g−1 for 38 compounds against 22) indicates that
the proposed method is more efficient than QuEChERS, The method was applied to monitoring these
compounds along the Turia River. In river waters, Paracetamol (175 ng L−1), ibuprofen (153 ng L−1)
and bisphenol A (41 ng L−1) were the compounds most frequently detected while in sediments were
vildagliptin (7 ng g−1) and metoprolol (31 ng g−1) and in fish, bisphenol A (33 ng g−1) or
sulfamethoxazole (13 ng g−1).
Separation of bioactive chamazulene from chamomile extract using metal-organic framework
Reda M. Abdelhameed, Hassan Abdel-Gawad, Mohamed Taha, Bahira Hegazi
ABSTRACT
Isolation of bioactive compounds from extracts of pharmaceutical plant is very important. In this work,
copper benzene-1,3,5-tricarboxylate metal organic framework (Cu-BTC MOF) has been synthesized.
It is used in separating of chamazulene from chamomile extract. The Cu-BTC MOF not only shows
good chamazulene adsorption but also maintains good desorption properties. However, the research on
this field is still new and the maturation of novel MOFs or the enhancements of known ones are
required.The chamomile extract obtained after each stage of the treatments was carefully characterized
by thin-layer chromatography (TLC), Fourier-transform infrared spectroscopy (FTIR), UV–vis
spectrometry and gas chromatography-mass spectrometry (GC–MS). The morphology and the
crystallinity of Cu-BTC MOF were investigated using scanning electron microscopy (SEM) and
powder X-ray diffraction (PXRD), respectively. Breakthrough experiments in a column was
investigated and the data was fitted with Bohart-Adams model. Monte Carlo simulation was conducted
to investigate the preferential adsorption sites of Cu-BTC for chamazulene molecules.
338
1H NMR based pharmacometabolomics analysis of urine identifies metabolic phenotype of
clopidogrel high on treatment platelets reactivity in coronary artery disease patients
Arwa M. Amin, Lim Sheau Chin, Chin-Hoe Teh, Hamza Mostafa, Baharudin Ibrahim
ABSTRACT
Clopidogrel high on treatment platelets reactivity (HTPR) has burdened achieving optimum
therapeutic outcome. Although there are known genetic and non-genetic factors associated with
clopidogrel HTPR, which explain in part clopidogrel HTPR, yet, great portion remains unknown, often
hindering personalizing antiplatelet therapy. Nuclear magnetic resonance (1H NMR)
pharmacometabolomics analysis is useful technique to phenotype drug response. We investigated
using 1H NMR analysis to phenotype clopidogrel HTPR in urine. Urine samples were collected from
71 coronary artery disease (CAD) patients who were planned for interventional angiographic
procedure prior to taking 600 mg clopidogrel loading dose (LD) and 6 h post LD. Patients' platelets
function testing was assessed with the VerifyNow® P2Y12 assay at 6 h after LD. Urine samples were
analysed using 1H NMR. Multivariate statistical analysis was used to identify metabolites associated
with clopidogrel HTPR. In pre-dose samples, 16 metabolites were associated with clopidogrel HTPR.
However, 18 metabolites were associated with clopidogrel HTPR in post-dose samples. The pathway
analysis of the identified biomarkers reflected that multifactorial conditions are associated with
clopidogrel HTPR. It also revealed the implicated role of gut microbiota in clopidogrel HTPR.
Pharmacometabolomics not only discovered novel biomarkers of clopidogrel HTPR but also revealed
implicated pathways and conditions.
Permeability through the Caco-2 cell monolayer of 42 bioactive compounds in the TCM formula
Gegen-Qinlian Decoction by liquid chromatography tandem mass spectrometry analysis
Qi Wang, Yi Kuang, Wei Song, Yi Qian, Min Ye
ABSTRACT
Caco-2 cell monolayer model was used to evaluate the intestinal permeability of 42 bioactive
compounds in the famous traditional Chinese medicine (TCM) formula Gegen-Qinlian Decoction
(GQD). These compounds include alkaloids, flavonoids and glycosides, triterpenoid saponins, and
coumarins. Their transportations across the cell monolayers in the forms of herb extract and formula
extract were monitored by liquid chromatography coupled with tandem mass spectrometry
(LC/MS/MS) analysis. Most alkaloids from Huang-Lian; flavonoid C-glycosides from Ge-Gen and
Huang-Qin; O-glycosides from Ge-Gen, Huang-Qin and Gan-Cao; O-glucuronides from Huang-Qin;
and coumarins from Gan-Cao exhibited favorable permeability. Their PAB values were > 1.05 × 10−5
cm/s, and efflux ratios (ER, PBA/PAB) were ≤ 1.0. In contrast, triterpenoid saponins showed poor
permeability (PAB ≤ 1.50 × 10−6 cm/s, ER ≤ 1.5), indicating a paracellular diffusion mechanism.
Furthermore, GQD could remarkably improve the intestinal transport of alkaloids in Huang-Lian,
flavonoid C-glycosides in Ge-Gen, as well as coumarins and flavonoid O-glycosides in Gan-Cao.
These results indicate herb–herb interactions in GQD.
339
Simultaneous quantification of arginine, alanine, methionine and cysteine amino acids in
supplements using a novel bioelectro-nanosensor based on CdSe quantum dot/modified carbon
nanotube hollow fiber pencil graphite electrode via Taguchi method
Sara Hooshmand, Zarrin Es‘haghi
ABSTRACT
A number of four amino acids have been simultaneously determined at CdSe quantum dot-
modified/multi-walled carbon nanotube hollow fiber pencil graphite electrode in different
bodybuilding supplements. CdSe quantum dots were synthesized and applied to construct a modified
carbon nanotube hollow fiber pencil graphite electrode. FT-IR, TEM, XRD and EDAX methods were
applied for characterization of the synthesized CdSe QDs. The electro-oxidation of arginine (Arg),
alanine (Ala), methionine (Met) and cysteine (Cys) at the surface of the modified electrode was
studied. Then the Taguchi‘s method was applied using MINITAB 17 software to find out the optimum
conditions for the amino acids determination. Under the optimized conditions, the differential pulse
(DP) voltammetric peak currents of Arg, Ala, Met and Cys increased linearly with their concentrations
in the ranges of 0.287–33670 μM and detection limits of 0.081, 0.158, 0.094 and 0.116 μM were
obtained for them, respectively. Satisfactory results were achieved for calibration and validation sets.
The prepared modified electrode represents a very good resolution between the voltammetric peaks of
the four amino acids which makes it suitable for the detection of each in presence of others in real
samples.
Simultaneous optimization of pH and binary organic composition by grid form modeling of the
retention behavior in reversed-phase ultra high-performance liquid chromatography
Tsukasa Sasaki, Kenichiro Todoroki, Toshimasa Toyo‘oka
ABSTRACT
A novel computer-assisted methodology for the simultaneous optimization of aqueous pH and binary
organic eluent composition through a broad range of analytical conditions of reversed-phase ultra
high-performance liquid chromatography is proposed. Two of nonlinear prediction models were
employed to fit into the retention time (tR) on a linear gradient elution with a predefined slope. One
model was derived from Bernoulli-type probability distribution to predict the value of tR against the
pH value of the aqueous eluent. This sigmoid-shaped model was successfully fitted for tR value shift
in the presence of three levels of organic eluent compositions (volumetric mixing of
acetonitrile/methanol ratios 1:0, 1:1, and 0:1). The resultant pH versus tR value models were
subsequently combined into grid form by quadratic multiple regression models based on the solubility
parameter theory and their binary organic composition axes. The predicted tR values afforded from
grid models were highly accurate for 13 different acidic non-steroidal anti-inflammatory drugs [root
mean square error (RMSE) ≤0.030] and 16 basic histamine H1-receptor blockers (RMSE ≤0.067) in a
pH ranging from 2.5 to 9.0 and an acetonitrile/methanol volumetric mixing ratio ranging from 1:0 to
0:1. Each compatibility score was defined as the indicator of the peak separation. Scores were
calculated for all combinations of aqueous pH values and binary organic compositions via the
predicted tR values. A colored map generated from the calculated scores was greatly effective in
determining optimal combinations of both mobile phase conditions. By employing this predictive data,
all analytes in both acidic and basic sample mixtures were finally separated at their respective
optimized conditions.
340
On-Line two dimensional liquid chromatography based on skeleton type molecularly imprinted
column for selective determination of sulfonylurea additive in Chinese patent medicines or
functional foods
Pengqi Guo, Xinya Xu, Guoning Chen, Kamran Bashir, Qiang Fu
ABSTRACT
Substandard and counterfeit anti-diabetic medicines directly influence the health and impose a great
danger to individual patients and to public health. Counterfeiting has become a serious and
underreported problem in the pharmaceutical industry. There are a large number of counterfeit
medicines flooded in anti-diabetic markets which effect human health directly and indirectly.
Therefore, some novel analytical techniques are necessary to be established for detecting these
counterfeit drugs. In this study, a novel skeleton type molecularly imprinted column was successfully
prepared. Based on the column, a simple, fast and reliable two-dimensional chromatography analytical
system was established for selective determination of the illegal sulfonylurea additive in traditional
Chinese patent medicines and functional foods. The developed method was validated. The linearitiesof
the method were tested with calibration curves using ten calibration points in the concentration range
of 0.25–12.5 μg/g. The LODs were 0.0125 μg/g and 0.01 μg/g for tolbutamide and glibenclamide
respectively. The five batches of Chinese patent medicines and dietary supplements obtained from
different markets and online websites were tested by the validated method. With good retention time
and spectral confirmation, chemical anti-diabetic substances were identified and quantified in
traditional Chinese medicine and in dietary supplements.
Impact of chemotherapy on metabolic reprogramming: Characterization of the metabolic profile
of breast cancer MDA-MB-231 cells using 1H HR-MAS NMR spectroscopy
Roberta M. Maria, Wanessa F. Altei, Heloisa S. Selistre-de-Araujo, Luiz A. Colnago
ABSTRACT
Doxorubicin, cisplatin, and tamoxifen are part of many chemotherapeutic regimens. However, studies
investigating the effect of chemotherapy on the metabolism of breast cancer cells are still limited. We
used 1H high-resolution magic angle spinning (HR-MAS) NMR spectroscopy to study the metabolic
profile of human breast cancer MDA-MB-231 cells either untreated (control) or treated with
tamoxifen, cisplatin, and doxorubicin. 1H HR-MAS NMR single pulse spectra evidenced signals from
all mobile cell compounds, including fatty acids (membranes), water-soluble proteins, and metabolites.
NMR spectra showed that phosphocholine (i.e., a biomarker of breast cancer malignant
transformation) signals were stronger in control than in treated cells, but significantly decreased upon
treatment with tamoxifen/cisplatin. NMR spectra acquired with Carr-Purcell-Meiboom-Gill (CPMG)
pulse sequence were interpreted only qualitatively because signal areas were attenuated according to
their transverse relaxation times (T2). The CPMG method was used to identify soluble metabolites
such as organic acids, amino acids, choline and derivatives, taurine, guanidine acetate, tyrosine, and
phenylalanine. The fatty acid variations observed by single pulse as well as the lactate, acetate,
glycine, and phosphocholine variations observed through CPMG 1H HR-MAS NMR have potential to
characterize both responder and non-responder tumors in a molecular level. Additionally, we
emphasized that comparable tumors (i.e., with the same origin, in this case breast cancer) may respond
totally differently to chemotherapy. Our observations reinforce the theory that alterations in cellular
metabolism may contribute to the development of a malignant phenotype and cell resistance.
341
An integrated strategy for rapid discovery and identification of the sequential piperine
metabolites in rats using ultra high-performance liquid chromatography/high resolution mass
spectrometery
Zhanpeng Shang, Wei Cai, Yanfeng Cao, Fei Wang, Jiayu Zhang
ABSTRACT
Piperine, one of the major bioactive constituents isolated from natural flavorings and medicinal-
culinary herbs, possesses various biological activities. In the present study, an integrated strategy based
on ultra high-performance liquid chromatography/high resolution mass spectrometry was established
to reveal piperine metabolism in rats. First of all, post-acquisition data-mining methods, including high
resolution extracted ion chromatograms (HREICs) and multiple mass defect filtering (MMDF), were
used to screen piperine metabolite candidates in a full-scan HRMS1 level. Then, parent ion list-
dynamic exclusion coupled with data-dependent data-acquisition method was utilized to acquire MSn
datasets. In addition, the established reverse molecular assembly (RMA) approach based on paired
diagnostic product ions (pDPIs) coupled with neutral loss fragments (NLFs) was used to ascertain and
identify the major-to-trace piperine metabolites efficiently. And then, the calculated ClogP values were
utilized to distinguish the positional isomers. As a result, a total of 148 piperine metabolites were
detected and characterized tentatively. The results demonstrated that piperine mainly underwent
hydrogenation, dehydrogenation, hydroxylation, glucuronide conjugation, sulfate conjugation, ring-
cleavage, and their composite reactions. Our results not only provided novel and useful data to better
understand the safety, toxicity and efficacy of this potential therapeutic agent, but also indicated that
the proposed strategy was reliable for a rapid discovery and identification drug-related constituents in
vivo.
***
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