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Spark-Holland Automated on-line DPS Extraction and Analysis with a Q-Exactive Orbitrap
Lecture 9, Page 41
Developing DPS Methods
•Normal regulated bioanalysis protocol:
› Centrifuge collected blood to get plasma
› Fortify control plasma (not blood) with standards and QC’s to create the calibration curve
› Fortify test plasma samples with IS at a fixed level
› Extract plasma standards and QC’s along with test samples and analyze by LC/MS
Lecture 9, Page 42
Towards Higher Throughput DPS
On-line SPE-LC-HRAMS DPS Extraction (Exactive Plus)
DPS Card
Exactive Plus
2 mm
Spark-Holland DPS autosampler
Lecture 9, Page 43
The Q Exactive: Hardware Innovations
Orbitrap Mass Analyzer
HCD Cell
S-lens Ion Source
Enhanced FT
C-Trap Quadrupole Mass Filter
Lecture 9, Page 44
RP = 15,000 RP = 56,700
High Resolution and Accurate Mass – Example In
ten
sity
(%
)
0
20
40
60
80
100
Mass [amu]
516.65 516.70 516.75 516.80 516.85 516.90
Inte
nsi
ty (
%)
0
20
40
60
80
100
Mass [amu] 516.65 516.70 516.75 516.80 516.85 516.90
516.77581 (observed)
516.76671
(correct) 516.78490
(correct)
516.78490 516.76671
Wrong Answer for Both Peptides
Peptide mixture: [Val5]-Angiotensin II Lys-des-Arg9-Bradykinin
Sequence: DRVYVHPF KRPPGFSPF
Formula: C49H69N13O12 C50H73N13O11
Exact mass: [M+2H]2+ = 516.76671 [M+2H]2+ = 516.78490
Dm (mmu): 18.2 mmu
Right Answer for Both Peptides
Lecture 9, Page 45
Representative XICs (5 ppm) with Orbitrap HRAMS
Lecture 9, Page 46
RBC Filtration Workflow 1
•Whole blood standards, QCs, and blanks were subjected to RBC filtration device to produce DPS
Standard Curve Prepared Using Filtration Device
Offline RBC Filtration card
Lecture 9, Page 47
RBC Filtration Workflow 1
• Acceptable accuracy and precision
Drawbacks
• Time-consuming to prepare all standards and QCs using filtration device
• What is the best way to approach this?
• Stay tuned
Accuracy and Precision
Guanfacine Concentration (ng/mL)
LLOQ QC LQC MQC HQC
0.50 0.75 200 400
Mean 0.49 0.65 211 428
CV (%) 10.6 14.5 2.9 5.1
RE (%) -1.4 -12.8 5.3 6.9
n 5 6 6 6
Lecture 9, Page 48
Summary
• Q-Exactive provides excellent selectivity in a small molecule quantitation assay providing a linear dynamic range over three orders of magnitude.
• A novel membrane-based device capable of filtering RBCs out of whole blood without the need for centrifugation has been presented for offline quantitative bioanalysis.
• Acceptable accuracy and precision for whole blood QCs subjected to our filtration device can be achieved using a fortified plasma standard curve.
• The DPS card is compatible with commercially available online-extraction dried blood spot robotic autosamplers.
RBC filtration device for generating dried plasma spots
Lecture 9, Page 49
Lecture 9, Page 50
Lecture 9, Page 51
Lecture 9, Page 52
Lecture 9, Page 53
The Advion TriVersa NanoMate A Robotic Workstation For Automated nano ESI
Lecture 9, Page 54
TriVersa + MS
MS sample orifice
Automated Direct ‘Infusion’ nano ESI of Protein Samples
Infusion can be for short or long times; the latter allowing for simultaneously screening for
hundreds of compounds! Lecture 9, Page 55
Lecture 9, Page 56
Lecture 9, Page 57
Lecture 9, Page 58
Another Form of Selectivity coupled with Mass Spectrometry
• Ion mobility Spectrometry
Lecture 9, Page 59
Differential Mobility Spectrometry (DMS) ‘Selexion’ from AB Sciex
(DMS is not mass spectrometry)
An orthogonal separation mechanism based upon charge-to-mass and
molecular shape
Lecture 9, Page 60
Mass Spec Vacuum draws ions and gas through
planar DMS electrodes
2.3 torr
2.8 L/min
1 mm
3 cm
760 torr
Millisecond flight times
Lecture 9, Page 61
Chemical Enhanced Separations Driven by Mobility Changes Due to Clustering / Declustering.
Low field
clustering-
mobility decreases
+
HV=3kV
Schneider BB, Covey TR, Coy SL, Krylov EV, and Nazarov EG, “Chemical Effects in the Separation Process of a Differential Mobility/Mass
Spectrometer System”, Anal. Chem., 2010, 82, 1867-1880.
High field
declustering;
mobility increases
L V=0.3 kV
F ~ 1 MHz, T= 10-6 s
Lecture 9, Page 62
DMS Hardware on AB Sciex 5500 QTRAP
Lecture 9, Page 63
Testosterone Female #3 Plasma Sample: LLE
DMS OFF/
289/109
DMS OFF/
289/97
DMS ON/
289/109 DMS ON/
289/97
Interference
Ion ratio is Peak of interest Interference
Peak of interest
Lecture 9, Page 64
Testosterone Female #3 Plasma Sample: PPT
DMS OFF/
289/109
DMS OFF/
289/97
DMS ON/
289/109
DMS ON/
289/97
Peak of interest
Peak of interest @ RT 3.37 min
Lecture 9, Page 65
•Question: If we consider the entire known organic chemical space, including proteins, peptides, RNA, DNA, as well as polar small molecule chemicals, what % of these are amenable to GC/MS?
•Answer: Let’s take a look….
GC/MS or LC/MS/MS Which Technique Handles the Wider Range of Chemical
Compounds?
Lecture 9, Page 66
GC/MS vs. LC/MS/MS for Range of Pesticides Detected
Lutz Alder,* Kerstin Greulich, Gu¨nther Kempe, and Ba¨rbel Vieth, RESIDUE ANALYSIS OF 500 HIGH PRIORITY PESTICIDES, Mass Spectrometry Reviews, 2006, 25, 838– 865
Lecture 9, Page 67
• This overview evaluates the capabilities of mass spectrometry (MS) in combination with gas chromatography (GC) and liquid chromatography (LC) for the determination of a multitude of pesticides. The selection of pesticides for this assessment is based on the status of production, the existence of regulations on maximum residue levels in food, and the frequency of residue detection. GC–MS with electron impact (EI) ionization and the combination of LC with tandem mass spectrometers (LC–MS/MS) using electrospray ionization (ESI) are identified as techniques most often applied in multi-residue methods for pesticides at present. Therefore, applicability and sensitivity obtained with GC–EI–MS and LC–ESI–MS/MS is individually compared for each of the selected pesticides. Only for one substance class only, the organochlorine pesticides, GC-MS achieves better performance. For all other classes of pesticides, the assessment shows a wider scope and better sensitivity if detection is based on LC–MS.
• Ref: (Mass Spec Rev 25:838–865, 2006)
GC/MS vs. LC/MS/MS
Lutz Alder,* Kerstin Greulich, Gu¨nther Kempe, and Ba¨rbel Vieth, RESIDUE ANALYSIS OF 500 HIGH PRIORITY PESTICIDES, Mass Spectrometry Reviews, 2006, 25, 838– 865
Lecture 9, Page 68
Smaller Mass Spectrometers
Mini 12
Expression CMS
Lecture 9, Page 69
Commercially Available Small Mass Spectrometers
Lecture 9, Page 70
Mini 12 mass spectrometer
Lecture 9, Page 71
Control Tablet
Electronics
Cassette
Loading
system
Bar Code Reader
Vacuum
System
Solvent
Container
Solvent Pump
Mini 12: Internal components
Lecture 9, Page 72
Paper Spray: Immediate Point-of-Care Analysis
kV
1. Prick Finger
2. Load Sample
3. Apply Solution 4. Apply HV
5. Acquire Data
0 s
15 s
30 s 40 s
50 s
60 s
[M+H]+
6. Report Results
• One drop of blood • 60 second analysis of biological samples
Lecture 9, Page 73
Atenolol
MW=266
m/z 267 Atenolol 10 ppm spiked in whole blood
0.4μl blood loaded for each sampling spot
10 μl methanol/H2O added for spraying
m/z 267
MS paper spray of blood spiked with
atenolol (4ng/spot)
MS/MS spectrum to identify atenolol
Paper spray/mini MS EESI (direct detection, Atenolol in blood)
251
225
208 190
145
173
180
He Wang, Jiangjiang Liu, Guangming Huang
CID
Lecture 9, Page 74
The Mini 12 From Purdue University A Miniature Mass Spectrometer for Clinical and Other
Applications Introduction and Characterization
Linfan Li,† Tsung-Chi Chen,† Yue Ren,† Paul I. Hendricks,‡ R. Graham Cooks,*,‡,§ and Zheng Ouyang*,†,§ dx.doi.org/10.1021/ac403766c | Anal. Chem. 2014, 86, 2909−2916 Lecture 9, Page 75
Mini 12 MS and MS/MS nano ESI Mass Spectra for Clenbuterol, Thiabendazole and Amitryptyline
Lecture 9, Page 76
Cart-Based MS System Advion, Inc.
Lecture 9, Page 77
• Little or no sample preparation • No chromatography • Data in < 1 minute The closed end of a glass capillary is dipped into the sample of interest or scraped on a solid surface, and then placed into the CMS for analysis. Ideal for: • Reaction monitoring • Compound ID • Food safety • Natural products
ASAP® – Atmospheric Solids Analysis Probe
Lecture 9, Page 78
SPME Sample Preparation for Drugs in Urine
SPME: Solid-Phase Micro Extraction. • Insert a fused-silica fiber coated
with an HPLC stationary phase into a biological sample
• Allow to equilibrate with the sample
• Remove and rinse with water • Place fiber containing extracted
sample into hot nitrogen gas • Ionize by APCI • Obtain mass sectra
Lecture 9, Page 79
Amlodipine in Urine
Amlodipine C20H25ClN2O5
MW: 408.87
Preparation of fortified urine sample The stock solution of amlodipine was 1.0 mg/ml in methanol by dissolving a 2.5 mg tablet in 2.5 mL Methanol. The 50ng/ul and 100ng/ul amlodipine in urine was made by spiking the stock solution in urine sample.
Lecture 9, Page 80
ASAP-SPME-MS Analysis of 50 ng/ul Amlodipine in urine Extract time: 5 mins, SPME LC Tips SCX/C18
Mass spectrum of Amlodipine in urine
XIC for m/z 406
Total ion current
[M+H]+
C20H25ClN2O5
MW: 408.87
Lecture 9, Pages 81
Copyright Jack Henion 2016
SPME-ASAP-SIM of Incurred urine, two hours after 2.5mg dose Extract time: 60 mins, SPME LC Tips C18
XIC for m/z 409 after background subtraction
Total ion current
Did not detect the m/z 409 in the full-scan mode. However, with the SIM mode, it clearly detected the m/z 409. SCX/C18 tips has similar result to C18 tips
Lecture 9, Pages 82
Real-Time Monitoring of Suzuki Reaction with ASAP®-MS
Changtong Hao, Ph.D Advion Inc.
Copyright Jack Henion 2016
Lecture 9, Pages 83
Experimental and Materials
Reactants A and B were mixed at equi-molar amounts in a round-bottom reaction flask and stirred at room temperature. 2mL aliquots was transferred to the capillary tip of ASAP® probe for ASAP®-MS analysis.
NaOH, PdCl2; 4 mol% EtOH/H2O 4:1 5ml Room temp NH2
BrB
OH
OH
NH2
+
A B
C D
Suzuki Reaction
Reactants and Catalyst Product
A B C D E
compound 4-bromaniline Phenyl Boronic acid
Sodium hydroxide
Palladium chloride
4-aminobiphenyl
Chemical Formula
C6H6BrN C6H7BO2 NaOH PdCl2 C12H11N
MW 171 122 40 176 169
moles 0.005 0.005 0.0065 0.005 0.005
mg 855 610 260 35 845
E
Chemicals were purchased from Sigma-Aldrich with a purity greater than 99%. MS solvent was LC/MS grade.
Copyright Jack Henion 2016
Lecture 9, Pages 84
Figure 1. Mass spectra of Suzuki reaction mixture at different time
BrNH2
4-bromaniline C6H6BrN
MW: 172.02
[M+H]+:
NH2
4-aminobiphenyl C12H11N
MW: 169.22
[M+H]+:
0 min 10 min 15 min 30 min 60 min 180 min
The bromine ‘doublet’ in the reactant at m/z 172/174 diminishes while the product ion increases at m/z 170!
m/z 79/81 doublet
NH2
Br
NH2
Copyright Jack Henion 2016
Lecture 9, Pages 85
Reactant is consumed while product is produced: Know when the reaction is finished!
Reaction Time(mins)
0 50 100 150 200
Rela
tive R
ati
o t
o T
oa
l P
eak H
eig
ht
of
Reac
tan
t an
d P
rod
uct
(%)
0
20
40
60
80
100
Product
Reactant
Suzuki Reaction Monitoring by ASAP/MS
NH2
Br
NH2
Copyright Jack Henion 2016
Lecture 9, Pages 86
Thin Layer Chromatography/Mass Spectrometry
TLC/MS
The Advion PlateExpressTM
Copyright Jack Henion 2016
Lecture 9, Pages 87
Thin Layer Chromatography
Wouldn’t it be nice to know what each of those spots really is?
Copyright Jack Henion 2016
Lecture 9, Pages 88
A Compact Mass Spectrometer (CMS) connected to the Plate Express™ sample extraction device
TLC-CMS set-up
Copyright Jack Henion 2016
Lecture 9, Pages 89
TLC-CMS Workflow Schematic
200 µL/min Methanol with 0.1% Formic Acid
SSI Isocratic Pump
6 port valve
Plate Express sample extraction device
TLC Plate
Sealing Face
Elution Head
• Controlled by MS operation software (Mass Express)
• Programmable sealing force for different media (eg TLC)
• Interchangeable heads for different bed heights
Copyright Jack Henion 2016
Lecture 9, Pages 90
TLC/MS for the detection of chemical compounds related to cannabis
Daniel Eikel and Simon Prosser
Advion Inc., Ithaca, NY, USA
Copyright Jack Henion 2016
Lecture 9, Pages 91
Cannabinoids used in this study
OH
OH CH3
CH3
CH3CH3
Cannabigerol Mw=316.24 (C21H32O2)
CH3 CH3O
OH
CH3
CH3
Cannabichromene Mw=314.22 (C21H30O2)
Cannabinol (CBN) Mw=310.19 (C21H26O2)
CH3
CH3OH
OH
CH3
HHCH2
OH
OCH3
CH3OH
OH
CH3
HHCH2 Cannabidiolic acid (CBD-A)
Mw=358.21 (C22H30O4) Cannabidiol (CBD) Mw=314.22 (C21H30O2)
CH3
CH3
CH3
CH3O
OH
CH3
CH3
CH3
CH3O
OHH
HCH3
CH3
CH3
CH3O
OHH
H
OH
O
-Δ9-THC acid (THC-A) Mw=358.21 (C22H30O4) -Δ9-THC (THC) Mw=314.22 (C21H30O2)
Copyright Jack Henion 2016
Lecture 9, Pages 92
93
6
TLC-FIA-MS (TIC)
TLC-CMS example:
THC (M-H)- at m/z 313.3
In-source CID-MS
CH3
CH3
CH3
CH3O
OHH
HCH3
CH3
CH3
CH3O
OHH
H
OH
O
(M+H)+
Copyright Jack Henion 2016
References: Mini 12
Copyright Jack Henion 2016
• Linfan Li,† Tsung-Chi Chen,† Yue Ren,† Paul I. Hendricks,‡ R. Graham Cooks,*,‡,§ and Zheng Ouyang*,†, Mini 12, Miniature Mass Spectrometer for Clinical and Other Applications Introduction and Characterization, Anal. Chem. 2014, 86, 2909−2916
Lecture 9, Pages 94
1. Ramagiri, S. and F. Garofolo, Large molecule bioanalysis using Q-TOF without predigestion and its data processing challenges. Bioanalysis, 2012. 4(5): p. 529-540.
2. Zhang, J., et al., Proteoform analysis of lipocalin-type prostaglandin D-synthase from human cerebrospinal fluid by isoelectric focusing and superficially porous liquid chromatography with Fourier transform mass spectrometry. Proteomics, 2014. 00: p. 1–9.
3. Ruan, Q., et al., Strategy and Its Implications of Protein Bioanalysis Utilizing High-Resolution Mass Spectrometric Detection of Intact Protein. Analytical Chemistry, 2011. 83(23): p. 8937-8944.
4. Li, H., et al., Native Top-Down Electrospray Ionization-Mass Spectrometry of 158 kDa Protein Complex by High-Resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Analytical Chemistry, 2013. 86(1): p. 317-320.
5. Sun, L., et al., Fast Top-Down Intact Protein Characterization with Capillary Zone Electrophoresis–Electrospray Ionization Tandem Mass Spectrometry. Analytical Chemistry, 2013. 85(12): p. 5989-5995.
6. Zhang, J., et al., Top-Down Mass Spectrometry on Tissue Extracts and Biofluids with Isoelectric Focusing and Superficially Porous Silica Liquid Chromatography. Analytical Chemistry, 2013. 85(21): p. 10377-10384.
7. Ref: Hongcheng Liu, Georgeen Gaza-Bulseco, and Chris ChumsaeJ Am Soc Mass Spectrom 2009, 20, 2258–2264.
Relevant Publications
Lecture 9, Pages 95
The End
Thank you
Jack Henion
Lecture 9, Pages 96