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Review ArticleDetermination of Strong Acidic Drugs in Biological MatricesA Review of Separation Methods

Lingli Mu1 Feifan Xie12 Sanwang Li2 and Peng Yu2

1 Medical College Hunan Normal University Changsha 410006 China2 School of Pharmaceutical Sciences Central South University Changsha 410013 China

Correspondence should be addressed to Peng Yu pengyucsueducn

Received 3 June 2014 Revised 17 September 2014 Accepted 17 September 2014 Published 29 September 2014

Academic Editor Toyohide Takeuchi

Copyright copy 2014 Lingli Mu et alThis is an open access article distributed under theCreativeCommonsAttributionLicense whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Strong acidic drugs are a class of chemical compounds that normally have high hydrophilicity and large negative charges suchas organophosphatic compounds and organosulphonic compounds This review focuses on sample preparation and separationmethods for this group of compounds in biological matrices in recent years A wide range of separation techniques especiallychromatographic method are presented and critically discussed which include liquid chromatography (eg ion-pair and ion-exchange chromatography) capillary electrophoresis (CE) and other types Due to the extremely low concentration level of targetanalytes as well as the complexity of biological matrices sample pretreatment methods such as dilute and shoot methods proteinprecipitation (PP) liquid-liquid extraction (LLE) solid-phase extraction (SPE) degradation and derivatization strategy also playimportant roles for the development of successful analytical methods and thus are also discussed

1 Introduction

The main members of strong acidic drugs include organo-phosphatic compounds and organosulphonic compoundswhich have emerged as promising candidates for treatinga wide range of diseases such as AIDS [1] cancer [2]osteoarthritis [3] Alzheimerrsquos disease and cardiovasculardisorders [4] For example nucleoside reverse transcriptaseinhibitors (NRTI) the first safe and effective agents for thetreatment of patients infected with HIV must be selectivelyphosphorylated to their active triphosphate moieties (NRTI-TP) within human peripheral blood mononuclear cells [1]Taurine a major metabolite of sulfur-containing amino acidin mammals plays a very important role in several essentialbiological processes and recently existence of taurine inurine was found to be an indicator of bladder cancer [5]Chondroitin sulfate (CS) and dermatan sulfate (DS) showimportant functions in central nervous system developmentwound repair infection growth factor signaling morpho-genes and cell division differentiation andmigration in addi-tion to osteoarthritis and their conventional structural roles

Furthermore CS is used and recommended by the Euro-pean League against Rheumatism (EULAR) as a SYSADOA(symptomatic slow-acting drug for osteoarthritis) drug inEurope in the treatment of knee and hand osteoarthritis [3]

Due to the high hydrophilicity there are numbers of diffi-culties and challenges for analyzing these strong acidic drugs(a) These compounds contain one or more strong acidicgroups and they are hard to be retained on common HPLCchromatographic columns like C

18or C8columns which

makes itmore difficult to separate them fromhydrophilic bio-logical interference material (b) Many of these compoundshave several pKa values which may span the entire pH rangemaking it more challenging to establish a chromatographicseparation method with a simple isocratic mobile phase (c)Some of these compounds have weak ultraviolet absorptionor fluorescence which makes it hard to inspect them whileseparating (d) To measure these sorts of analytes at low con-centration levels sensitive quantitative methods are requiredespecially when applied to calculate pharmacokinetic andpharmacodynamic parameters (e) For the complexity andchallenges experienced during sample preparation of these

Hindawi Publishing CorporationChromatography Research InternationalVolume 2014 Article ID 469562 10 pageshttpdxdoiorg1011552014469562

2 Chromatography Research International

compounds in biological fluids and tissues special techniquesare required to extract the target analytes from biologicalmatrices to separate them from interfering materials andthen to quantify and characterize them On related topics theseparation methods for bisphosphonate active pharmaceu-tical ingredients in pharmaceuticals and biological materialwere reviewed in 2008 by Zacharis and Tzanavaras [6] andchromatographic separation methods for the determinationof therapeutic oligonucleotides were reviewed by McGinniset al in 2012 [4]

Trying to find proper separation methods to resolve theaccurate and precise quantification of strong acidic drugsin complex biological matrix samples we searched andanalyzed the related information in dozens of academicarticles ranging from 2002 to date In this review we focus onbioanalyticalmethods (especially chromatographicmethods)for analyzing strong acidic compounds mainly organophos-phatic compounds and organosulphonic compounds Thereview covers analytical methods using liquid chromatogra-phy (eg ion-pair ion-exchange and hydrophilic interactionchromatography) capillary electrophoresis (CE) and otheranalytical methods Due to the extremely low concentrationlevels of target analytes as well as the complexity of biolog-ical matrices sample pretreatment methods such as diluteand shoot method protein precipitation (PP) liquid-liquidextraction (LLE) solid-phase extraction (SPE) degradationand derivatization strategy are also explored

2 Sample Preparation

Generally biological samples such as urine serumor plasmaand tissues should not be directly injected into a separationsystem due to matrix complexity To resolve the problema sequence of sample treatment steps is performed priorto analysis The main purposes of sample pretreatment areto remove unwanted interfering substances suppress matrixeffects and enhance selectivity convert the analytes to a com-patible form for analysis and if possible increase sensitivitythrough preconcentration [6]

Dilute and shoot method is one of the simplestapproaches for sample preparation Using this methoda fast sensitive and noninvasive LC-MSMS techniquewas developed and validated to separate and quantitativelydetermine taurine and related biomarkers in urine matricesby Gamagedara et al [5] This method is simpler and moretime-saving than other sample preparation methods and itis much suitable for the biological fluids like urine whichcontains relatively high concentration levels of target analytesas well as a small quantity of proteins and other endogenoussubstances But dilute and shoot method is hard to apply toother biological samples such as plasma and serum

Protein precipitation is a simple procedure to removeproteins from plasma and other biological matrices Brieflythe reagents that are used to precipitate proteins frombiological samples mostly include inorganic acids organicacids and organic solvents such as perchloric acid (PCA)[7 8] trichloroacetic acid (TCA) [9] acetonitrile (ACN)[10ndash15] and methanol [2 16 17] After adding protein

precipitating reagent the mixture is usually needed to becentrifuged to remove the denatured protein [18] Usingtrichloroacetic acid as protein precipitation reagent Losa etal isolated gemcitabine di- and triphosphate in peripheralblood mononuclear cells (PBMC) [9] In this study thetemperature was controlled at 4∘C during sample preparationtomaintain the stability of nucleotide triphosphates and afterprotein precipitation Freon (112-trichlorotrifluoroethane)-trioctylamine (4 1 vv) was used to yield clean extractsand it neutralizes the samples Protein precipitation can alsoattain other objectives simultaneously For instance sodiumoctanoate was used as binding competition to replace p-cresyl sulfate and indoxyl sulfate from albumin followedby protein precipitation for sample preparation [19] Thismethod allowed direct quantification of indoxyl sulfate andp-cresyl sulfate which are protein-bound marker moleculesin chronic kidney disease Chen et al [14] used acetonitrile toprecipitate protein while extracting indocyanine green fromdog plasma and bile The high efficiency of protein precipi-tation has also been proved in many studies while preparingsorts of strong acidic drug samples with different biologicalmatrices including serum [16 19 20] plasma [7 10 12 14ndash17 21] cerebrospinal fluid [7] urine [12 13 16] bile [20]brain tissue [2 7 21] PBMC [9] and liver [10 16] Howeverthe dilution effects of adding protein precipitation reagentssometimes become a problem when the concentration levelsof target analytes in biological matrices are extremely lowUnder these conditions the samples are often needed to befurther concentrated by using procedures such as liquid-liquid extraction (LLE) and solid-phase extraction (SPE) toimprove method sensitivity before sample analysis

LLE is a simple and efficient method for the separationand concentration of relatively hydrophobic compoundsHowever most of strong acidic drugs in biological fluidsand tissues present in phosphorylated sulfated or sulfonatedforms so the strong acidic drugs are usually classified ashighly hydrophilic compounds In many cases LLE is not aneffective sample preparation method for these compoundsThus it is nearly impossible to extract them with organicsolvent from biological samples A strategy to this problem isto add certain strong acid to adjust the pH value so that theirionization is suppressed and they can be treated as hydropho-bicmolecules For exampleDu andEddington [22] describeda LLE method using trifluoroacetic acid as ionization-suppressing reagent for the extraction of chondroitin sulfatedisaccharides Actually controlling the pH values is notalways an effective way to suppress ionization for stronglyacidic compounds Therefore adding proper counterion toperform the ion-pair extraction becomes a better choice inwhich the target analytes and the counterions are combinedinto ion-pair complexes which can be easily extracted withusual organic solvents Briefly adding a special ion-pairreagent into the sample solution allows target analytes to formion-pair complexes with the counterions of opposite chargeand then the ion-pairs possess higher partition coefficientsthan the original analytes and as a result the transferringof the target drugs into the extractant (organic) phase isenhanced Xu et al [23] applied an ion-pair liquid-liquidextractionmethod while successfully extracting nonsteroidal

Chromatography Research International 3

anti-inflammatory drugs from water-based samples In thisstudy using tetrabutylammonium hydrogen sulfate as theion-pair reagent the formed ion-pairs had much highersolubility in organic phase (ethyl acetate) than the originalcompounds which facilitated the extraction process Butthe difficulty during selecting and optimizing the operationconditions is the main drawback of this method To the bestof our knowledge till today only few studies applying ion-pair extraction method to extract strong acidic compoundsin biological matrix have been published

Usually one of the major challenges in developing amethod for the simultaneous quantitative analysis of targetcompound and its metabolites is that some of the targetanalytes are highly water-soluble while others are highlyhydrophobic Thus there is usually loss of some analytesusing the standard two-phase LLE methods which couldsacrifice the recovery and the method sensitivity for some ofthose target analytes So protein precipitationwas sometimesused for the extraction of sphingosine and sphingosine 1-phosphate prior to the LC-MSMS analysis because of itssimplicity [24] However Emotte et al [25] found that forsamples originating from blood protein precipitation wasnot always compatible with LC-MSMS analysis as theyobserved some sensitivity loss and MS clogging during thesample analysis Consequently they used C

18SPE columns

for the extraction of fingolimod and fingolimod-phosphatefrom biological matrices and the SPE step was automatedand operated online with LC-MSMS detection offeringlower limit of detection (LOD) by sample preconcentrationwhile saving time Another example is that Satonin et al[26] applied a single-step SPE method to separate and con-centrate 5-hydroxy-6-methoxy duloxetine sulfate in plasmain which the average extraction recovery of 5-hydroxy-6-methoxy duloxetine sulfate in plasma was approximately97 Huang et al [10] described an analytical procedurefor monitoring bile acids and their sulfate metabolites inmouse bile and urine using Supelclean LC-18 SPE using one-step sample preparation method which could also achievehigh extraction recovery Furthermore SPE has been suc-cessfully used for sample pretreatment of various strongacidic compounds such as the phosphorylated metabolitesof nucleoside [27 28] sulfate metabolites of bile acids[10 11] ATP ADP AMP and guanine nucleotides [29]Compared with the protein precipitation and LLE SPEshows the following advantages (a) Because the separa-tion mechanism of SPE is based on interaction betweenthe functional groups of analyte and packing material themethod is reproducible and easily realizes autostandardiza-tion procedure (b) SPE can be automated and operatedonline with LC-MSMS detection thus offering lower limitof detection (LOD) by sample preconcentration while savingtime

In addition to those traditional methods for samplepreparation of strong acidic drugs some special methodsare also used Robbins et al [27] described an anionexchange solid-phase extraction combined with degrada-tion method for extracting triphosphate metabolites ofzidovudine lamivudine and abacavir This research suc-cessfully separated triphosphate metabolites of zidovudine

lamivudine and abacavir from their mono- and diphos-phates and degraded these phosphorylated metabolites tothe parent drugs which were more suitable for chromato-graphic analysis Huang et al [30] reported that sulfategroup losses were quite commonwhile applying tandemmassspectrometry as detector for analyzing acidic compoundssuch as glycosaminoglycans which made the analysis oforganosulphonic mixture highly problematic due to thesimultaneous presence of many product ions from sulfatedand nonsulfated drugs Sufficient fragmentation informationfor definitive assignment of sulfation sites is not alwaysavailable even under optimized conditions which makescertain methods (eg LC-MSMS and CE-MS) more diffi-cult for identifying sulfation position of glycosaminoglycansoligosaccharides with high degrees of heterogeneity So theydeveloped a chemical-derivatization strategy to convert thechondroitin sulfate oligosaccharides to compatible forms forchromatographic separation in which chondroitin sulfatehexasaccharides were first premethylated to protect freehydroxyl and carboxyl groups and then the sulfate groupswere removed and the resulting active hydroxyl groupswere acetylated The purpose of the chemical-derivationstrategy is to use a more stable and distinguishable groupthat can be differentiated from premethylated groups byboth mass spectrum and chromatographic retention timeto replace the labile sulfate groups This strategy allowsthe chromatographic separation and structural identifica-tion of different sulfated glycosaminoglycans oligomers Inanother study chondroitin sulfate disaccharides in dog andhorse plasma were extracted using LLE method and thendigested by chondroitinase followed by derivatization with1 dansylhydrazine in ethanol at 40∘C for 3 h and finallyseparation on a C

18column and detection using a fluo-

rescence detector [22] The degradation or derivatizationmethodology can transform strong hydrophilic analytes intothe hydrophobic forms thereby increasing their retentiontime on the reverse-phase chromatographic column whichoften improves the stability as well as analytical sensitivityof analytes But these special methods are usually labor-consuming and time-costing which may bring inevitableerrors

3 Reversed-Phase Liquid Chromatography

Reversed-phase high-performance liquid chromatography(RP-HPLC) continues to dominate the chromatographic sep-aration of chemical drugs and other small molecules despitethe availability of other HPLC modes such as ion-exchangeand normal phase chromatography Lu et al [31] developedand validated a novel stability-indicating RP-HPLC methodusing two oxocyclic organic modifiers in the mobile phasewhich was used to separate a total of 32 potential impuritiesanddegradation products frombetamethasone sodiumphos-phate and betamethasone acetate In this study special selec-tivity was achieved by the combination of two oxocyclic sol-vents namely tetrahydrofuran (THF) and 14-dioxane whichwere used as the organic modifiers of the mobile phasesFurther improvement of peak symmetry and separation


efficiency was achieved by using two chaotropic agents (tri-fluoroacetic acid and potassium hexafluorophosphate) in themobile phases Melendez et al [32] developed and validateda sensitive and specific method using high-performanceliquid chromatography-tandem mass spectrometry (HPLC-MSMS) for the determination of ribavirin monophosphate(RBV-MP) and ribavirin triphosphate (RBV-TP) in cellsIn this method ribavirin phosphorylated metabolites wereextracted using anion exchange solid-phase extraction (SPE)prior to the simultaneous HPLC-MSMS determination ofRBV-MP and RBV-TP Gamagedara et al [5] systematicallyinvestigated and optimized the conditions that could affectLC separation and MSMS detection to establish a methodfor separation and quantitative determination of taurineand related biomarkers in urine matrices This method cancompletely separate the target compounds (using a phenyl-hexyl column) within 10min De Loor et al [19] employedRP-HPLC based on SunFire C

18column with fluorescence

detector (FLD) setting at 120582 ex 260 nm120582 em 288 nm for p-cresyl sulfate and 120582 ex 280 nm120582 em 390 nm for indoxylsulfate to determine indoxyl sulfate and p-cresyl sulfate in theserum samples from healthy subjects and uremic patients

Totally due to strong hydrophilicity the target analyteshave poor retention on the chromatographic column so inmany cases common RP-HPLC method cannot successfullyseparate these ionized compounds and thus restricts itsapplication in this kind of bioanalysis

4 Ion-Pair Liquid Chromatography

In some cases pH adjustment of the mobile phase in RP-HPLC fails to separate mixtures of very hydrophilic organiccompounds with ionic character while ion-pair liquid chro-matography (IPLC) is one of the most popular approachesto achieve efficient separations of such species IPLC can beeasily performed by modifying the mobile phases used in theRP-HPLC on the same analytical columns An amphiphilicanion or cation usually an alkyl sulphonic acid or saltand alkyl quaternary amine respectively is added to themobile phases to enhance the retention of analytes bearingopposite charges Briefly tetrabutylammonium salt [9 20 2933ndash36] hexylamine [37ndash39] dimethylhexylamine (DMHA)[25] and tributylamine [40] were usually used as ion-pairreagents for measuring strong acidic compounds in IPLCmethodology With adequate selectivity this method allowsuniversal UV detection for quantitative analysis and is moreeasily interfaced with mass spectrometry to provide exactmass data and MSMS characterization

Lefebvre et al [34] reported a simple rapid and repro-ducible analyticalmethod for the simultaneous quantificationof zidovudine (AZT) and its monophosphate (AZT-MP) incell extracts by IPLC Due to the relatively low octanol-water partition coefficients (log P) of AZT-MP it is quitechallenging to establish a chromatographic method becausethis highly water-soluble compound shows extremely poorretention on ordinary reversed-phase columns Thus a RP-HPLC approach with addition of triethylammonium acetateas ion-pairing reagent to the mobile phase was developed

to improve the chromatographic selectivity of AZT AZT-MP and the internal standard on an analytical RP-HPLCcolumn Calaf et al [20] developed a rapid simple and verysensitive ion-pair liquid chromatographymethod to quantifyseveral uremic solutes in a unique sample of biologicalfluid phenol indole-3-acetic acid p-cresol indoxyl sulfateand p-cresol sulfate The chromatographic separation wassuccessfully obtained by using tetrabutylammonium iodideas ion-pair reagent Moreover in this study an isocraticflow was employed instead of gradient flow which avoidedtime-consuming column reequilibrium Emotte et al [25]established a reliable method for the simultaneous deter-mination of fingolimod (FTY720) and its active metaboliteFTY720-phosphate (FTY720-P) in human blood sample inwhich the mobile phases consisting of dimethylhexylamine(DMHA) solution and acetonitrile-isopropanol (8020 vv)were optimized for the separation of fingolimod phos-phate and fingolimod from the interfering materials Thechromatographic separation condition showed that ion-pairreagents with more volatility such as DMHA could alsobe successfully employed to retain ionizable compoundson analytical column Xie et al [41] presented an IPLCmethod using evaporative light-scattering detection (ELSD)for identification and simultaneous determination of fourbisphosphonates (alendronate pamidronate zoledronic acidand etidronate) which were not retained on hydrophobiccolumn and lack chromophore for detection In this studyorganic amines including n-butylamine n-hexylamine andn-octylamine were investigated as ion-pair reagents for sep-aration of the four compounds and were evaluated alongwith n-amylamine in order to find an appropriate retentionof the four compounds And as a result n-amylamine wasselected as the most suitable volatile additive agent Thisnewly developedmethod sufficiently separated analytes fromeach other on a Phenomenex C

18column and enabled direct

analysis of bisphosphonates without any derivatizationIPLC has been widely used to selectively separate ion-

izable and ionic organic compounds in various samplesHowever this technique is generally not compatible with ESI-MS because of the use of nonvolatile mobile phase solventsbuffers or salts Even given the availability of some relativelyvolatile ion-pairing reagents ion suppression and other issuesrelated to method robustness are still insolvable problemswith LC-MS analysis Researches on new ion-pair agents aremainly focused on ensuring that the ion-pairing buffer iscompatible with electrospray ionization-mass spectrometry

5 Ion-Exchange Liquid Chromatography

Ion-exchange liquid chromatography is a separation tech-nique used to analyze anions and cations in solution Ion-exchange chromatography with UV and other detectorsis an excellent method for separating charged moleculeswhich means it is also amenable to multiply charged strongacidic compounds Motoyama et al [42] reported an onlinemultidimensional LC method using an anion and cationexchange (ACE) mixed bed for the first separation of pep-tides and phosphopeptides The mixed-bed ion-exchange


resin improved peptide recovery over strong cation-exchange(SCX) resins alone and showed better orthogonality thanreversed-phase separations in two-dimensional separationsThe application of this method to phosphopeptide-enrichedsamples increased phosphopeptide identifications by 94over SCX alone Unlike previous methods that used anionexchange to change selectivity or enrich phosphopeptidesthe proposed format was unique for it works with typicalacidic buffer systems which could be used with electrosprayionization making it feasible for online multidimensionalLC-MSMS applications In this study the proposed anionand cation exchange mixed-bed system was effective simpleto implement and useful for a variety of analyses includingonline chromatographic enrichment of acidic phosphopep-tides Liu et al [43] developed and validated a rapid directand stability-indicating method for analysis of etidronatea bisphosphonate compound without UV chromophore inwhich amixed-mode columnwas used to separate etidronatefrom its impurities in an 8min gradient method and acharged aerosol detector (CAD) was used for detectionIn this study various columns with strong ion-exchangecharacteristics were explored and the mixed-mode columnsprovided satisfactory separation and retention for all peaks ofinterest Etidronate exhibited four pKa values (135 287 703and 113) that spanned the entire pH range which resultedin multiple charged ions in solution and caused poor peakshapeThe peak shapes were significantly improved when thenew mixed-mode anion-exchange reversed-phase columnPrimesep SB was used This might be attributed to the factthat the Primesep SB packing particles were bound using abulky basic group with a pKa value around 13 and there wasmore space between ligands Compared with other publishedmethods for separation of etidronate [41 44] this methoddemonstrated sufficient sensitivity under the condition thatneither ion-pair reagents nor time-consuming derivatizationoperation was used

The capability of ion-exchange liquid chromatographyto separate certain ionized analytes from other hydrophilicmolecules in biological matrices as well as its high tolerancefor salts significantly simplifies the extraction of biologicalsamples These unique advantages confer on ion-exchangechromatography an indispensable role in the pharmacoki-netic and pharmacodynamic studies of strong acidic drugsBut this technique has the same problem as that of ion-pair liquid chromatography that is to say it is generally notcompatible with ESI-MS because of the use of nonvolatilemobile phase solvents buffers or salts

6 Hydrophilic Interaction LiquidChromatography

Hydrophilic interaction chromatography (HILIC) may be aninteresting alternative to reversed-phase liquid chromatog-raphy for the separation of strong hydrophilic compoundsThe HILIC mode was introduced by Alpert in 1990 [45]and later used in tandem with ESI-MS to separate andcharacterize hydrophilic low-molecular-weight compoundssuch as amino acids peptides glycoconjugates and organic

acids without derivatization [46 47] In the HILIC mode anaqueous-organic mobile phase (containing high proportionof organic solvent) is used with a hydrophilic stationaryphase to provide seemingly normal-phase retention behavior[45 48] and hydrophilic compounds are retained longerthan hydrophobic ones and the hydrophilic mobile phasecomponent (usually water) is the stronger solvent

Goutier et al [49] developed and validated a method forthe determination of cAMP ATP (adenosine triphosphate)and other nucleotides in a biological system by combiningzwitterionic hydrophilic interaction liquid chromatographyand tandem mass spectrometry (MSMS) Because of theextreme hydrophilicity of nucleotides especially the pres-ence of (multiple) phosphate groups most LC methodsuse anion-exchange chromatography or reversed station-ary phases combined with ion-pair agents Unfortunatelyboth of the two methods are not compatible to be usedin combination with MSMS analysis Instead hydrophilicinteraction liquid chromatography is able to separate veryhydrophilic compounds without the addition of ion-pairagents or other nonvolatile modifiers into the mobile phasewhich makes it compatible with mass spectrometry analysisMost of the biologically active cyclic nucleotides can beused as zwitterions for their purine base and phosphategroups and potentially be analyzed in positive or negativeelectrospray ionizationmode De Person et al [50] developedand validated a procedure based on hydrophilic interactionchromatography coupled to tandem mass spectrometry forthe simultaneous determination of underivatized taurineand methionine in fluid samples Satisfactory separationwas obtained on an Astecap Hera NH

2column (150mm

times 46mm 5 120583m) with methanol-water (60 40 vv) as themobile phaseThis method could be applied to other types ofbiological samples such as plasma or urine In another study asensitive liquid chromatography tandem mass spectrometry(LC-MSMS)method based on aWaters Atlantis HILIC silicacolumn equipped with a Phenomenex C

18(ODS) column

was developed to simultaneously quantify amino acids andmyoinositol (eg taurine and phosphocholine) in mousebrain [2] A HPLC method based on HILIC was developedby Mora et al [51] for the simultaneous analysis of adenosinetriphosphate (ATP) adenosine diphosphate (ADP) adeno-sinemonophosphate (AMP) inosinemonophosphate (IMP)inosine (Ino) hypoxanthine (Hx) and nicotinamide ade-nine dinucleotide (NAD+) in meat samples A comparisonbetween the concentrations of the compounds measured byHILIC using a ZIC-pHILIC column and by IP-RP-HPLCusing a Zorbax Eclipse XDB-C

18column was also made in

which very good agreement between the two sets of datawas obtained Thus being a valid and reliable method toanalyze ATP and its metabolites HILIC can be considered asan interesting alternative to other methodologies

7 Capillary Electrophoresis

The versatility and a variety of modes of capillary elec-trophoresis (CE) imply that almost all molecules and evenwhole organisms can be separated using sorts of powerful


CE modes such as capillary zone electrophoresis (CZE)isotachophoresis micellar electrokinetic chromatography(MEKC) isoelectric focusing and capillary electrochro-matography (CEC) This makes CE quite useful in situationswhere other liquid phase separation techniques are limited orimpracticalThemain advantages of capillary electrophoretictechniques include high separation efficiencies low sampleconsumption short analysis time low reagents consumptionapplicable automation and low volumes of waste [52 53]

Metabolite identification and metabolite profiling areof major importance in the pharmaceutical and clinicalresearches However highly hydrophilic and ionic substancesare rarely included in most of these researches only becauseof the lack of applicable analytical methods Bunz et al[54] presented a method for the determination of urinarysulfates sulfonates phosphates and other anions of strongacids The method comprised a CE separation using anacidic butyl glycidyl ether (BGE) solution (pHle 2) andanodic detection by MS via negative ESI In this waysulfates and sulfonates were detected in the first part of theelectropherogram followed by phosphates and potentiallyhighly acidic carboxylates This method could be comple-mentary to the previous existing methods for metabolitecharacterization in urine Zinellu et al [55] reported anultrafast method to detect adenosine 51015840-triphosphate adeno-sine 51015840-diphosphate and adenosine 51015840-monophosphate in redblood cells using pressure-assisted capillary electrophoresis20120583mmolsdotLminus1 sodium acetate buffer at pH 380 was used asrunning electrolyte and the separation was performed usingthe condition of a CE voltage of 25 kV and an overimposedpressure of 02 psi from inlet to outlet In 2007-2008 theadulteration of raw heparin with oversulfated chondroitinsulfate (OSCS) produced a global crisis which resulted inextensive revisions to the pharmacopeia monographs andprompted the FDA to recommend the development of addi-tional methods for the analysis of heparin purity As a con-sequence a wide variety of innovative analytical approacheswere developed for the quality assurance and purity anal-ysis of unfractionated and low-molecular-weight heparinsElectrophoresis techniques were preferred for the sensitiveseparation detection and partial structural characterizationof heparin contaminants For the proposed CE methodprovides only partial separation of oversulfated chondroitinsulfate (OSCS) contaminant from heparin Somsen et al [56]developed an improved CEmethod that was especially usefulfor the reliable quantification of OSCS and dermatan sulfate(DS) impurities in heparin In this study parameters such astype and concentration of background electrolyte capillarytemperature sample concentration and injection volumewere all investigated and optimized Using high concentra-tions of Tris phosphate (pH 30) as background electrolyte incombination with a 25120583m internal diameter (ID) capillarythe presented method provided good separations of OSCSand DS from heparin without causing excessive currents andJoule heating Because of the possibility of injecting relativelylarge sample volumes and high concentrations of heparinOSCS could be detected down to the 01 level despite theweak UV absorbance of these sulfated glycosaminoglycansLoegel et al [57] developed a CE method for the separation

of heparin dermatan sulfate chondroitin sulfate (or hyaluro-nan) and the impurity of OSCS using polyamine-containingelectrolyte ethylenediamine (EDA) It was possibly one of thefirst CE methods which successfully made use of a 50 120583m IDfused silica capillary for analysis of OSCS impurity in heparinsamples Using the design optimization software the CErun buffer containing 200mM EDA and 455mM phosphatewas the best for peak resolution The migration time ofOSCS was the longest instead of shortest while compared topreviousmethods using Tris or lithium phosphate containingelectrolytes

A CE method for the determination of the ethanolconsumption marker (ethyl sulfate EtS) in human urinewas developed and validated [58] Analysis was performedin negative polarity mode with a background electrolytecomposed of 15mM maleic acid 1mM phthalic acid and005mM cetyltrimethylammonium bromide (CTAB) at pH25 This buffer system provided selective separation for EtSand vinylsulfonic acid (employed as internal standard) fromurine matrix components The proposed method seeminglywas a convenient and valuable alternative for the comparablyexpensive and tedious LC-MSmethods for the determinationof EtS in human urine In another study Caslavska et al[59] reported a confirmation analysis of ethyl glucuronide(EtG) and EtS in human serum and urine after intake ofalcoholic beverages using CZE coupled to sheath liquid-based electrospray ionization (ESI) and multiple-stage iontrap mass spectrometry (MSn) Electrophoretic separationswere performed in uncoated fused-silica capillaries using apH 95 ammonium acetate background electrolyte in normalpolarity mode CZE-MS and CZE-MS2 results obtained afterinjection of solid-phase extracts for EtG and EtS and ofdiluted urine confirmed the presence of EtG and EtS in sam-ples whose concentration levels were previously determinedby CZE with indirect UV detection

Tseng et al [13] demonstrated a simple and efficientmethod for the simultaneous separation and stacking of neu-rotransmitters in capillary electrophoresiswithUVdetectionBy using poly diallyldimethylammonium chloride (PDDAC)as a buffer additive the fast and reversed electroosmotic flow(EOF) was observed Moreover the mobility of indolaminesand catecholamines decreased as the PDDAC concentrationincreased Based on the difference of mobility betweenthe presence and absence of PDDAC the analytes weresimply stacked between the boundary of the sample zoneand the background electrolyte containing PDDAC For thedetermination of taurine in body fluids as well as in humanskin extract da Silva et al [60] developed simple methodsusing CE and the qualitative and quantitative comparisonsbetween fluorescence detection after precolumn derivatiza-tion and direct detection using integrated pulse amperometrywere performed

8 Discussion and Conclusion

For bioanalytical researchers it is really a difficult problem toseparate and analyze strong acidic compounds in biologicalmatrices The existence of phosphonic sulphonic groups in


their chemical structures brings strongly ionic charactersand remarkably increased hydrophilicity Moreover somemembers of this kind of compounds lack chromophoresfunctional groups thus convenient direct UV detection isoften impracticable Therefore resolving the problems inbioanalysis of strong acidic compounds may greatly promotethe pharmacokinetic and pharmacodynamic studies of thiskind of drugs as well as clinical applications of these com-pounds This review attempts to highlight the experienceswith successful establishment and application of separationmethods of strong acidic drugs and the challenges theresearchers are facing

In this review the methods using RP-HPLC IPLC ion-exchange LC HILIC CE and so forth for analysis of strongacidic compounds are reviewed Among these methodsRP-HPLC is the most powerful and popular method forthe analysis of moderately hydrophilic and hydrophobiccompounds but it cannot always satisfactorily separate thesehighly hydrophilic compounds due to the poor retention ofthese analytes on common reversed-phase chromatographiccolumns IPLC has been extensively used to selectivelyseparate ionizable and ionic organic compounds in varioussamples However this technique is generally not compatiblewith ESI-MS because of the use of nonvolatile mobile phasesolvents buffers or salts In addition even considering theavailability of relatively volatile ion-pairing reagents ionsuppression and other issues related to method ruggednessare still major problems when IPLC is combined with LC-MSfor the analysis of hydrophilic analytes in biological matricesIon-exchange chromatography is an excellent method forseparating charged molecules and accordingly is amenableto large multiple charged strongly hydrophilic compoundsThe unique advantages of ion-exchange chromatography(eg high tolerance for salts and significantly simplifiedsample extractions) make it an indispensable role in thebioanalysis of strong acidic compounds But this techniquehas the same problem as that of ion-pair liquid chromatog-raphy namely it is generally not compatible with ESI-MSHydrophilic interaction chromatography is able to separatevery hydrophilic compounds without the addition of ion-pairing agents and it can use mobile phases which arecompatiblewith ESI-MS Itmay be an interesting and efficientalternative technique to other methods (eg ion-pair andion-exchange chromatography) for the separation of stronglyhydrophilic compounds CE is an efficient technique forthe separation and quantification of both ionic and neutralspecies but the technology of interface between CE and MSstill needs to be improved Furthermore the high concentra-tions of salt in the electrolytes used in CE often lead to theloss of ESI-MS detection sensitivity

On the other hand sample preparation is of key impor-tance as it influences both selectivity via effective matrixremoval and sensitivity via preconcentration Dilute andshoot method is a simpler and time-saving approach forsample preparation But it is only suitable for the biologicalfluids (eg urine) which contain low concentrations ofproteins and high concentrations of target analytes and itcannot apply to most biological samples such as plasmaand serum In addition the direct dilute and shoot method

may lead to poor separation and detection sensitivity of theanalytes Protein precipitation is a widely accepted methodfor pretreatment of strong acidic compounds-containingbiological samples prior to liquid chromatographic analysisThis method has been proven quite effective but the solutionobtained after pretreatment often needs to be further concen-trated using other procedures such as LLE or SPE Liquid-liquid extraction is also a simple and efficient method forthe separation and concentration of relatively hydrophobiccompounds However for highly hydrophilic compoundssuch as the strong acidic compounds LLE is often not aneffective extraction procedure A strategy to the problem is touse certain strong acid tomodify the pH of the samplematrixor to add ion-pair reagents Alternative approaches includeSPE or combination of SPE and protein precipitation whenhigher selectivity is required Compared with protein precip-itation and LLE SPE is more reproducible easier to transferbetween the laboratories and more suitable for procedurestandardization and automation Additionally this methodhas better selectivity and enrichment capability higher pre-cision and accuracy and other prominent advantages Forsome highly hydrophilic compounds with poor stabilityor without chromophores they must be firstly degradedandor derivatized and then separated and quantified Thedegradation or derivatization methodology can transformhighly hydrophilic analytes into the moderately hydrophilicor hydrophobic forms thereby prolonging their retentiontime on the chromatographic column and improving thestability and analytical sensitivity of analytes But thesespecial methods are generally labor-consuming and time-costing and the more complex sample procedures may bringsome inevitable errors

Although LC-MS and LC-MSMS nowadays have thedominating role in bioanalysis applications of these tech-niques to the determination of highly hydrophilic com-pounds are still limited there are especially somany unsolvedproblems in the field of separation and determination ofstrong acidic drugs in biological samples In our opinionthe coming research needs to focus on creating some noveloriginal and pioneering theories which can bring completelydifferent separation modes The second best way is todevelop more salt-tolerant and sensitive detection methodsfor example making ion-pair and ion-exchange chromatog-raphy compatible with mass spectrometry Since samplepreparation plays an indispensable role in the bioanalysisof strong acidic drugs finding simpler and more effectivesample extraction procedures should not be ignored in thefuture

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (no 81102499) and Hunan Science andTechnology Project (no 2011SK3261)


References

[1] T King L Bushman P L Anderson T Delahunty M Rayand C V Fletcher ldquoQuantitation of zidovudine triphosphateconcentrations from human peripheral blood mononuclearcells by anion exchange solid phase extraction and liquidchromatography-tandemmass spectroscopy an indirect quan-titation methodologyrdquo Journal of Chromatography B AnalyticalTechnologies in the Biomedical and Life Sciences vol 831 no 1-2pp 248ndash257 2006

[2] S P Bathena J Huang A A Epstein H E Gendelman MD Boska and Y Alnouti ldquoRapid and reliable quantitation ofamino acids and myo-inositol in mouse brain by high perfor-mance liquid chromatography and tandemmass spectrometryrdquoJournal of Chromatography B Analytical Technologies in theBiomedical and Life Sciences vol 893-894 pp 15ndash20 2012

[3] N Volpi ldquoHigh-performance liquid chromatography and on-line mass spectrometry detection for the analysis of chon-droitin sulfateshyaluronan disaccharides derivatized with 2-aminoacridonerdquo Analytical Biochemistry vol 397 no 1 pp 12ndash23 2010

[4] A CMcGinnis B Chen andMG Bartlett ldquoChromatographicmethods for the determination of therapeutic oligonucleotidesrdquoJournal of Chromatography B Analytical Technologies in theBiomedical and Life Sciences vol 883-884 pp 76ndash94 2012

[5] S Gamagedara H Shi and Y Ma ldquoQuantitative determi-nation of taurine and related biomarkers in urine by liquidchromatography-tandem mass spectrometryrdquo Analytical andBioanalytical Chemistry vol 402 no 2 pp 763ndash770 2012

[6] C K Zacharis and P D Tzanavaras ldquoDetermination of bispho-sphonate active pharmaceutical ingredients in pharmaceuticalsand biological material a review of analytical methodsrdquo Journalof Pharmaceutical and Biomedical Analysis vol 48 no 3 pp483ndash496 2008

[7] POeckl andB Ferger ldquoSimultaneous LC-MSMS analysis of thebiomarkers cAMP and cGMP in plasma CSF and brain tissuerdquoJournal of Neuroscience Methods vol 203 no 2 pp 338ndash3432012

[8] E J C M Coolen I C W Arts E L R Swennen ABast M A C Stuart and P C Dagnelie ldquoSimultaneousdetermination of adenosine triphosphate and its metabolites inhuman whole blood by RP-HPLC and UV-detectionrdquo Journalof Chromatography B Analytical Technologies in the Biomedicaland Life Sciences vol 864 no 1-2 pp 43ndash51 2008

[9] R Losa M I Sierra M O Gion E Esteban and J MBuesa ldquoSimultaneous determination of gemcitabine di- andtriphosphate in human blood mononuclear and cancer cells byRP-HPLC and UV detectionrdquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol840 no 1 pp 44ndash49 2006

[10] J Huang S P R Bathena I L Csanaky and Y AlnoutildquoSimultaneous characterization of bile acids and their sulfatemetabolites in mouse liver plasma bile and urine using LC-MSMSrdquo Journal of Pharmaceutical and Biomedical Analysisvol 55 no 5 pp 1111ndash1119 2011

[11] Y Alnouti I L Csanaky and C D Klaassen ldquoQuantitative-profiling of bile acids and their conjugates in mouse liver bileplasma and urine using LC-MSMSrdquo Journal of Chromatogra-phy B vol 873 no 2 pp 209ndash217 2008

[12] A Liu Y Chen Z Yang et al ldquoNew metabolites of fenofi-brate in Sprague-Dawley rats by UPLC-ESI-QTOF-MS-basedmetabolomics coupled with LC-MSMSrdquo Xenobiotica vol 39no 4 pp 345ndash354 2009

[13] W-L Tseng S-M Chen C-Y Hsu andM-M Hsieh ldquoOn-lineconcentration and separation of indolamines catecholaminesand metanephrines in capillary electrophoresis using highconcentration of poly(diallyldimethylammonium chloride)rdquoAnalytica Chimica Acta vol 613 no 1 pp 108ndash115 2008

[14] C Y Chen RM FancherQ Ruan PMarathe AD Rodriguesand Z Yang ldquoA liquid chromatography tandem mass spec-trometrymethod for the quantification of indocyanine green indog plasma and bilerdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 47 no 2 pp 351ndash359 2008

[15] G Corona C Elia B Casetta et al ldquoLiquid chromatographytandemmass spectrometry assay for fast and sensitive quantifi-cation of estrone-sulfaterdquo Clinica Chimica Acta vol 411 no 7-8pp 574ndash580 2010

[16] I Bobeldijk M Hekman J de Vries-van der Weij et alldquoQuantitative profiling of bile acids in biofluids and tissuesbased on accurate mass high resolution LC-FT-MS compoundclass targeting in a metabolomics workflowrdquo Journal of Chro-matography B Analytical Technologies in the Biomedical and LifeSciences vol 871 no 2 pp 306ndash313 2008

[17] S J Mao S X Hou Z Liang et al ldquoIon-pair reversed-phaseHPLC assay validation of sodium tanshinone IIA sulfonatein mouse plasmardquo Journal of Chromatography B AnalyticalTechnologies in the Biomedical and Life Sciences vol 831 no 1-2pp 163ndash168 2006

[18] J Lai J Wang and Z Cai ldquoNucleoside reverse transcriptaseinhibitors and their phosphorylated metabolites in humanimmunodeficiency virus-infected human matricesrdquo Journal ofChromatography B vol 868 no 1-2 pp 1ndash12 2008

[19] H de Loor B K I Meijers T W Meyer et al ldquoSodiumoctanoate to reverse indoxyl sulfate and p-cresyl sulfate albuminbinding in uremic and normal serum during sample prepara-tion followed by fluorescence liquid chromatographyrdquo Journalof Chromatography A vol 1216 no 22 pp 4684ndash4688 2009

[20] R Calaf C Cerini C Genovesio et al ldquoDetermination ofuremic solutes in biological fluids of chronic kidney diseasepatients by HPLC assayrdquo Journal of Chromatography B Analyti-cal Technologies in the Biomedical and Life Sciences vol 879 no23 pp 2281ndash2286 2011

[21] J R Zgoda-Pols S Chowdhury M Wirth M V MilburnD C Alexander and K B Alton ldquoMetabolomics analysisreveals elevation of 3-indoxyl sulfate in plasma and brain duringchemically-induced acute kidney injury in mice investigationof nicotinic acid receptor agonistsrdquo Toxicology and AppliedPharmacology vol 255 no 1 pp 48ndash56 2011

[22] J Du and N Eddington ldquoDetermination of the chondroitinsulfate disaccharides in dog and horse plasma by HPLC usingchondroitinase digestion precolumn derivatization and fluo-rescence detectionrdquo Analytical Biochemistry vol 306 no 2 pp252ndash258 2002

[23] L Xu M Jiang and G Li ldquoInjection port derivatizationfollowing sonication-assisted ion-pair liquid-liquid extractionof nonsteroidal anti-inflammatory drugsrdquo Analytica ChimicaActa vol 666 no 1-2 pp 45ndash50 2010

[24] T LanH BiW Liu X Xie S Xu andHHuang ldquoSimultaneousdetermination of sphingosine and sphingosine 1-phosphatein biological samples by liquid chromatography-tandem massspectrometryrdquo Journal of Chromatography B Analytical Tech-nologies in the Biomedical and Life Sciences vol 879 no 7-8 pp520ndash526 2011

[25] C Emotte F Deglave O Heudi F Picard and O Kretz ldquoFastsimultaneous quantitative analysis of FTY720 and itsmetabolite


FTY720-P in human blood by on-line solid phase extractioncoupled with liquid chromatography-tandem mass spectrome-tryrdquo Journal of Pharmaceutical and Biomedical Analysis vol 58no 1 pp 102ndash112 2012

[26] D K Satonin J D McCulloch F Kuo and M P KnadlerldquoDevelopment and validation of a liquid chromatography-tandem mass spectrometric method for the determination ofthe major metabolites of duloxetine in human plasmardquo Journalof Chromatography B vol 852 no 1-2 pp 582ndash589 2007

[27] B L Robbins P A Poston E F Neal C Slaughter andJ H Rodman ldquoSimultaneous measurement of intracellulartriphosphate metabolites of zidovudine lamivudine and aba-cavir (carbovir) in human peripheral blood mononuclear cellsby combined anion exchange solid phase extraction and LC-MSMSrdquo Journal of Chromatography B Analytical Technologiesin the Biomedical and Life Sciences vol 850 no 1-2 pp 310ndash3172007

[28] L R Bushman J J Kiser J E Rower et al ldquoDeterminationof nucleoside analog mono- di- and tri-phosphates in cellularmatrix by solid phase extraction and ultra-sensitive LC-MSMSdetectionrdquo Journal of Pharmaceutical and Biomedical Analysisvol 56 no 2 pp 390ndash401 2011

[29] P Yeung L Ding and W L Casley ldquoHPLC assay with UVdetection for determination of RBC purine nucleotide concen-trations and application for biomarker study in vivordquo Journal ofPharmaceutical and Biomedical Analysis vol 47 no 2 pp 377ndash382 2008

[30] R Huang V H Pomin and J S Sharp ldquoLC-MSn analysis ofisomeric chondroitin sulfate oligosaccharides using a chemicalderivatization strategyrdquo Journal of the American Society forMassSpectrometry vol 22 no 9 pp 1577ndash1587 2011

[31] J Lu YWei andAMRustum ldquoA stability-indicating reversed-phase high performance liquid chromatography method forsimultaneous assay of two corticosteroids and estimation oftheir related compounds in a pharmaceutical injectable formu-lationrdquo Journal of Chromatography A vol 1217 no 44 pp 6932ndash6941 2010

[32] MMelendez O Rosario B Zayas and J F Rodrıguez ldquoHPLC-MSMS method for the intracellular determination of ribavirinmonophosphate and ribavirin triphosphate in CEMss cellsrdquoJournal of Pharmaceutical and Biomedical Analysis vol 49 no5 pp 1233ndash1240 2009

[33] S zur Nedden R Eason A S Doney and B G FrenguellildquoAn ion-pair reversed-phase HPLC method for determinationof fresh tissue adenine nucleotides avoiding freeze-thaw degra-dation of ATPrdquo Analytical Biochemistry vol 388 no 1 pp 108ndash114 2009

[34] I Lefebvre J-Y Puy C Perrin andC Perigaud ldquoQuantificationof zidovudine and its monophosphate in cell extracts by on-line solid-phase extraction coupled to liquid chromatographyrdquoJournal of Chromatography B Analytical Technologies in theBiomedical and Life Sciences vol 858 no 1-2 pp 2ndash7 2007

[35] D P Bhatt X Chen J D Geiger and T A Rosenberger ldquoAsensitive HPLC-based method to quantify adenine nucleotidesin primary astrocyte cell culturesrdquo Journal of ChromatographyB Analytical Technologies in the Biomedical and Life Sciencesvol 889-890 pp 110ndash115 2012

[36] M G Volonte G Yuln P Quiroga andA E Consolini ldquoDevel-opment of an HPLC method for determination of metaboliccompounds in myocardial tissuerdquo Journal of Pharmaceuticaland Biomedical Analysis vol 35 no 3 pp 647ndash653 2004

[37] A M Brustkern L F Buhse M Nasr A Al-Hakim andD A Keire ldquoCharacterization of currently marketed heparinproducts reversed-phase ion-pairing liquid chromatographymass spectrometry of heparin digestsrdquo Analytical Chemistryvol 82 no 23 pp 9865ndash9870 2010

[38] L Coulier H Gerritsen J J A van Kampen et al ldquoCompre-hensive analysis of the intracellularmetabolism of antiretroviralnucleosides and nucleotides using liquid chromatography-tandemmass spectrometry and method improvement by usingultra performance liquid chromatographyrdquo Journal of Chro-matography B Analytical Technologies in the Biomedical and LifeSciences vol 879 no 26 pp 2772ndash2782 2011

[39] L Coulier R Bas S Jespersen E Verheij M J van der Werfand T Hankemeier ldquoSimultaneous quantitative analysis ofmetabolites using ion-pair liquid chromatography-electrosprayionizationmass spectrometryrdquoAnalytical Chemistry vol 78 no18 pp 6573ndash6582 2006

[40] H Kojima M Inagaki T Tomita T Watanabe and S UchidaldquoImproved separation and characterization of lipopolysac-charide related compounds by reverse phase ion pairing-HPLCelectrospray ionization-quadrupole-mass spectrometry(RPIP-HPLCESI-Q-MS)rdquo Journal of Chromatography B Ana-lytical Technologies in the Biomedical and Life Sciences vol 878no 3-4 pp 442ndash448 2010

[41] Z Xie Y Jiang and D-Q Zhang ldquoSimple analysis of fourbisphosphonates simultaneously by reverse phase liquid chro-matography using n-amylamine as volatile ion-pairing agentrdquoJournal of Chromatography A vol 1104 no 1-2 pp 173ndash1782006

[42] A Motoyama T Xu C I Ruse J A Wohlschlegel andJ R Yates III ldquoAnion and cation mixed-bed ion exchangefor enhanced multidimensional separations of peptides andphosphopeptidesrdquo Analytical Chemistry vol 79 no 10 pp3623ndash3634 2007

[43] X-K Liu J B Fang N Cauchon and P Zhou ldquoDirect stability-indicating method development and validation for analysis ofetidronate disodium using a mixed-mode column and chargedaerosol detectorrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 46 no 4 pp 639ndash644 2008

[44] A Ismail S Aldous E J Triggs B A Smithurst and HD Barry ldquoGas chromatographic analysis of Didronel tabletsrdquoJournal of Chromatography A vol 404 pp 372ndash377 1987

[45] A J Alpert ldquoHydrophilic-interaction chromatography for theseparation of peptides nucleic acids and other polar com-poundsrdquo Journal of Chromatography vol 499 pp 177ndash196 1990

[46] M A Strege ldquoHydrophilic interaction chromatography-electrospray mass spectrometry analysis of polar compoundsfor natural product drug discoveryrdquo Analytical Chemistry vol70 no 13 pp 2439ndash2445 1998

[47] H Schlichtherle-CernyM Affolter and C Cerny ldquoHydrophilicinteraction liquid chromatography coupled to electrospraymass spectrometry of small polar compounds in food analysisrdquoAnalytical Chemistry vol 75 no 10 pp 2349ndash2354 2003

[48] T Yoshida ldquoPeptide separation by hydrophilic-interactionchromatography a reviewrdquo Journal of Biochemical and Biophys-ical Methods vol 60 no 3 pp 265ndash280 2004

[49] WGoutier P A SpaansMAW van derNeut A CMcCrearyand J H Reinders ldquoDevelopment and application of an LC-MSMSmethod formeasuring the effect of (partial) agonists oncAMP accumulation in vitrordquo Journal of Neuroscience Methodsvol 188 no 1 pp 24ndash31 2010


[50] M de Person A Hazotte C Elfakir and M LafosseldquoDevelopment and validation of a hydrophilic interactionchromatography-mass spectrometry assay for taurine andmethionine in matrices rich in carbohydratesrdquo Journal ofChromatography A vol 1081 no 2 pp 174ndash181 2005

[51] LMora A S Hernandez-Cazares M-C Aristoy and F ToldraldquoHydrophilic interaction chromatographic determination ofadenosine triphosphate and its metabolitesrdquo Food Chemistryvol 123 no 4 pp 1282ndash1288 2010

[52] K D Altria A Marsh and C Sanger-van de Griend ldquoCapillaryelectrophoresis for the analysis of small-molecule pharmaceu-ticalsrdquo Electrophoresis vol 27 no 12 pp 2263ndash2282 2006

[53] D S Hage ldquoAn overview of CE in clinical analysisrdquoMethods inMolecular Biology vol 919 pp 3ndash10 2013

[54] S-C Bunz W Weinmann and C Neusuess ldquoThe selectivedetermination of sulfates sulfonates and phosphates in urineby CE-MSrdquo Electrophoresis vol 31 no 7 pp 1274ndash1281 2010

[55] A Zinellu S Sotgia B Scanu et al ldquoUltra-fast adenosine51015840-triphosphate adenosine 51015840-diphosphate and adenosine 51015840-monophosphate detection by pressure-assisted capillary elec-trophoresis UV detectionrdquo Electrophoresis vol 31 no 16 pp2854ndash2857 2010

[56] G W Somsen Y H Tak J S Torano P M J M Jongenand G J de Jong ldquoDetermination of oversulfated chondroitinsulfate and dermatan sulfate impurities in heparin by capillaryelectrophoresisrdquo Journal of Chromatography A vol 1216 no 18pp 4107ndash4112 2009

[57] T N Loegel J D Trombley R T Taylor and N D DanielsonldquoCapillary electrophoresis of heparin and other glycosamino-glycans using a polyamine running electrolyterdquo AnalyticaChimica Acta vol 753 pp 90ndash96 2012

[58] F A Esteve-Turrillas W Bicker M Lammerhofer T Kellerand W Lindner ldquoDetermination of ethyl sulfatemdasha markerfor recent ethanol consumptionmdashin human urine by CE withindirect UV detectionrdquo Electrophoresis vol 27 no 23 pp 4763ndash4771 2006

[59] J Caslavska B Jung andWThormann ldquoConfirmation analysisof ethyl glucuronide and ethyl sulfate in human serum andurine by CZE-ESI-MS119899 after intake of alcoholic beveragesrdquoElectrophoresis vol 32 no 13 pp 1760ndash1764 2011

[60] D L P da Silva Y Mrestani H H Ruttinger J Wohlraband R Neubert ldquoComparison between fluorescence and pulsedintegrated electrochemical detectors for the determination oftaurine in human skin urine and plasma by CErdquo Chro-matographia vol 67 no 9-10 pp 813ndash817 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

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Analytical Methods in Chemistry

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Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Medicinal ChemistryInternational Journal of


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ElectrochemistryInternational Journal of



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compounds in biological fluids and tissues special techniquesare required to extract the target analytes from biologicalmatrices to separate them from interfering materials andthen to quantify and characterize them On related topics theseparation methods for bisphosphonate active pharmaceu-tical ingredients in pharmaceuticals and biological materialwere reviewed in 2008 by Zacharis and Tzanavaras [6] andchromatographic separation methods for the determinationof therapeutic oligonucleotides were reviewed by McGinniset al in 2012 [4]

Trying to find proper separation methods to resolve theaccurate and precise quantification of strong acidic drugsin complex biological matrix samples we searched andanalyzed the related information in dozens of academicarticles ranging from 2002 to date In this review we focus onbioanalyticalmethods (especially chromatographicmethods)for analyzing strong acidic compounds mainly organophos-phatic compounds and organosulphonic compounds Thereview covers analytical methods using liquid chromatogra-phy (eg ion-pair ion-exchange and hydrophilic interactionchromatography) capillary electrophoresis (CE) and otheranalytical methods Due to the extremely low concentrationlevels of target analytes as well as the complexity of biolog-ical matrices sample pretreatment methods such as diluteand shoot method protein precipitation (PP) liquid-liquidextraction (LLE) solid-phase extraction (SPE) degradationand derivatization strategy are also explored

2 Sample Preparation

Generally biological samples such as urine serumor plasmaand tissues should not be directly injected into a separationsystem due to matrix complexity To resolve the problema sequence of sample treatment steps is performed priorto analysis The main purposes of sample pretreatment areto remove unwanted interfering substances suppress matrixeffects and enhance selectivity convert the analytes to a com-patible form for analysis and if possible increase sensitivitythrough preconcentration [6]

Dilute and shoot method is one of the simplestapproaches for sample preparation Using this methoda fast sensitive and noninvasive LC-MSMS techniquewas developed and validated to separate and quantitativelydetermine taurine and related biomarkers in urine matricesby Gamagedara et al [5] This method is simpler and moretime-saving than other sample preparation methods and itis much suitable for the biological fluids like urine whichcontains relatively high concentration levels of target analytesas well as a small quantity of proteins and other endogenoussubstances But dilute and shoot method is hard to apply toother biological samples such as plasma and serum

Protein precipitation is a simple procedure to removeproteins from plasma and other biological matrices Brieflythe reagents that are used to precipitate proteins frombiological samples mostly include inorganic acids organicacids and organic solvents such as perchloric acid (PCA)[7 8] trichloroacetic acid (TCA) [9] acetonitrile (ACN)[10ndash15] and methanol [2 16 17] After adding protein

precipitating reagent the mixture is usually needed to becentrifuged to remove the denatured protein [18] Usingtrichloroacetic acid as protein precipitation reagent Losa etal isolated gemcitabine di- and triphosphate in peripheralblood mononuclear cells (PBMC) [9] In this study thetemperature was controlled at 4∘C during sample preparationtomaintain the stability of nucleotide triphosphates and afterprotein precipitation Freon (112-trichlorotrifluoroethane)-trioctylamine (4 1 vv) was used to yield clean extractsand it neutralizes the samples Protein precipitation can alsoattain other objectives simultaneously For instance sodiumoctanoate was used as binding competition to replace p-cresyl sulfate and indoxyl sulfate from albumin followedby protein precipitation for sample preparation [19] Thismethod allowed direct quantification of indoxyl sulfate andp-cresyl sulfate which are protein-bound marker moleculesin chronic kidney disease Chen et al [14] used acetonitrile toprecipitate protein while extracting indocyanine green fromdog plasma and bile The high efficiency of protein precipi-tation has also been proved in many studies while preparingsorts of strong acidic drug samples with different biologicalmatrices including serum [16 19 20] plasma [7 10 12 14ndash17 21] cerebrospinal fluid [7] urine [12 13 16] bile [20]brain tissue [2 7 21] PBMC [9] and liver [10 16] Howeverthe dilution effects of adding protein precipitation reagentssometimes become a problem when the concentration levelsof target analytes in biological matrices are extremely lowUnder these conditions the samples are often needed to befurther concentrated by using procedures such as liquid-liquid extraction (LLE) and solid-phase extraction (SPE) toimprove method sensitivity before sample analysis

LLE is a simple and efficient method for the separationand concentration of relatively hydrophobic compoundsHowever most of strong acidic drugs in biological fluidsand tissues present in phosphorylated sulfated or sulfonatedforms so the strong acidic drugs are usually classified ashighly hydrophilic compounds In many cases LLE is not aneffective sample preparation method for these compoundsThus it is nearly impossible to extract them with organicsolvent from biological samples A strategy to this problem isto add certain strong acid to adjust the pH value so that theirionization is suppressed and they can be treated as hydropho-bicmolecules For exampleDu andEddington [22] describeda LLE method using trifluoroacetic acid as ionization-suppressing reagent for the extraction of chondroitin sulfatedisaccharides Actually controlling the pH values is notalways an effective way to suppress ionization for stronglyacidic compounds Therefore adding proper counterion toperform the ion-pair extraction becomes a better choice inwhich the target analytes and the counterions are combinedinto ion-pair complexes which can be easily extracted withusual organic solvents Briefly adding a special ion-pairreagent into the sample solution allows target analytes to formion-pair complexes with the counterions of opposite chargeand then the ion-pairs possess higher partition coefficientsthan the original analytes and as a result the transferringof the target drugs into the extractant (organic) phase isenhanced Xu et al [23] applied an ion-pair liquid-liquidextractionmethod while successfully extracting nonsteroidal




18SPE columns





























2column (150mm


18(ODS) column






















Acknowledgments



References









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




18SPE columns





























2column (150mm


18(ODS) column






















Acknowledgments



References









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






















2column (150mm


18(ODS) column






















Acknowledgments



References









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of








2column (150mm


18(ODS) column






















Acknowledgments



References









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

















Acknowledgments



References









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of









Acknowledgments



References









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


References









































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of
















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Documents

Review Article Determination of Strong Acidic Drugs in ...downloads.hindawi.com/archive/2014/469562.pdf · p-cresyl sulfate which are protein-bound marker molecules inchronickidneydisease.Chenetal.[