Chiralcapillaryelectrophoresis–electrospraymassspectrometry
couplingusingvancomycinaschiralselector
SalvatoreFanalia,ClaudiaDesiderioa,GeorgSchulteb,StefanHeitmeierb,
bb,1b,DirkStrickmann,BezhanChankvetadze,GottfriedBlaschke*
aIstitutodiCromatografiadelC.N.R.,AreadellaRicercadiRoma,P.O.Box10,00016MonterotondoScalo,Rome,Italy
b¨¨InstituteofPharmaceuticalChemistry,UniversityofMunster,Hittorfstrasse58-62,48149Munster,Germany
Abstract
Capillaryelectrophoresis–electrospraymassspectrometrycoupling(CE–ESI-MS)usingvancomycin(VC)asachiral
selectorisdescribedinthisstudy.TheselfelectrophoreticmobilityofVC,asapositivelychargedchiralselectoratlowpH,allowsustoavoidthecontaminationoftheionsourceoftheESI-MSwiththechiralselectorinasimpleway.Theusefulnessofthistechniqueisillustratedfortheanalysisofchiralanioniccompoundsofpharmaceuticalimportance.Additionally,examplesofastereospecificdeterminationofibuprofenanditsphaseImetabolitesaswellasetodolacanditsmetabolitesinurinesamplesaredescribed.TheadvantagesofMSforpeakidentification,peakpuritytestingandforselectivemonitoringofoverlappingpeaksaredemonstrated.©1998ElsevierScienceB.V.
Keywords:Enantiomerseparation;Chiralselectors;ChiralCE–ESI-MS;Vancomycin;Arylpropionicacids;Etodolac;Ibuprofen
1.Introduction
Theseparationofenantiomericcompoundscanbeeasilyachievedbycapillaryelectrophoresis(CE)usingthedirectseparationmethod,e.g.,byadditionofthechiralselectortothebackgroundelectrolyte(BGE).
AwidenumberofchiralselectorshavebeenusedinCE[1–3]suchascyclodextrinsandtheirderiva-tives,chiralsurfactants,proteins,metal–aminoacidcomplexes,crownethers,andrecentlyantibiotics[4–11].
ChiralCE–massspectrometry(MS)couplingisdevelopingwithinthelastfewyears[12–14].MSis
*Correspondingauthor.1Permanentaddress:DepartmentofChemistry,TbilisiStateUniversity,ChavchavadzeAve1,380028,Tbilisi,Georgia.sensitive,specificandatthesametimeauniversaldetectionsystem.Additionally,MSprovidesimpor-tantinformationaboutthestructureofanalyteswhichisespeciallyusefulfortheidentificationofmetabolitesinclinicalandbiopharmaceuticalanaly-ses.On-lineMScouplingisadvantageousinCEbecausefractioncollectionforfurtheranalysisisverytime-consumingandmoredifficultinthistechniquecomparedwithhigh-performanceliquidchromatography(HPLC).
InchiralCE,thepresenceofchiralselectorsintheBGEmaycausesomeproblemsduetotheinterfer-encewithanalytes(UV)orcontaminationofthedetector(MS).TheappearanceofthechiralselectorintheionsourceoftheMSmaydecreasethesensitivityduetothecompetitionwithananalyteforavailablechargeaswellasduetotheincreaseofthebackgroundnoise[12,13].Toavoidtheappearance
0021-9673/98/$19.00©1998ElsevierScienceB.V.Allrightsreserved.PIIS0021-9673(97)00978-3
70S.Fanalietal./J.Chromatogr.A800(1998)69–76ofchiralselectorintheionsourcetheuseofcolumn-couplingandmultiplevoltageswitchingtechniquehasbeenproposedrecently[13].Analternativeapproachcanbebasedontheoppositemigrationdirectionofananalyteandachiralselector.ThisprinciplewasproposedforUV-adsorbingchiralselectorsbyValtchevaetal.[15]andinvolvedfillingonlypartoftheseparationcapillarywiththechiralselector.Aslightlymodifiedversionofthistech-niqueallowingthefillingofthewholecapillarywithachiralselectorwasdescribedinRef.[16].TheusefulnessofthepartialfillingtechniqueinchiralCEwithUVdetectionhasbeendemonstratedinanumberofstudies[8,9,11,17].Thetechniquepro-posedinRef.[16]hasbeenrecentlyusedalsoinchiralCE–electrospray(ESI)-MScouplinginordertoavoidtheappearanceofchargedcyclodextrinsintheionsource[14].
InthispaperachiralCE–ESI-MScouplingfortheseparationofseveralracemicarylpropionicacids
(APAs)employingvancomycin(VC)asachiralselectorisreported.
2.Experimental
2.1.Chemicalsandreagents
Aceticacid,ammoniumacetate,ammoniumhy-droxideandmethanolwerepurchasedfromMerck(Darmstadt,Germany).Racemicflurbiprofen(FP),ketoprofen(KP)andcarprofen(CP)werefromSigma(Daisenhofen,Germany).(1)-and(2)-Nap-roxen(NP)werekindlyprovidedbyDr.CeciliaBartolucci,IstitutodiStrutturisticaChimica,C.N.R.(Montelibretti,Rome,Italy).
Racemicetodolac(ET)anditsphaseImetabo-lites,7-hydroxyetodolac(7-HET)and8-(19-hydroxy-ethyl)etodolac(8-HET)weregiftsfromWyeth
¨Pharma(Munster,Germany).Racemicibuprofen
Fig.1.Chemicalstructuresofthecompounds.
S.Fanalietal./J.Chromatogr.A800(1998)69–7671
(IB)anditsphaseImetabolites,2-hydroxyibuprofen(HIB)andcarboxyibuprofen(CIB)weregiftsfrom
¨Kanoldt(Hochstadt/Donau,Germany).Thestruc-turesofthechiralcompoundsusedinthisstudyare
showninFig.1.
VC,methacrylicacid3-trimethoxysilylpropylester,ethylenediaminetetraaceticacid,acrylamide,sodiumpersulfateandN,N,N9,N9-tetramethyl-ethylenediaminewereobtainedfromFluka(Buchs,Switzerland).
Stocksolutionsofracemicsamples(1mg/ml)werepreparedinmethanol,storedat48Canddilutedto50–100mg/mlinacetatebufferpH4.8beforetheanalysis.50mMAceticacid–ammoniumacetate,pH4.8,containingvariousamountsofVCwastheBGEusedfortheenantioseparationofthestudiedAPAs,ETortheirmetabolites;theBGEscontainingthechiralselectorwerefreshlyprepareddaily.
operatedinaconstantvoltagemodeandtheelectro-phoreticrunswereperformedinapolyacrylamidecoatedcapillary[18]of44cm350mmI.D.filledeitherwith50mMaceticacid–ammoniumacetatebufferpH4.8orthesamebuffersupplementedwiththeappropriateamountofVC.Hydrostaticinjection(10cm)for5–10satthecathodicendofthecapillarywasusedinallexperiments.Theotherendoftheseparationcapillarywasprotrudedtotheionspraytipwhereavoltageof2.6kVwasmaintained.ALCQiontrapmassspectrometer(Finnigan,Bran-ford,CT,USA)equippedwithanelectrosprayinterfacewasusedinthenegativeionmodeforthedetectionofanalytes[12–14].Thesheathliquidconsistingofmethanol–water–ammonia(50:48:2)wasdeliveredataflow-rateof6ml/minusingasyringepump.
2.2.Apparatus
AGromcapillaryelectrophoresissystem100highvoltagepowersupply(Herrenberg,Germany)was
3.Resultsanddiscussion
VCisoneofthemostpowerfulchiralselectorsfortheseparationofcompoundscontainingfreecar-
Fig.2.Schemeofthecounterflowelectrophoreticseparationoftheanionicanalytesusingvancomycinasthechiralselector.
72S.Fanalietal./J.Chromatogr.A800(1998)69–76Fig.3.CE–ESI-MSelectropherogramsofracemicnon-steroidal-antiinflammatorydrugs:(a)(6)-carprofen(CP),(b)(6)-flurbiprofen(FP),(c)(6)-ketoprofen(KP)and(d)(6)-naproxen(NP)inthepresenceof5mMofvancomycinintheBGE.Capillary(polyacrylamidecoated)44cm350mmI.D.;appliedvoltage,20kV;BGE,50mMaceticacid–ammoniumacetatepH4.8;injection,hydrostatic(10cm)310satthecathodicendofthecapillary.
S.Fanalietal./J.Chromatogr.A800(1998)69–7673
boxylicgroups[4,8–11].However,thehighab-sorbanceintheUVrangemaycausesignalinterfer-encewiththeanalyteandconsequentlysomesen-sitivityrelatedproblemsinCEwithUVdetection.Recently,thepartialfillingtechniquehasbeenproposedasthesolutiontothisproblem[8–11,17].AninterferencebetweenthesignalsofVCandtheanalytesisnottheprobleminCEwithMSdetection.However,theappearanceofthechiralselectorintheMSionsourcemaycauseadecreaseofdetectionsensitivitybycompetitionwiththeanalyteforanavailablechargeaswellasbyanincreaseofthebaselinenoiseduetothecontaminationoftheionsource[12,13].Therefore,itisdesirabletoavoidthepenetrationofnonvolatilebuffercomponentsintheMS.
AsrepresentedinFig.2thetechniquesimilartopartialfillingtechniquemayalsobeusefulinchiralCEwithMSdetection[14].Thistechniqueallowsustoavoidinasimplewayacontaminationoftheionsourcewiththechiralselectorandconsequentlyimprovesthesensitivityandstabilityoftheresponse.ThishasbeendemonstratedrecentlyforchiralCE–ESI-MScouplingusingchargedcyclodextrins[14].Aceticacid–ammoniumacetatebufferatpH4.8wasselectedfortheexperiments.ThisbuffermeetsseveralimportantrequirementsforchiralCE–MScoupling,namely:(i)itisvolatileandthuscompat-iblewiththeMSdetector,(ii)thestudiedanalytesmoveasanionsinarelativelyshorttimeand(iii)VC,whichispositivelychargedatthispH,migratesintheoppositedirectionofanalytesawayfromtheESI-MSsource.
InordertoconfirmthatthechiralselectordoesnotappearintheMSinterfacethefollowingexperimentwasperformed.TheseparationcapillarywasfilledwiththeBGEcontaining12.5mMVCandhighvoltagewasappliedsothattheanodewasontheMSsideofthecapillary.Thedetectionwasperformedalternativelyinbothinthepositiveandinthenegativeionmode.Onlytheminorpeakswithm/zrangingfrom700.6to760.5weredetectedinbothmodes.ItseemsworthnotingthatalthoughVCispositivelychargedintheseparationcapillarythesheathliquidusedfortheMSdetectionoftheanionicanalytesisalkalinewhichmaytransformthechiralselectortoitsanionicform.Thus,thisexperi-mentconfirmedundoubtedlythatthechiralselectordoesnotcontinuouslyentertheMS.ThetracesfoundareprobablyduetothepenetrationintotheMSduringfillingtheseparationcapillarywiththeBGEcontainingVC.
TheCE–ESI-MSelectropherogramsofracemicCP,FP,KPandNPusingtheBGEwith5mMVC
Fig.4.CE–ESI-MSelectropherogramsofracemic(a)etodolac(ET)anditsmetabolites,(b)7-hydroxyetodolac(7-HET)and(c)8-(19-hydroxyethyl)etodolac(8-HET)inthepresenceofvancomycinintheBGE.Experimentalconditions:5mMvancomycininaandband12mMinc;appliedvoltage15kVinaandband12kVinc.ForotherexperimentalconditionsseeFig.3.
74S.Fanalietal./J.Chromatogr.A800(1998)69–76areshowninFig.3.Verygoodenantiomericres-olutionwasachievedforallarylpropionicacids.Similarresultshavebeenobtainedfortheenantio-mericseparationofracemicETandtwoofitsphaseImetabolites(Fig.4).Theanalysistimeoftheseparatedenantiomerswasrelativelyshort(lessthan15min)andthepeakefficiencyhigh(90000–110000plates/m).Onlytwoamongfourpossible8-HETstereoisomerswereresolved.
Themasstrackscorrespondingtomolecularmass-esofIB(m/z204.5–205.5),anditsphaseImetabo-lites,2-HIB(m/z220.5–221.5)andCIBs(m/z234.5–235.5),fromthefull-scanCE–ESI-MSspec-traoftheirmixtureareshowninFig.5.Theadditionof5mMofVCintheBGEallowedtheenantio-separationofIB,2-HIBandallfourstereoisomersofCIB.However,peakoverlappinghasbeenobservedforIBand2-HIB.Inspiteofthesevereoverlappingofthemigrationzonestheselectionofcorrespondingmasstracksallowsustoperformpeakidentificationofthecompoundpossessingdifferentmolecularmasses.ThisisoneoftheimportantadvantagesofMSasadetectionsystemwhensamplesofcomplexoriginareanalyzed.Anadditionaladvantageofthis
Fig.5.CE–ESI-MSelectropherograms(selectedmasstracks)andfullscanESI-MSspectra(b)ofselectedpeaksofracemicibuprofen(IB),2-hydroxyibuprofen(2-HIB)andcarboxyibuprofens(CIB).ExperimentalconditionsasinFig.3.
S.Fanalietal./J.Chromatogr.A800(1998)69–7675
detectionprincipleistheinformationaboutpeakpurity.ThiscanbedonebyscanningthefullMSspectraofeachpeak.AsindicatedbytheMSspectra(Fig.5b)thepeakofIBenantiomerwiththemigrationtime12.63containstheimpurityofHIB(m/z5221)andthefirstpeakofthelatterone(t512.69)ismarkedlycontaminatedwithIB(m/z5205).Thesecondpeaksofbothcompounds(t513.36and13.15)seemtoberelativelypure.
AnotheradvantageofCE–MScouplingistheon-lineidentificationofresolvedpeakswithoutanysamplecollection.Forinstance,itisobviousthatallfourpeakswithm/z5234.5–235.5belongtothestereoisomersofCIB.
Becker-ScharfenkampandBlaschke[19]studiedthephaseIandthephaseIImetabolismofthenonsteroidalantiinflammatorydrugET.EvidenceforastereoselectiveeliminationofET-acylglucuronideand7-hydroxyetodolac-acylglucuronide(7-HETG)werefoundbyindirectdeterminationofthediffer-encesoftheconcentrationsofETand7-HETbeforeandafteralkalinehydrolysisoftheconjugatesusingHPLC.Later,BerendesandBlaschke[20]developedaHPLCmethodwhichinvolvestheacetylationofhydroxygroupsandmethylationofcarboxygroupsinordertoincreasethelipophilicityofthepolaracylglucuronidesofETand7-HET.Bothofthesetechniquesinvolveatediousandtime-consumingsampleextractionandpretreatment.Therefore,thedirectanalysisoftheurinesamplewithoutanysampleextractionandpretreatmentwasattempted.AsshowninthechiralCE–ESI-MSelec-tropherogramofaurinesampleofavolunteerreceivingasingle400mgdoseofracemicET(Fig.6)thesinglepeaksofETG(m/z61.9),oneoftheisomericHETs(m/z5302.1)andoneoftheglucuro-
Fig.6.CE–ESI-MSelectropherograms(selectedmass-tracks)(a)andfullscanESI-MSspectra(b)correspondingtoeachpeakofaurinesampleofavolunteerreceivinga400mgsingleoraldoseofracemicetodolac.Thecumulativeurinesamplewascollectedin3–7hafterdrugadministrationandwasdirectlyintroducedintotheseparationcapillarybyhydrodynamicinjection(10cm)for10s.Theconcentrationofvancomycinwas12.5mM;theappliedvoltage15kV;otherexperimentalconditionswereasinFig.3.
76S.Fanalietal./J.Chromatogr.A800(1998)69–76nidesoftheisomericHETs(m/z77.9)couldbeobservedintheurinetogetherwithendogenoushippuricacid(m/z5178.2).Accordingtopreviousresults[19,20]thepeakwithm/z5302.1mostlikelybelongstooneoftheenantiomersof7-HETandthepeakwithm/z77.9tothecorrespondingglucuro-nide.Furtherstudiesonthissubjectareinprogressnow.
Thus,asshowninthispreliminarystudychiralCE–ESI-MScouplingrepresentsaveryeffectivealternativeforexpensiveandtime-consumingHPLCmethodsfortheseparation,identificationandproba-blyalsoforthequantificationofchiraldrugsandtheirphaseIandphaseIImetabolitesinbiologicalmatrices.Insomecasesthebiologicalfluidscanbedirectlyinjectedintotheseparationcapillarywithoutanysamplepretreatment.
Acknowledgements
B.C.thankstheRectorateoftheUniversityofMunster¨forthefinancialsupportofhisstayasavisitingprofessorattheInstituteofPharmaceuticalChemistryofthisuniversity.Theshort-termstayofS.F.attheInstituteofPharmaceuticalChemistry,
UniversityofMunster,¨wasfinanciallysupportedby
DAAD(GermanSocietyforAcademicExchange,Bonn,Germany).
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