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Rapid quantification of the metabolite of valacyclovir hydrochloride in human plasma by liquid chromatography-tandem mass spectrometry

By Joseph Ramirez,2014-01-11 16:07
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Rapid quantification of the metabolite of valacyclovir hydrochloride in human plasma by liquid chromatography-tandem mass spectrometry

    Rapid quantification of the metabolite of

    valacyclovir hydrochloride in human plasma by liquid chromatography-tandem

    mass spectrometry

    AcademicJournalofXi'anJiaotongUniversity

    Volume22,Number2,May2010,PP8390

    Rapidquantificationofthemetaboliteofvalacyclovirhydrochloridein humanplasmabyliquidchromatographytandemmassspectrometry

    YuanTian'.HuiLin',Xue.YuZhang,,ZunJianZhang'.,Guo-GuangMao.

    1.KeyLaboratoryofDrugQualityControlandPharmacoVigi1ance(ChinaPharmaceuticalUniversity),

    MinistryofEducation,Nanjing210009;2.CenterforInstrumentalAnalysis,.ChinaPharmaceuticalUni?

    versity,Nanjing210009;3.DepartmentofClinicalPharmacology,WannanMedicalCollege,Wuhu

    241000,China.

    ABSTRA0T:ObjectiveToestablisharapid,sensitiveandselectiveIiquidchromatography-tandemmassspectrometry

    (LC.MS/MS)methodforthedeterminationofacyclovir(themetaboliteofvalacyclovirhydrochloride)inhumanplasma.

    MethodsAfteradditionofganciclovirasinternalstandard(IS),plasmasampleswerepreparedbyonmstepproteinprecipita-

    tionusingacetonitrileasprecipitant.foflowedbyanisocraticelutionwith0.1%formicacidsolution.methanol(95:5,v/v)

    onanAgilentZORBAXSB-C18(150mm×

    2.1mmi.d.,3.5m)column.Detectionwasperformedonatriple-quadrupole

    massspectrometerutilizingelectrosprayionization(ESI)interfaceoperatinginpositiveionandselectedreactionmonitoring

    (SRM)modewiththeprecursortoproductiontransitionsm/z226.2--,-152.1f0racyclovirandm/z256.2?152.1f0rthe

    IS.ResultsTheanalyticalresultsdemonstratedagoodlinearityovertherangesfrom0.005to4/~g/mL(r0.9999)for

    valacyclovirhvdrochloride.Therelativestandarddeviations(RSD)ofintra-batchandinter.batchwereIessthan4.06%and

    9.23%,respectively.Thelimitofdetectionandlowerlimitofquantificationinhumanplasmawere2ng/mLand5ng/mL,

    respectively.Conclusion111emethodwassimple,sensitive,accurateandreproducibleandhasbeensuccessfullyappliedto

    abioequivalencestudvofvalacyclovirhydrochloridecapsulesinChinesehealthymalevolunteers.

    KEYW0RDS:valacyclovirhydrochloride;acyclovir;liquidchr0matography_tandemmassspectrometry;methodvalida

    tion:humanplasma

    1IntrOducti0n

    Valacyclovir(Fig.1A),theL-valylesterof

    acyclovir,isanoralprodrugthatundergoesarapid

    andextensivefirstpassmetabolismtoyieldacyclov-

    irandessentialaminoacidL-valine.Acyclovir

    (Fig.1B),theactiveantiviralcomponentofvalacy-

    clovir,hasbeendemonstratedactivityagainstthe

    herpesviruses,suchasherpessimplexvirus(HSV)-

    1.HSV-2andvaricellazostervirus(VZV)[.Ac

    cordingtothefavorabletoxicityandpoorabsorp-

    tion,manypotentialapplicationsofacyclovirare

    limited.Acyclovirisneitherhighlylipidnoraque-

    OUSsoluble,anditsoralbioavailabilityisreportedto

    bebetween15%and30%.ThebioaVailabi1itvof acyclovirfromoraladministrationofvalacycloviris threetofivetimeshigherthanthatfromoralad- ministrationofacyclovir[.Thereby,theplasma concentrationofacyclovirisdeterminedduringthe pharmacokineticstudyofvalacyclovir.

    Themechanismofacyclovirinvolvesthehigh- lyselectiveinhibitionofherpesvirusDNAreplica- tion,viaenhanceduptakeinherpesviru~infected cellsandphosphorylationbyviralthymidinekinase (TK).DNApolymerasecontributestothesubstrate specificityofacyclovirtriphosphateforviralrather thancellular.Duetoitsaffinitytotheenzymes TK,whichisencodedbyHSVandVZV,thein-

    hibitoryactivityofacyclovirishighlyselective. Neithervalacyclovirnoracyclovirismetabolizedby cytochromeP450enzymes[.

    Sinceacyclovirisprimarilyeliminatedbythe kidney,theplasmaconcentrationmayexceedthe therapeuticrange,andfinallyleadstoaseriousneu rologicaltoxicityandimpairsrenalfunction.On theotherhand,tOOlowplasmaconcentrationmay causefailureintreatingofthepatientswhoaresuf- feringfromherpessimplexandherpeszoster[_5]. Forthisreason,asimpleandsensitivemethod, whichcanbeusedtodetermineacyclovirinhuman plasmafortherapeuticdrugmonitoring(TDM) rapidly,maybeefficienttoassessthecomplianceor evaluatetherisksofthetoxicity.

    Previousstudieshavereportedthequantifica-

Correspondingauthor:Zun

    JianZhang,professor.E-mail:zunjianzhangcpu@hotmail.corn

    Biography:YuanTian,female,bornin1975,master'sdegree,researchassistant,specializingi

    nresearchonpharmaceuticalanalysis

    E-mail:tiancpu@sina.eom

    ?

    84?

    tionofacyclovirinbiologicalfluidsbyhighper- formanceliquidchromatography(HPLC)with UV,fluorescence(F),massspectrometry(MS) andamperometric(A)detectors,high-performance capillaryelectrophoresis(HPCE),immunological techniquesandnearinfraredspectroscopy(NIRS). However.theHPLC-UVE"]andHPLC-F[meth

    odshadsomecommonlimitations:lowsensitivity, longchromatographicruntimeand/orlengthyex- tractionprocedure.Inaddition,becauseoftheuse ofion-pairingagentsandgradientelution,these methodshavehadlongturnaroundtimeintheir proceduresandalimitedcolumnlife.Kishinoet a1.[.]determinedganciclovirand/oracyclovirinhu- manplasmabyHPLC-ADandYueta1.[103deter

    minedacyclovirinplasmabyNIRS,whilethe methodusedinbiologicalsampleshigh-throughput analysiswasdisputed.Afewmethodologieswhich includeradioimmunoassayandenzyme-linkedim

    munosorbentassayhavebeenreportedtoquantify theantiviraldruginbiologicalfluids[_.Dueto therequirementofmanytediousstepstoobtainthe finalquantitativeresults,.

thesetechniqueshaveob

    viousdisadvantages.

    Nowadays,LC-MS/MSmethodshavebeen

    widelyspreadedduetotheirhighspecificity,sensi

    tivityandselectivity.Besides,manymethodologies inthisregardhavebeenreportedtoemployLC-MS/ MStodetermineacyclovirinhumanplasma.Maes eta1.[.]usedagradientmobilephasemodetodeter- mineacyclovirinhumanplasma,whichwastime- consuming.Stacyeta1.[appliedmassspectrome

    tricdetectiontoplasma,whiletheyusedhydrophil- icinteractionliquidchromatography(HILIC)in- steadofHPLCforchromatographicseparation.Ka- siarieta1.[153determinedacyclovirinhumanplasma withasimilarLC-MS/MSmethod.However,the lowerlimitofquantification(LLOQ)ofthemeth- odwas100ng/mLinplasma,whichwasnotsensi- tiveenoughtofullyenableapharmacokineticprofi- lingofacyclovir.

    Theproposedstudyfocusedonexploringthe highselectivityandsensitivityoftriplequadrupole MSsystemwithelectrosprayinterfaceforthedevel- opmentandvalidationofarobustreversedphase LG-MS/MSmethodinSRMmodetoquantifyacy- clovirinhumanplasma.Thiswascarriedoutbyits AeadJXi'anJiaotongUniv,Vo1.22,No.2,May2010 structuralanalogue,ganciclovir(Fig.1C),asinter- nalstandard(IS).Chromatographicseparationwas achievedwithin3.5minunderisocraticconditions

onanAgilentZORBAXSB-C18(150mm×2.1mm

    i.d.,3.5m)analyticalcolumn.Withoutatime- consumingstepofevaporation-concentration,the samplepreparationofthismethodinvolvedasimple proteinprecipitation.Themethodwasvalidated conformingtotheUnitedStatesFoodandDrugAd- ministration(USFDA)guidelines.Owingtoits simplicity,rapidity,selectivityandsensitivity,the mainadvantagesofthevalidatedmethodpresented inthispaperwouldmakeitanattractiveprocedure inhigh-throughputbioanalysisofacyclovir. O

    ][

    H2NN

    O

    L

    HNN

    H:N

    0

    NH2

    3

    (A)

    (B)

    (C)

    Fig.IChemicalstructureofvalacyclovir(A),acyclovir (B)andganciclovir(C)

    2Experimental

    2.1Chemicalsandreagents

    Valacyclovirhydrochloridetestcapsules(150 mg/capsule;batchNo.081201)werepurchased

    fromSuzhouChangzheng-XinkaiPharmaceutical Factory(Suzhou,China);thereferencecapsulesof valacyclovirhydrochloride(150mg/capsule;batch No.080801)wereobtainedfromthesamemanufac

    turer.Acyclovirreferencestandard(batchNo. 140630200602,98.4%purity)andganciclovirref- erencestandard(IS,batchNo.100380200301,

    99.4%purity)weresuppliedbytheChineseNa

    tionalInstitutefortheControlofPharmaceutical andBiologicalProducts(Beijing,China). Methano1(HPLCgrade)waspurchasedfrom

    .)

    .N

    YuanTian,eta1.Rapidquantificationofthemetaboliteofvalacyclovirhydrochloride

    VWRInternationalCompany(Darmstadt,Gcrma- ny).Acetonitrile(HPLCgrade)waspurchased fromHoneywellInternationalInc.(Muskegon, USA).Formicacid(analyticalgrade)waspur

    chasedfromNanjingChemicalReagentNo.1Facto

    rv.Waterwasdistillatedtwicebeforeuse. 2.2Preparationofthestandardstockand workingsolutions

    Thestandardstocksolutionsofacyclovir (1mg/mL)andganciclovir[theinternalstandard (IS),1mg/mL-]werepreparedbydissolving10mg ofeachin10mLwater,respectively.Thestandard stocksolutionofacyclovirwasthendilutedwith watertogetintermediateconcentrationsof40,30, 20,10,5,1,0.5,0.1and0.05/~g/mLforthecal-

    ibrationandqualitycontrol(Qc)purposes.The stocksolutionofganciclovirwasfurtherdiluted withwatertopreparetheworkinginternalstandard solutioncontaining10/~g/mLofganciclovir.The workingsolutionswereprepareddailyandthestock solutionswerepermanentlystoredat4?

    2.3LC.MS/MSinstrumentationandcondi

    tions

    Theliquidchromatographicseparationandmass spectrometricdetectionwereachievedbyemploying theFinniganMTSQQuantumDiscoveryMAXM LC-MS/MSsystem.ItconsistedofaFinniganSur- veyorLCpumpandaFinniganSurveyorauto-sam_ pler.ItwascoupledtoatriplequadrupoleTSQ quantummassspectrometer(ThermoElectronCor

    poration).Thechromatographyrunswerecarried outonanAgilentZORBAXSB-C18(150mm×2.1

    mmi.d.,3.5"m)analyticalcolumnat30?.The

    isocraticmobilephasewascomposedofmethanol

    0.1%formicacidsolution(5:95,v/v),being pumpedthroughthecolumnataflowrateof0.2 mL/min.

    ThetandemMSsystemwasequippedwithan ESIsourceandtheXcalibur2.0software(Thermo ElectronCorporation).Tuningoftheinstrument wasdonebycontinuousinfusionofacyclovir(10 t~g/mL)withaTSQQuantumelectronicallycon

    trolledintegratedsyringefollowingtheTSQQuan

    tumTuneprogram.Themassspectrometerwasop' eratedinpositiveionandSRMmodewithprecursor

    toproductqualifiertransitionm/z226.2152.1 foracyclovirandm/z256.2152.1forganciclovir. ?

    85?

    Sprayvoltagewasoptimizedat4000V.Thetrans. fercapillarytemperaturewas350?.Thesheathgas

    andauxiliarygas(nitrogen)pressuresweresetat40 and8arbitraryunites(setbytheLCQsoftware, ThermoElectronCorporation),respectively.Ar

    gonwasusedascollisiongasatapressureof1.5 mTorr.Thecollisionenergywas15Vforacyclovir and11Vforganciclovir.ThescanwidthforSRM was0.01m/zandscantimewas0.3s.Thepeak widthsettings(FWHM)were0.7m/zforbothQ1 andQ3.

    2.4Preparationofplasmasamples

    Thesampleswerepreparedbyasinglestepof proteinprecipitationwithacetonitrile.Aliquotsof 200Lplasmawerepipedintoa1.5mLplasticcen

    trifugetube,andthenvortexeduponanadditionof 20LworkingsolutionofIS(10g/mL).After additionof500Lacetonitrile,thesampleswere vortexedfor2minandcentrifugedfor5minat 14000g.200uLupperlayerwastransferredintoa 1.5mLplasticcentrifugetubeanddilutedwith200 uLwater.Thislatermixturewasthenvortexedfor 30sandcentrifugedat14000gfor8min.Only10 Lofthesupernatantwas

    MSsystem.

    injectedintotheLC-MS/

2.5Bioanalytiealmethodvalidation

    Themethodvalidationassayswerecarriedout accordingtotheUnitedStatesFoodandDrugAd- ministration(FDA)bioanalyticalmethodvalidation guidance(FoodandDrugAdministration,2001). Theblankplasmasamplesofhealthyhuman usedfortestingspecificityofthemethodwasob

    tainedfromsixdifferentsources.Thevisibleinter

    ferencesweretestedusingtheblankplasmasamples andtheplasmasampleswithacyclovirconcentra

    tionsclosetoLLOQ.

    Thelinearityofthemethodwasdeterminedby theanalysisofstandardplotsassociatedwitha9

    pointstandardcalibrationcurve.Thecalibration curveswerepreparedinfivedifferentdaysbyspi

    kingtheblankplasmawithappropriatevolumeof oneoftheworkingsolutionsmentionedaboveto producecurvepointsequivalentto0.005,0.01' 0.05,0.1,0.5,1,2,3and4/~g/mLofacyclovir. Thefollowingassayprocedureswerethesameas thosehaddescribedabove.Ineachrun,ablank plasmasample(processedwithouttheIS)wasana

    ?

    86?

    lyzedtoconfirmtheabsenceofinterferencesbutnot usedtoconstructthefunctionofthecalibration. Thecalibrationcurvesweregeneratedbytracingthe ratiosoftheanalytepeakareatotheISpeakarea versustheconcentrationofacyclovirandwerefitted

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