AnalyticalBiochemistryjournalhomepage:www.elsevier.com/locate/yabioAnalysisoftestosteroneanddihydrotestosteroneinmousetissuesbyliquidchromatography–electrosprayionization–tandemmassspectrometry
YanWenga,1,FangXiea,LiXua,2,DmitriZagorevskib,DavidC.Spinka,XinxinDinga,*abWadsworthCenter,NewYorkStateDepartmentofHealth,andSchoolofPublicHealth,StateUniversityofNewYorkatAlbany,Albany,NY12201,USACenterforBiotechnologyandInterdisciplinaryStudies,RensselaerPolytechnicInstitute,Troy,NY12180,USAarticleinfoabstract
Anovelmethodwasestablishedforsimultaneousquantitationoftestosterone(T)anddihydrotestoster-one(DHT)inmurinetissueandserumsamples.EndogenousTandDHT,togetherwiththeinternalstan-dards17a-methyl-Tand17a-methyl-DHT,wereextractedfromtissuesandthenderivatizedbyreactionwith2-hydrazino-4-(trifluoromethyl)-pyrimidine(HTP).Analysisbyliquidchromatography–electro-sprayionizationtandemmassspectrometry(LC–ESI–MS/MS)resultedinproductionspectraofHTPderivativesofbothTandDHTthatshowedanalyte-specificfragmentations;thelatterfragmentationswerecharacterizedbytheuseofhigh-resolutionOrbitrapMS/MS.ThesespecificfragmentationsenabledquantitationofTandDHTinthemultiple-reactionmonitoring(MRM)mode.Themethodwasvalidatedwithcharcoal-strippedserumasthematrix.Thelowerlimitofquantitation(LLOQ)was0.10ng/mlforTand0.50ng/mlforDHT.ThemethodwasthenusedfordeterminationofserumandtissuelevelsofTandDHTintransgenicmicecarryingahypomorphicNADPH–cytochromeP450reductasegene(Cpr-lowmice).Remarkably,ovarianTlevelsinCpr-lowmicewerefoundtobe25-foldhigherthanthoseinwild-typemice,afindingthatatleastpartlyexplainsthefemaleinfertilityseenintheCpr-lowmice.Inconclusion,ourmethodprovidesexcellentsensitivityandselectivityfordeterminationofendogenouslevelsofTandDHTinmousetissues.Ó2010ElsevierInc.Allrightsreserved.Articlehistory:Received5November2009Receivedinrevisedform17February2010Accepted26March2010Availableonline31March2010Keywords:LC–MSSteroidsTestosteroneDihydrotestosteroneMetabolismCytochromeP450MiceTestosterone(T)3istheprimarycirculatingandrogeninbothmalesandfemales[1,2].AbnormallevelsofThavebeenassociatedwithmanydiseaseconditions,includinghypogonadisminmales[3]andexcessiveandrogensyndrome[4]andandrogeninsufficiencyinfemales[1,5].Dihydrotestosterone(DHT),abiologicallymoreac-tiveformofandrogenthanT,isproducedmainlybytheactionof
*Correspondingauthor.Fax:+15184738722.E-mailaddress:xding@wadsworth.org(X.Ding).1Presentaddress:PfizerGlobalResearchandDevelopment,Groton/NewLondonLaboratories,Pfizer,Groton,CT06340,USA.2Presentaddress:AnalyticalDevelopment/PharmaceuticalandQualityServices,AlbanyMolecularResearch,Albany,NY12212,USA.3Abbreviationsused:T,testosterone;DHT,dihydrotestosterone;RIA,radioimmu-noassay;GC–MS,gaschromatography–massspectrometry;LC,liquidchromatogra-phy;HMP,2-hydrazino-1-methylpyridine;FMP,2-fluoro-1-methylpyridinium-p-toluenesulfonate;MS/MS,tandemmassspectrometry;HTP,2-hydrazino-4-(trifluo-romethyl)-pyrimidine;NADPH,reducednicotinamideadeninedinucleotidephos-phate;Cpr,NADPH–cytochromeP450reductasegene;P450,cytochromeP450;DHEA,dehydroepiandrosterone;D5-T,2,2,4,5,6-2H5-labeledtestosterone;MT,17a-methyl-testosterone;MDHT,17a-methyldihydrotestosterone;CS–FBS,charcoal-strippedfetalbovineserum;HPLC,high-performanceliquidchromatography;ESI,electrosprayionization;MRM,multiplereactionmonitoring;CE,collisionenergy;CXP,collisioncellexitpotential;WT,wild-type;SPE,solid-phaseextraction;LLOQ,lowerlimitofquantitation;S/N,signal/noise;APCI,atmosphericpressurechemicalionization;CID,collision-induceddissociation;LOD,limitofdetermination;QC,qualitycontrol;RSD,relativestandarddeviation.
0003-2697/$-seefrontmatterÓ2010ElsevierInc.Allrightsreserved.doi:10.1016/j.ab.2010.03.034steroid5a-reductases(SRD5A1/2)onTinandrogen-targettissues.DHTplayscriticalrolesinmaledevelopment[2].Furthermore,DHTisimplicatedinmanypathologicalprocesses,includingbenignprostatichyperplasia[6].AnabnormalratioofDHTtoT,asaresultofgeneticdefectsintheconversionofTtoDHT,couldcausemalepseu-dohermaphroditism[7].TheratioofDHTtoTisalsoavaluableclin-icalmeasureforevaluationoftheefficaciesof5a-reductaseinhibitors[8,9]andandrogenreplacementtherapy[10,11].There-fore,thecapabilitytosimultaneouslydeterminelevelsofTandDHTisdesirablebothinthediagnosisofsexsteroidhormone-re-lateddiseasesandinthemonitoringoftheeffectsofandrogenreplacementtherapy.
Radioimmunoassay(RIA)iscommonlyusedfordeterminationofserumlevelsofTandDHT[12–15].However,anincreasingnumberofstudieshaveshownthat,duetoinherentlimitationsinspecificity,RIAmethodsareunsuitablefordeterminationoftheverylowlevelsofTpresentinfemalesandchildrenorinhypogonadalmen[16–19].Gaschromatography–massspectrome-try(GC–MS)providesexcellentspecificityandsensitivityfordeter-minationofneutralsteroids[17,20,21].However,methodsbasedonthisinstrumentationusuallyrequireextrastepsforsamplepreparationandcleanup,andproblemswiththermalstabilityofthesteroidderivativesareoftenencountered.ThedeterminationofDHTisevenmorechallengingthanthatofTgiventhatDHTis122AnalysisofTandDHTinmousetissues/Y.Wengetal./Anal.Biochem.402(2010)121–128presentatmuchlowerlevelsinmostbiologicalsamples[13–15,22,23].Duringrecentyears,severalliquidchromatography(LC)–MS-basedmethods,allofwhichusechemicalderivatizationtoin-creasethedetectionsensitivity,havebeendevelopedforsimulta-neousdeterminationofTandDHTinvivo[24–30].Anumberofreagents,including2-hydrazino-1-methylpyridine(HMP)[24–26],2-fluoro-1-methylpyridinium-p-toluenesulfonate(FMP)[28,29],andhydroxylamine[27],producederivativesthatenhancesensitivityforLC–MSdeterminationofTandDHTinvariousbiologicalsamples.However,mostofthesereagents,includingHMP,2,3-pyridine-dicarboxylicanhydride,andhydroxylamine,formtwoisomeric(cisandtrans)productswitheitherTorDHT[25,27,30].ThechromatographicseparationoftheseisomersoftheTandDHTderivativesnotonlycomplicatesquantitativeanal-ysisbutalsocausesincreasedsusceptibilitytointerferencesbyendogenousorexogenouscompounds.Moreover,ionsderivedfromthederivatizationreagentmoieties,butnotanalyte-specificions,dominatetheproductionspectraofHMPderivatives,there-bycompromisingtheselectivityofthemethod[24–26].ThehydroxylaminederivativeofTprovidesstructurallyrelevanttan-demmassspectrometry(MS/MS)transitionsforthequantitationofTbutnotforDHT[27].TheFMPderivativesofTandDHTeacheluteasasinglepeakunderoptimizedLCconditions;however,thereactionofFMPwithTandDHThasalowefficiency,therebyrequiringanexcessiveamountofFMP.Consequently,additionalcleanupstepsarerequiredforFMPderivativesbeforetheycanbeanalyzedbyLC–MS[25,28,29].Inthecurrentstudy,anovelderivatizationmethod,whichusesaslittleas10lgofthecommerciallyavailablereagent2-hydra-zino-4-(trifluoromethyl)-pyrimidine(HTP),hasbeendeveloped.Wedescribetheadvantagesofthisprocedureoverotherscurrentlyinuse,wepresentproofofvalidationfortheaccuracyandpreci-sionofthemethod,andwedescribeanapplicationofthenewmethod,namely,anassessmentoftheimpactofareductioninexpressionoftheNADPH–cytochromeP450reductase(Cpr)geneoncirculatingandtissuelevelsofTandDHT.Thelatterstudyem-ployedatransgenicmousemodel(knownastheCpr-lowmouse);inthismouse,theCprgeneishypomorphic,leadingtodecreasedexpressionofCPRproteinandconsequentdecreasesintheactivi-tiesofmicrosomalcytochromeP450(P450)enzymesinallorgansexamined[31].MaterialsandmethodsMaterialsT,DHT,dehydroepiandrosterone(DHEA),androsterone,andHTPwerepurchasedfromSigma–Aldrich(St.Louis,MO,USA).2,2,4,5,6-2H5-labeledtestosterone(D5-T)waspurchasedfromCambridgeIsotopeLaboratories(Andover,MA,USA).17a-Methyl-testosterone(MT)and17a-methyldihydrotestosterone(MDHT)werepurchasedfromSteraloids(Wilton,NH,USA).Charcoal-strippedfetalbovineserum(CS–FBS)waspurchasedfromHyclone(Logan,UT,USA).High-performanceliquidchromatography(HPLC)-gradehexanewaspurchasedfromJ.T.Baker(Phillipsburg,NJ,USA).EthylacetatewaspurchasedfromAnachemia(Sparks,NV,USA).LC–MS-gradeacetonitrile,methanol,andwaterwerepurchasedfromFisherScientific(Pittsburgh,PA,USA).AceticacidwaspurchasedfromMallinckrodt(Hazelwood,MO,USA).DerivatizationAliquotsofTandDHTstandards(inmethanol)andtissueorserumextractswereevaporatedtodrynessinglasstubesunderN2.Theresidueswereredissolvedin50llofacetonitrileand50llofHTPreagent(0.2mg/mlindryacetonitrilecontaining0.05%trifluoroaceticacid).Thereactionmixturesweretrans-ferredto2-mlglassvials(NationalScientific,Rockwood,TN,USA),sealed,andincubatedat60°Cfor1h.Thereactionswerestoppedbycoolingonicefor5min.ThereactionmixtureswerethenevaporatedtodrynessunderN2.Thederivativesweredis-solvedin50llofdryacetonitrileandtransferredto250-llin-sertsintheautosamplervialsforLC–electrosprayionization(ESI)–MS/MSanalysis.ThereactionofHTPwithTisdepictedinFig.1.LC–ESI–MS/MSAllsampleswereanalyzedonanAppliedBiosystems/MDSSciexAPI4000Q-TrapmassspectrometerequippedwithaturboionspraysourceandinterfacedwithanAgilent1200seriesli-quidchromatograph.Thesystemwasoperated,anddatawereanalyzed,withAnalystsoftware(version1.4.2,AppliedBiosys-tems/MDSSciex,FosterCity,CA,USA).Themassspectrometerwasoperatedinthepositiveionizationmode.Instrumentalparameterswereoptimizedduringdirectinfusionofstandardswithsolventconsistingof10%A(0.1%aceticacidinwater/ace-tonitrileat9:1[v/v])and90%B(acetonitrile)ataflowrateof0.2ml/min.The[M+H]+ionsofT,DHT,theT–HTPderivative,andtheDHT–HTPderivativewereidentifiedbyLC–ESI–MS,withQ1operatedinfullscanningmodeintherangeofm/z200–1000.Aproductionspectrumwasobtainedforeachcompound.Multi-plereactionmonitoring(MRM)wasusedforquantitativeanaly-sis.Nitrogenwasusedasthecurtaingas(settingat35),gas1(settingat35),gas2(settingat60),andthecollisiongas(settinghigh).Theionsprayvoltagewassetat5500,andthegastem-peraturewassetat500°C.Thedeclusteringandentrancepoten-tialswere80and10V,respectively.Thecollisionenergy(CE)andcollisioncellexitpotential(CXP)foreachMRMtransitionaregiveninTable1.ALunaPhenyl-Hexylcolumn(2.0Â150mm,3lmparticlesize,Phenomenex,Torrance,CA,USA)wasusedforHPLC.Themobilephaseconsistedof0.1%aceticacidin90%H2O/10%acetonitrile(A)and100%acetonitrile(B).Alineargradientwasperformed,withtheinitialholdat30%Bfor1min,increasingto90%Bover8min,aholdat90%Bfor4min,andareturnto30%Bover1min.Thecolumnwasequilibratedattheinitialconditionfor8minpriortothenextinjection.Theinjectionvolumewasrou-tinelysetat10ll,theflowratewas0.2ml/minthroughout,andthecolumnwasatambienttemperature.ForLC–MS/MSanalyses,datawerecollectedbetween7and14min;columneffluentbefore7minandafter14minwasdivertedtowaste.High-resolutionOrbitrapMSHigh-resolutionMSwasperformedwithaThermoElectronLTQXLOrbitrapMSdeviceoperatinginthepositiveionelectrosprayMS/MSmode.High-resolutionproductionspectrawererecordedoverthem/zrangeof120–600witharesolutionof60,000atm/z400andamassaccuracywithin2ppm.The[M+H]+ionofbis(2-ethylhexyl)phthalateatm/z391.2843servedasthelockmass.Thenormalizedcollisionenergywas24eV,theioninjectiontimewas500ls,thecapillarytemperaturewas300°C,andthesheathgasflowratewasatsetting13withoutauxiliaryflow.Theelectro-spraysourcewasat4.0kV,thecapillaryvoltagewas46,thetubelensvoltagewas99,andthesourcecurrentwas100lA.SampleswereintroducedintotheMSdeviceinamobilephaseconsistingof0.2%formicacid/acetonitrile(30:70)viaanAgilent1200nano-flowHPLCsystemataflowrateof50ll/min.AnalysisofTandDHTinmousetissues/Y.Wengetal./Anal.Biochem.402(2010)121–128123Fig.1.ProposedschemefortheformationofT–HTPviareactionofHTPwithT.Table1
CEandCXPforMRMtransitionsemployedforthedetectionofHTPderivativesofT,DHT,MT,andMDHT.CompoundDHT–HTPDHT–HTPT–HTPT–HTPMDHT–HTPMDHT–HTPMT–HTPMT–HTPMS/MStransition451?288451?260449?257449?269465?302465?274463?257463?269CE(eV)3849505038495050CXP(V)1816211418162114PreparationofTandDHTstandardsolutionsandcalibrationcurvesMethanolicstocksolutionsofTandDHT(at1mg/ml)wereusedtoprepareworkingstandardsofTandDHT(at0.1,1,10,100,and1000ng/ml)byserialdilutionintomethanol.AllstandardswerestoredinambervialsatÀ80°C.CS–FBSwasusedasthematrixforpreparationofthecalibrationcurves.Thecalibratorswerepre-paredbytheadditionofappropriateamountsofworkingstandardsofTandDHTto0.1mlofCS–FBStogivethefinalconcentrationsforT(at0.1,0.25,0.5,1.5,5,15,50,and150ng/ml)andforDHT(at0,0.5,1,2,5,10,20,and40ng/ml).AnimalsMaleandfemalewild-type(WT)C57BL/6andCpr-lowmice[31],at2–3monthsofage,wereobtainedfrombreedingstocksmaintainedattheWadsworthCenter.AnimaluseprotocolswereapprovedbytheinstitutionalanimalcareandusecommitteeoftheWadsworthCenter.TheanimalsweresacrificedbyeuthanasiawithCO2.Bloodsamples,collectedthroughcardiacpuncture,werekeptonicefor1hpriortocentrifugationat13,000gfor10minat4°C.TissueandserumsampleswerestoredatÀ80°Cuntiluse.SamplepreparationBrain,seminalvesicle,andtestissampleswerethawed,weighed,andhomogenizedinphysiologicalsalineataconcentra-tionof100mg/ml.Ovariesandprostateswerethawedandthenhomogenizedin1mlofsalineperovaryorprostate.APolytron(Kinematica,modelGT10-35)wasusedfortissuehomogenization.A20-llaliquotofeachhomogenatewassavedforproteindetermi-nationbyuseofthebicinchoninicacidmethod(Pierce,Rockford,IL,USA).FordeterminationofTandDHTintissuesamples,1mlofthehomogenatewasused.Forserumsamplesandcalibrators,0.1mlofmouseserumorCS–FBSwasaddedto0.9mlofsaline.Allsam-pleswerefortifiedwith200pgofMTandMDHTandthenex-tractedwith8mlof60%hexane/40%ethylacetate.Thetubesweregentlyshakeninahorizontalshakerfor1hatroomtemper-ature.Aftercentrifugationat1000gfor10min,theorganicphasewastransferredtoanew15-mlglasstubeandthenevaporatedtodrynessunderN2.Theresidueswereredissolvedin0.4mlofmethanol.Thesampleswerefurtherdilutedwith1.6mlofwaterandthenpurifiedbysolid-phaseextraction(SPE)onIsoluteC18cartridges(200mg/3ml,Biotage,Charlottesville,VA,USA).TheC18cartridgeswerefirstactivatedwith5mlofmethanolandthenequilibratedwith3mlofH2O.Afterloadingofthedilutedsamples(2mlcontaining20%methanol),thecartridgeswerewashedwith2mlof10%methanol.Theanalyteswereelutedin2mlofmetha-nolandevaporatedtodrynessunderN2.TheresiduesafterSPEwereresuspendedin50llofdryacetonitrile,combinedwith50llofHTPreagentforderivatization(asdescribedabove),andthenanalyzedbyLC–MS/MS.LLOQs,recovery,andmatrixeffectLowerlimitsofquantitation(LLOQs)fortheanalysesofTandDHTasHTPderivativeswereevaluatedaccordingtotheU.S.FoodandDrugAdministrationLLOQguidelines(signal/noise[S/N]P5,precisionof20%,andaccuracyof80–120%).FordeterminationoftherecoveriesofTandDHT,knownamountsofTandDHTwereaddedtoCS–FBSeitherbeforethehexane/ethylacetateextractionoraftertheSPE,andtheratios(before/after)oftheamountsde-tectedforeachanalyteinthetwosampletypeswerecalculated.InternalstandardswereaddedtoCS–FBSbeforeextraction.Poten-tialionsuppressioneffectsofthematrixwereevaluatedbycom-paringpeakareasofknownamountsofTorDHTspikedinCS–FBSextract(afterSPEasdescribedabove)withthepeakareasofthesameamountsofTorDHTspikedinsolvent.MethodvalidationThemethodfordeterminationofTandDHTintissueandserumsampleswasvalidatedintermsofinterbatchaccuracyandpreci-sion.Foreachbatch,0.1mlofCS–FBSwasdilutedwith0.9mlofsaline,fortifiedwithafixedamountofinternalstandards(200pgeachofMTandMDHT),andcombinedwithappropriateamountsofTandDHTworkingstandardstoachieveTandDHTconcentra-tionsof0.15and0.75ng/ml(lowlevel),2and4ng/ml(mediumle-vel),and8and8ng/ml(highlevel),respectively.Thesampleswereextracted,derivatized,andanalyzedbyLC–ESI–MS/MS(usingMRM).Threevalidationbatcheswerepreparedforevaluationoftheaccuracyandprecisionofthemethod;eachbatchwasanalyzedonadifferentday.Eachbatchincludedadoubleblank(noanalyteorinternalstandard),ablank(noanalyte),asetofcalibrationstan-dardswithduplicatesateachTandDHTconcentration,andfourreplicatesofsamplesspikedwithlow,medium,andhighlevelsofTandDHT.124AnalysisofTandDHTinmousetissues/Y.Wengetal./Anal.Biochem.402(2010)121–128ResultsanddiscussionDerivatizationofTandDHTforLC–MSanalysesTheformationofT–HTPviaderivatizationofTwithHTPisshowninFig.1;theformationofDHT–HTPfromDHTandHTPwasanalogous.Ascomparedwithpositiveatmosphericpressurechemicalionization(APCI),positiveESIprovidedbetterionizationefficiencyforbothTandDHTderivatives(datanotshown).ThemassspectrumofT–HTPisdominatedbytheprotonatedmolecule,[M+H]+,atm/z449(datanotshown).TheproductionspectrumofT–HTPundercollision-induceddissociation(CID)showedforma-tionoftwodominantproductionsatm/z257and269(Fig.2A);thefragmentationpathwaysappearedtoinvolvecleavagesofthesteroidAandBrings,consistentwiththepathwayreportedforthefragmentationofunderivatizedT[32].ThefragmentationpathwayproposedinFig.2Aisfurthersup-portedbythefactthationsatm/z257and269arealsoobservedintheproductionspectrumofthe[M+H]+moleculeofMT–HTPatm/z463(seeSupplementalFig.S1Ainsupplementarymaterial).FragmentationbyneutrallossoffragmentscontainingthesteroidDring,withorwithoutaC17methylgroup,couldbeexpectedtogiverisetoionsofthesamem/zvaluesinthespectraofT–HTPandMT–HTPiftheionsobservedarerepresentativeofportionsofthesteroidAandBrings,aswehaveproposed.High-resolutionproductionspectraofthe[M+H]+ionsofT–HTPandMT–HTPwereconsistentwiththeproposedfragmentationsthroughthesteroidAandBrings,producingionsconsistentwiththeempiricalformulasC12H12N4F3andC11H12N4F3(Table2).Theionsatm/z257and269,arisingfromthe[M+H]+ofT–HTP,showedahighdegreeofstabil-ity;attemptsatfurthercollisionalactivationwereineffective.Whenthem/z449?269and449?257MS/MStransitionswereFig.2.ProductionspectraofT–HTP(A)andDHT–HTP(B)derivatives.Proposedfragmentationpatternsofthe[M+H]+ionoftheT–HTP(A)andDHT–HTP(B)derivativesunderCIDarealsoshown.Table2
High-resolutionMS/MSanalysisofHTPderivativesofT,MT,DHT,andMDHT.FormulaMeasuredmassTheoreticalmassDelta(ppm)ProductionsofT–HTP[M+H]+ionatm/z449.25C12H12N4F3269.1010269.10090.54C11H12N4F3257.1010257.10090.44C5H5N3F3164.0429164.0430À0.56ProductionsofMT–HTP[M+H]+ionatm/z463.27C12H12N4F3269.1008269.1009À0.06C11H12N4F3257.1008257.1009À0.10C5H5N3F3164.0428164.0430À1.02ProductionsofDHT–HTP[M+H]+ionatm/z451.27C19H30ON288.2318288.2322À1.33C17H26ON260.2007260.2009À0.77C5H5N3F3164.0427164.0430À1.75ProductionsofMDHT–HTP[M+H]+ionatm/z465.28C20H32ON302.2476302.2478À0.71C18H28ON274.2166274.21650.35C5H5N3F3164.0427164.0430À0.85monitoredinthequantitativeanalysisofserumsamples,thesen-sitivitiesobtainedforthetwotransitionsweresimilar.However,whentissuesampleswereanalyzed,lesserextentsofmatrix-de-rivedinterferenceintheMS/MSionchromatogramswereobservedforthem/z449?257transitionthanforthem/z449?269tran-sition.Therefore,them/z449?257transitionwasusedforquan-titationandthem/z449?269transitionwasusedforconfirmationofTlevelsintissueandserumsamples.ThemassspectrumofDHT–HTPisalsodominatedbythepro-tonatedmolecule,[M+H]+,atm/z451.AsshowninFig.2B,m/z288,260,and164arethemostprominentfragmentionsintheproductionspectrumofDHT–HTP.Weproposethattheionatm/z164representstheprotonatedtrifluoromethylpyrimidinemoi-ety,whereastheionsatm/z288and260arisefromthesteroidringsystem.Theionatm/z288couldbeformedthroughheterolyticcleavageofthehydrazoneN–Nbond,withthechargecarriedbythesteroidfragment.Them/z260fragmentcouldarisefromtheneutrallossofC2H4fromthem/z288ion,aninterpretationsup-portedbytheobservationofm/z302and274ionsintheproductionspectrumproducedbycollisionalactivationofthe[M+H]+forMDHT–HTPatm/z465(SupplementalFig.S1B).Theshiftsof14Daoverthecorrespondingionsinthespectraofthenonmethy-latedsteroidsindicatethatthemethylgroupatC17ispresentinthesefragments.Thisevidenceoftheintactmethyl-substitutedsteroidDringstronglysupportsthecontentionthattheionatm/z302arisesfromcleavageofthehydrazoneN–Nbond,apath-wayanalogoustothefragmentationproposedfortheproductionspectrumofDHT–HTP.Thehigh-resolutionproductionspectraofthe[M+H]+ionsofDHT–HTPandMDHT–HTPareconsistentwiththeoccurrenceoffragmentationattheN–Nbond,neutrallossofC2H4,andcleavageoftheprotonatedtrifluoromethylpyrimidinemoiety,givingrisetoionsconsistentwiththeempiricalformulasC19H30ON,C17H26ON,andC5H5N3F3(Table2).ForquantitativeanalysisofDHT,morematrixinterferenceswereobservedwhenmonitoringthem/z451?288transitionthanwhenmonitoringthem/z451?260transition.Therefore,them/z451?260transitionwasusedforquantitationandthem/z451?288transitionwasusedforconfirmationbothformeth-odvalidationandfordeterminationofDHTlevelsintissueandser-umsamples.OptimizationofderivatizationreactionTrifluoroaceticacid(atafinalconcentrationof0.025%)isre-quiredforthederivatizationreaction.Thederivatizationefficien-ciesweresimilarwheneitherethanoloracetonitrilewasusedasAnalysisofTandDHTinmousetissues/Y.Wengetal./Anal.Biochem.402(2010)121–128125thereactionsolvent.Thereactiontimecoursewasinvestigatedovertherangeof5–120min.Thereactionwascompletedby1h.Therefore,theoptimizedconditionsforHTPderivatizationcon-sistedofanincubationofthereactionmixtureindryacetonitrilecontaining0.025%trifluoroaceticacidat60°Cfor1h.Ascomparedwiththelimitofdetermination(LOD)forunderivatizedT,theLOD(S/N>5)forderivatizedTwas10-foldlower(0.2vs.2pgoncol-umn).TheLODforderivatizedDHT(1pgoncolumn)was40timeslowerthanthatforunderivatizedDHT(40pgoncolumn).StabilityofHTPderivativesTopreventpossiblehydrolysisofthederivative,weremovedthetrifluoroaceticacidbyevaporatingthereactionmixturetototaldrynessunderN2andthenresuspendedtheresiduesindryaceto-nitrile.TheamountsofunderivatizedDHTandTdetectedinthisfi-nalpreparationwerelessthan5%oftherespectivetotalamountsoforiginallyaddedTandDHT(datanotshown).TheHTPderiva-tivesstoredindryacetonitrilewerestableat4°C.Theamountsremainingafter24hwere100%forT–HTPand88%forDHT–HTP.LC–MSanalysisInpreliminarystudies,severalnewderivatizationreagents(notshown)weretestedtoidentifythosethatshowbothsatisfactoryionizationefficiencyandadequatechromatographicbehaviorsfordeterminationofTandDHT.Allofthederivativestestedformedcisandtransisomers.ThepairsofisomersoftheHTPderivativesofTorDHTweredetectedassinglepeaksinLC–MSanalysis(Fig.3AandB),whereasthepairsofisomersforallothertypesofderivativesofTorDHTweredetectedasseparatepeaks(notshown).Therefore,HTPwaschosenforfurtherstudies.ItwasdifficulttoachievebaselineseparationoftheHTPderiv-ativesofTandDHTundertheHPLCconditionstested.Weestimatethat,with2ngofderivatizedToncolumn,thesignaldetectedasDHT–HTPwasequivalenttoapproximately8pg(thepeakareara-tioform/z451?260fromthesameamountofseparatelyinjectedT–HTP[SupplementalFig.S2B]andDHT–HTP[SupplementalFig.S2A]was$0.4%).Conversely,with2ngofderivatizedDHToncolumn,thesignaldetectedasT–HTPwasequivalenttoapprox-imately2pg(thepeakarearatiosform/z449?257fromthesameamountofT[SupplementalFig.S2A]andDHT[SupplementalFig.S2C]derivativeswas$0.1%).BecausephysiologicallevelsofTareusuallyhigherthanthelevelsofDHT,thecoelutionofDHTwithTisunlikelytointerferewithdetectionofT,butsuchcoelu-tioncouldresultinanoverestimationofDHTlevelsinthosetissueswhereTispresentatmuchhigherlevelsthanDHT.DHEA,anabundantendogenoussteroidwiththesamemolecu-larweightasthatofT,isamajorsourceofpotentialinterferenceinLC–MS/MSmeasurementofT[27,30,33].Therefore,wedeterminedthepotentialinterferencebyDHEAinourassayforT.DHEA–HTPandT–HTPwerenotbaselineseparatedundertheHPLCconditionstested.With2ngofderivatizedDHEAoncolumn,thesignalde-tectedasT–HTPwasequivalenttoapproximately1.2pg(thepeakarearatioform/z449?257fromthesameamountofDHEAandTderivativeswas$0.06%).Therefore,DHEAisunlikelytointerferewithdeterminationofTintissuesorserum.Notably,androster-one–HTPandDHT–HTPwerebaselineseparatedundertheLCcon-ditionsused;thus,therearenoconcernsaboutpotentialinterferencebyandrosteroneforthedeterminationofDHT.A20000449→25715000T-HTPB12000100008000DHT-HTP451→260Intensity (cps)Intensity (cps)10000600040002000050000891011121314891011121314Retention time (min)Retention time (min)C30000MT-HTP463→257D6000500040003000200010000465→274MDHT-HTP25000Intensity (cps)150001000050000891011121314Intensity (cps)20000891011121314Retention time (min)Retention time (min)Fig.3.MS/MSionchromatogramsforHTPderivativesofT(A:449/257)andDHT(B:451/260),aswellastheirinternalstandardsMT(C:463/257)andMDHT(D:465/274),obtainedfrommediumQCsamples.TheminorpeaksinpanelsBandDrepresentbackgroundsignals(notproducedbythestandard).126AnalysisofTandDHTinmousetissues/Y.Wengetal./Anal.Biochem.402(2010)121–128AnalysisofTandDHTintissueandserumsamplesMTandMDHTwereusedastheinternalstandardsforquantita-tionofTandDHT,respectively.D5-TwasalsoroutinelyincludedforconfirmationoftheretentiontimeofT;however,itwasnotusedforquantitationofTbecausethedeuteriumlabelsofD5-Twereunstableundertheconditionsforderivatization.TherelativeretentiontimesofMTandMDHT,comparedwiththoseofTandDHT,areshowninFig.3CandD.NoendogenousMTorMDHTwasdetectedinanyofthebiologicalsamples.ForquantitativeanalysisofTandDHTinbiologicalsamples,calibrationcurves(overtherangeof0.10–150ng/mlforTand0.5–40ng/mlforDHT)wereprepared,withtheuseof0.1mlofCS–FBSasthematrix.Thecalibrationcurveswereplottedwiththeuseofconcentrationratiosofanalytestointernalstandards(T/MTorDHT/MDHT)asthexaxisandpeakarearatiosoftheana-lytestointernalstandardsastheyaxis.ThecalibrationcurvesforbothTandDHTshowedexcellentlinearityovertheconcentrationrangeused(R2>0.998)(SupplementalFig.S3).ThereconstructedionchromatogramsdonotshowanysignificantbackgroundpeaksintheunspikedCS–FBSsamples(Fig.4AandC).TherecoveriesofTandDHTwerenearlyidenticaltothoseoftheirinternalstandardsunderavarietyofextractionconditions.Thematrixeffects(ionsuppression)oftheCS–FBSonthemeasurementsofTandDHTweredeterminedtobe23%and25%,respectively(n=4).Theresultsofinterbatchvalidationassaysforthequalitycon-trol(QC)samples(spikedat0.15and0.75ng/ml,at2and4ng/ml,andat8and8ng/mlforTandDHT,respectively)areshowninSupplementalTableS1inthesupplementarymaterial.Theaccu-racyofthemethodwasevaluatedbydeterminationofthemeanrelativeerrors.Theprecisionofthemethodwasevaluatedbydeterminationoftherelativestandarddeviation(RSD).Theintra-batchaccuracyvaluesofthespikedsamplesatallthreelevelstestedwereintherangeofÀ7.8%to0.92%,whereastheRSDvalueswere67.4%.TheLLOQforTwasfoundtobe0.10ng/ml(asdeterminedbyanalysisofCS–FBSsamplesspikedwithTatvariousconcentra-tions)withanRSDof15.6%,ameanrelativeerrorofÀ6.5%,andanS/Nratiofortheanalyteexceeding5(n=6determinations).TheLLOQforDHTwas0.50ng/mlwithanRSDof3.9%,ameanrel-ativeerrorofÀ4.6%,andanS/Nratiofortheanalyteexceeding5(n=6determinations).TheMS/MSionchromatogramsofTandDHTatLLOQareshowninFig.4BandD.Notably,theLLOQachievedinourstudyforDHT(0.5ng/ml),throughtheuseoftheHTPderivatizationtechniqueandanAPI4000Q-TRAPinstrument,ishigherthanthatreportedbyShiraishiandcoworkers($0.02ng/ml),whousedthemoresensitiveAPI5000LC–MSinstrumentandmicroborechromatography[33].Undoubtedly,thesensitivityofourassaycanbefurtherincreasedbyimprovementsinLCcondi-tionsandbyutilityofanMSinstrumentwithgreatersensitivity.Furthermore,theabilityofourtechniquetoprovideaconfirmatoryMS/MStransitionisvaluablefortheidentificationofpotentialinterferencesincomplexsamplematricessuchastissueextracts.Asafirstapplicationofthenewassaymethod,wedeterminedthelevelsofTandDHTintheserum,brain,andovaryoffemaleCpr-lowandWTmiceaswellasintheserum,testis,seminalves-icle,andprostateofmaleCpr-lowandWTmice(Tables3and4andSupplementalFigs.S4andS5).DHTwasnotdetectedinanyofthesamplesfromfemales,whereasTwasdetectedinbothmalesandfemales.Tlevelsweresignificantlyhigher(by25-fold)intheovary,aswellasintheserumandbrain(bothby$2.5-fold),oftheCpr-lowfemalesthaninthecorrespondingtissuesoftheWTfemales.A25002000Blank CS-FBS 449→257C160014001200Blank CS-FBS 451→260Intensity (cps)Intensity (cps)15001000500089101112131410008006004002000891011121314(T-HTP)(DHT-HTP)Retention time (min)Retention time (min)B25002000449→257LLOQ sampleT-HTP (0.1 ng/ml)D160014001200LLOQ sample451→260Intensity (cps)Intensity (cps)1500100050008910111213141000800600400200089DHT-HTP(0.50 ng/ml)1011121314Retention time (min)Retention time (min)Fig.4.MS/MSionchromatogramsforT–HTPandDHT–HTPinblankCS–FBS(AandC)andLLOQ(BandD)samples.AnalysisofTandDHTinmousetissues/Y.Wengetal./Anal.Biochem.402(2010)121–128Table3
LevelsofTinbrain,ovary,andserumoftheWTandCpr-lowfemalemice.StrainTlevelSerum(pg/ml)WT(n=4)Cpr-low(n=5)a127Brain(pg/mgprotein)0.77±0.162.00±0.94cOvary(pg/mgprotein)36±28900±525c100±18a250±67bForsampleswithTlevelsbelowtheLLOQ,asecondinjectionwasmadeaftertheremainingsampleswereconcentrated.bP<0.01comparedwiththeWTmice(Student’sttest).cP<0.05comparedwiththeWTmice(Student’sttest).Asexpected,serumTlevelsweremuchhigherinmalesthaninfe-males;themeanserumTlevelsinfemales(100pg/ml)wereattheLLOQoftheTassay.Ingeneral,serumlevelsofT,andtissuelevelsofTandDHT,appearedtobehigherintheCpr-lowmalesthanintheWTmales.However,onlyserumlevelsofTandtissueslevelsofDHTintheseminalvesicleweresignificantlyhigherintheCpr-lowmalesthanintheWTmales.Inmammals,Tissynthesizedmainlybythetestisinmales[2]andbytheovaryinfemales[1];Tistransportedfromthetestisortheovarytootherorgansviathecirculatorysystem.ItisstillnotclearhowthesystemicandtissuelevelsofTareregulated;com-plexnetworks,includingeventsthatmodulatetheratesofsteroidbiosynthesisanddegradation[34–36]aswellastheabundanceofandrogen-bindingproteins[37],areassumedtobeinvolved.IntheCpr-lowmice,theexpressionofCPRwasdown-regulatedinallorganstested[31].Therefore,allCPR-dependentactivities,includingthoseoftheP450enzymes,weresuppressed.However,becausemicrosomalP450enzymesareinvolvedinboththebio-synthesis(e.g.,CYP17)anddegradation(e.g.,CYP19,CYP3A,CYP2A)ofT,theimpactofanorganism-widesuppressionofP450activitiesontissuelevels,aswellasonsystemicTlevels,willlikelyreflectthecollectiveeffectsonbiosyntheticaswellasmeta-bolicP450enzymesinvarioustissues.Thus,ourfindingthatthesuppressionofCPR-dependentenzymeactivitiesintheovaryre-sultedindramaticallyhigherTlevelsinthisorgan(Table3)sug-geststhattheTdegradationpathways(particularlyCYP19)weremorestronglyaffectedthantheTbiosynthesispathwaysintheovaryoftheCpr-lowmice.AlesslikelyalternativeisthatthehighlyelevatedTlevelsintheovaryresultfromincreasedTstorageand/orreducedTsecretiongiventhehighersystemiclevelsofTintheseanimalsascomparedwithWTmice.Incontrasttothe25-foldhigherTlevelsseenintheovary,theserumlevelofTwasonly2.5-foldhigherintheCpr-lowfemalesthanintheWTfemales(Table3).ThisrelativelysmalleffectofCprsuppressiononserumTlevelscouldbeduetotherelativelyhighresidualP450-mediatedTdegradationenzymeactivitiesintheliveroftheCpr-lowmouse[31];inthatregard,systemicdeg-radationofTisthoughttobecarriedoutmainlybyhepaticen-zymes,includingP450s[38,39].ThelevelsofTinthebrainwerealsoapproximately2.5-foldhigherintheCpr-lowfemalesthanintheWTfemales(Table3).Thisresultisconsistentwithaprevi-ousreportthatTcouldfreelycrosstheblood–brainbarrier[40];thus,thehighersystemicTwouldleadtoproportionallyhigherTlevelsinthebrain.ThecurrentfindingofveryhighTlevelsintheovaryoftheCpr-lowmice,alongwithourpreviousfindingofhighserumTlevelsinthesemice,couldexplaintheinfertilityphenotypeobservedprevi-ouslyinCpr-lowfemales[31].Notably,elevatedlevelsofTintheovariesofwomencouldalsocausepolycysticovarysyndromeandinfertility[4].SimilartothefindingforfemaleCpr-lowmice,theserumlevelsofTweresignificantlyhigherintheCpr-lowmalesthanintheWTmales(Table4).However,thetissuelevelsofTinthetestis(themainorganofsynthesis),seminalvesicle,andprostatewerenotsignificantlydifferentbetweentheCpr-lowandWTmales;thisobservationsuggeststhatthetissuelevelsofTinthemalemiceareregulatedbothbylocalmechanismsandbysystemicclearance.DHTlevelsinthetestisandprostatewerenotsignificantlydif-ferentbetweentheCpr-lowandWTmales;however,DHTlevelsintheseminalvesicleoftheCpr-lowmalesweresignificantlyhigher(1.6-fold)thanthoseintheseminalvesicleoftheWTmales(Ta-ble4).IncontrasttotheinfertilityobservedfortheCpr-lowfe-males,noobviousreproductivedysfunctionswereobservedfortheCpr-lowmales.Therefore,thephysiologicaleffectoftheele-vatedtissuelevelsofDHTintheseminalvesicleoftheCpr-lowmalesnecessitatesfurtherinvestigation.ConclusionsAnovelmethodhasbeendevelopedandvalidatedforsimulta-neousquantitativeanalysisofTandDHT,asHTPderivatives,invariousmousetissues.Comparedwithpreviouslydescribedderiv-atizationmethodsfordeterminationofTandDHT,ourmethodismoreattractiveforquantitationofTandDHTintissuesintwoways.First,theproductionspectraoftheHTPderivativesofbothTandDHTaredominatedbystructurallyinformativeanalyte-spe-cificfragmentionsthatprovidebothprimaryandconfirmatoryMRMtransitionsforquantitativeanalysis.Second,theHTPderiva-tivesofTandDHTareelutedassinglepeaks,therebyimprovingthesensitivityandselectivityofthemethod.AninitialapplicationofthismethodtodeterminationsofthephysiologicallevelsofTandDHTintheserum,brain,ovary,testis,seminalvesicle,andprostateofCpr-lowandWTmiceledtotheintriguingfindingofdramaticallyhigherTlevelsintheovaryofthefemaleCpr-lowmiceascomparedwiththeovarianlevelsintheWTfemales.Thus,ourmethod,whichallowsdeterminationofTandDHTlevelsinvarioustissuesamplesfromindividualmice,shouldhavebroadapplicationforthecharacterizationoftransgenic/knockoutmousemodels.AcknowledgmentsWethankAdrianaVerschooroftheWadsworthCenterforread-ingthemanuscript,WeizhuYangfortechnicalassistance,andSar-ahMordan-McCombsandJoEllenWelshoftheUniversityatAlbanyforassistancewithprostatedissection.Thisworkwassup-Table4
LevelsofTandDHTinserum,testis,seminalvesicle,andprostateoftheWTandCpr-lowmalemice.StrainSteroidlevelSerum(pg/ml)TWT(n=6)Cpr-low(n=6)aTestis(pg/mgprotein)T978±8101660±980DHT120±85193±81Seminalvesicle(pg/mgprotein)T4.0±4.06.0±2.0DHT34.4±18.154.3±9.2aProstate(pg/mgprotein)T52.5±61.7113.0±57.0DHT81.7±62.0130.0±47.03700±313013700±8500aP<0.05comparedwiththeWTmice(Student’sttest).128AnalysisofTandDHTinmousetissues/Y.Wengetal./Anal.Biochem.402(2010)121–128portedinpartbygrantCA081243(toD.C.S.)fromtheNationalCan-cerInstitute,NationalInstitutesofHealth,andgrantES007462(toX.D.)fromtheNationalInstituteofEnvironmentalHealthSciences,NationalInstitutesofHealth.AppendixA.SupplementarydataSupplementarydataassociatedwiththisarticlecanbefound,intheonlineversion,atdoi:10.1016/j.ab.2010.03.034.References[1]M.A.Papalia,S.R.Davis,Whatistherationaleforandrogentherapyforwomen?,TreatEndocrinol.2(2003)77–84.[2]J.D.Wilson,M.W.Leihy,G.Shaw,M.B.Renfree,Androgenphysiology:unsolvedproblemsatthemillennium,Mol.Cell.Endocrinol.198(2002)1–5.[3]S.Basaria,A.S.Dobs,Hypogonadismandandrogenreplacementtherapyinelderlymen,Am.J.Med.110(2001)563–572.[4]R.J.Derman,Effectsofsexsteroidsonwomen’shealth:implicationsforpractitioners,Am.J.Med.98(1995)137S–143S.[5]G.Bachmann,J.Bancroft,G.Braunstein,H.Burger,S.Davis,L.Dennerstein,I.Goldstein,A.Guay,S.Leiblum,R.Lobo,M.Notelovitz,R.Rosen,P.Sarrel,B.Sherwin,J.Simon,E.Simpson,J.Shifren,R.Spark,A.Traish,Femaleandrogeninsufficiency:thePrincetonconsensusstatementondefinition,classification,andassessment,Fertil.Steril.77(2002)660–665.[6]G.Andriole,N.Bruchovsky,L.W.Chung,A.M.Matsumoto,R.Rittmaster,C.Roehrborn,D.Russell,D.Tindall,Dihydrotestosteroneandtheprostate:thescientificrationalefor5a-reductaseinhibitorsinthetreatmentofbenignprostatichyperplasia,J.Urol.172(2004)1399–1403.[7]J.Imperato-McGinley,Y.S.Zhu,Androgensandmalephysiology:thesyndromeof5a-reductase-2deficiency,Mol.Cell.Endocrinol.198(2002)51–59.[8]R.V.Clark,D.J.Hermann,G.R.Cunningham,T.H.Wilson,B.B.Morrill,S.Hobbs,Markedsuppressionofdihydrotestosteroneinmenwithbenignprostatichyperplasiabydutasteride,adual5a-reductaseinhibitor,J.Clin.Endocrinol.Metab.89(2004)2179–2184.[9]R.S.Rittmaster,E.Stoner,D.L.Thompson,D.Nance,K.C.Lasseter,EffectofMK-906,aspecific5a-reductaseinhibitor,onserumandrogensandandrogenconjugatesinnormalmen,J.Androl.10(1989)259–262.[10]C.Steidle,S.Schwartz,K.Jacoby,T.Sebree,T.Smith,R.Bachand,AA2500testosteronegelnormalizesandrogenlevelsinagingmaleswithimprovementsinbodycompositionandsexualfunction,J.Clin.Endocrinol.Metab.88(2003)2673–2681.[11]R.S.Swerdloff,C.Wang,G.Cunningham,A.Dobs,A.Iranmanesh,A.M.Matsumoto,P.J.Snyder,T.Weber,J.Longstreth,N.Berman,Long-termpharmacokineticsoftransdermaltestosteronegelinhypogonadalmen,J.Clin.Endocrinol.Metab.85(2000)4500–4510.[12]L.J.Funderburgh,W.B.Zipf,J.F.Sotos,Directmeasurementoftestosteroneinapediatriccenter,withuseofaradioimmunoassaykitandunextractedserum,Clin.Chem.29(1983)1796–1798.[13]M.Pazzagli,G.Forti,A.Cappellini,M.Serio,Radioimmunoassayofplasmadihydrotestosteroneinnormalandhypogonodalmen,Clin.Endocrinol.(Oxf.)4(1975)513–520.[14]K.M.Pirke,P.Doerr,Plasmadihydrotestosteroneinnormaladultmalesanditsrelationtotestosterone,ActaEndocrinol.(Copenh.)79(1975)357–365.[15]C.Wang,A.Iranmanesh,N.Berman,V.McDonald,B.Steiner,F.Ziel,S.M.Faulkner,R.E.Dudley,J.D.Veldhuis,R.S.Swerdloff,Comparativepharmacokineticsofthreedosesofpercutaneousdihydrotestosteronegelinhealthyelderlymen:aclinicalresearchcenterstudy,J.Clin.Endocrinol.Metab.83(1998)2749–2757.[16]M.L.Cawood,H.P.Field,C.G.Ford,S.Gillingwater,A.Kicman,D.Cowan,J.H.Barth,Testosteronemeasurementbyisotopedilution–liquidchromatography–tandemmassspectrometry:validationofamethodforroutineclinicalpractice,Clin.Chem.51(2005)1472–1479.[17]J.Taieb,B.Mathian,F.Millot,M.C.Patricot,E.Mathieu,N.Queyrel,I.Lacroix,C.Somma-Delpero,P.Boudou,Testosteronemeasuredby10immunoassaysandbyisotopedilution–gaschromatography–massspectrometryinserafrom116men,women,andchildren,Clin.Chem.49(2003)1381–1395.[18]U.Turpeinen,S.Linko,O.Itkonen,E.Hamalainen,Determinationoftestosteroneinserumbyliquidchromatography–tandemmassspectrometry,Scand.J.Clin.Lab.Invest.68(2008)50–57.[19]C.Wang,D.H.Catlin,B.Starcevic,A.Leung,E.DiStefano,G.Lucas,L.Hull,R.S.Swerdloff,Testosteronemetabolicclearanceandproductionratesdeterminedbystableisotopedilution/tandemmassspectrometryinnormalmen:influenceofethnicityandage,J.Clin.Endocrinol.Metab.89(2004)2936–2941.[20]T.K.Kwan,D.J.Trafford,H.L.Makin,A.I.Mallet,D.B.Gower,GC–MSstudiesof16-androstenesandotherC19steroidsinhumansemen,J.SteroidBiochem.Mol.Biol.43(1992)549–556.[21]G.Moneti,M.G.Giovannini,A.Guarna,G.Forti,R.Salerno,A.Magini,M.Serio,Determinationoftestosteroneanditstissuemetabolites(DHTand3a-diol)inhumanplasmaandprostatictissuebyisotopicdilutionmassspectrometry,J.SteroidBiochem.27(1987)53–59.[22]G.E.Abraham,F.S.Manlimos,M.Solis,A.C.Wickman,Combinedradioimmunoassayoffoursteroidsinonemlofplasma:II.Androgens,Clin.Biochem.8(1975)374–378.[23]S.Werawatgoompa,N.Dusitsin,P.Sooksamiti,S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