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首页 维生素D2 D3候选参考方-1

维生素D2 D3候选参考方-1.pdf

维生素D2 D3候选参考方-1

bekiliang
2012-11-27 0人阅读 举报 0 0 0 暂无简介

简介:本文档为《维生素D2 D3候选参考方-1pdf》,可适用于高等教育领域

CandidateReferenceMeasurementProceduresforSerumHydroxyvitaminDandHydroxyvitaminDbyUsingIsotopeDilutionLiquidChromatography–TandemMassSpectrometryHedwigCMStepman,AnVanderroost,KatleenVanUytfanghe,andLindaMThienpont*BACKGROUND:hydroxyvitaminD(OH)DassaysarecharacterizedbypoorbetweenassaycomparabilityThisresultemphasizestheneedforreferencemeasurementprocedures(RMPs)toestablishcalibrationtraceabilityandassistinmethodvalidationWeaimedatdevelopingcandidateRMPsonthebasisofisotopedilutionliquidchromatography–tandemmassspectrometry(IDLCMSMS)forseparatequantificationofserum(OH)Dand(OH)DMETHODS:Hexadeuterated(OH)DDwasaddedtoserumThismixturewasextractedwithnhexaneandfractionatedonSephadexLHbeforedimensionalLCMSMSInthefirstdimension,bothproceduresusedaCcolumnhowever,intheseconddimension,the(OH)DprocedureusedaCandthe(OH)DprocedureusedaZorbaxSBCNcolumnCalibrationwastraceabletotheNISTStandardReferenceMaterial(SRM)ValidationcomprisedassessmentofinterferenceandlimitofquantificationdetectionImprecisionandtruenesswerevalidatedbyanalysisoftheSRMagainstspecifications(CV�andbias�)TheexpandeduncertaintyforquadruplicatemeasurementswasestimatedRESULTS:TestingofpotentiallyinterferingsubstanceswasnegativeInterferencebyepi(OH)DwasresolvedbysufficientchromatographicresolutionThelimitsofquantificationdetectionwerenmolLandpmolLfor(OH)DandnmolLandpmolLfor(OH)DMeantotalCVsanddifferencesfromtheSRMtarget(�sidedCI)wereand�(OH)Dandand�(OH)D,respectivelyTherespectiveexpandeduncertaintieswereandCONCLUSIONS:Fromthevalidationdata,weconcludethatweachievedourobjectiveofstateoftheartcandidateRMPsforserum(OH)Dand(OH)D©AmericanAssociationforClinicalChemistryThereisevidenceformajorpublichealthissuessurroundingthevitaminDstatusinhumans()VitaminDexistsinformsandisobtainedfromsourcesVitaminDisformedbyexposureoftheskintosunlight,whereastheDformisobtainedfromthedietFortifiedfoodanddietarysupplementscontainbothformsThecirculatinglivermetaboliteshydroxyvitaminDandD(OH)Dand(OH)DareusedasmarkersofthevitaminDstatus()Severedeficiencyhasrecentlybeenimplicatedinawiderangeofclinicaldisordersotherthanskeletaldeformities(–)Asaresultofthisemergingconsensus,physiciansincreasinglymonitorthevitaminDstatusinindividualsThistestingisdonewithavarietyofmethodsbasedondifferentmeasurementprinciples,ofwhichthetypicalperformancecharacteristicsandlimitationsrecentlyhavebeenreviewed(,)ImmunoassaysmainlymaybelimitedbycrossreactivityoftheantibodiesandnonequimolarrecognitionoftheDandDformsofthe(OH)metabolite,whereasinchromatographicmethods,itistheresolvingpoweranddetectionthataredeterminantsforsufficientspecificityInthisregard,liquidchromatography(LC)coupledtotandemmassspectrometry(MSMS)inherentlyhasthebetterpotentialNotethatinchromatographicmethods,incontrasttoimmunoassays,equimolarityofmeasurementisnotanissuebecausethesemethodsseparatetheDandD(OH)metabLaboratoryforAnalyticalChemistry,FacultyofPharmaceuticalSciences,GhentUniversity,Ghent,Belgium*Addresscorrespondencetothisauthorat:LaboratoryforAnalyticalChemistry,FacultyofPharmaceuticalSciences,GhentUniversity,Harelbekestraat,BGhent,BelgiumFax�emaillindathienpontugentbeReceivedJune,acceptedDecember,PreviouslypublishedonlineatDOI:clinchemNonstandardabbreviations:(OH)D,hydroxyvitaminD(OH)D,hydroxyvitaminDLC,liquidchromatographyMSMS,tandemmassspectrometrySI,Syste`meInternationald’Unite´sSRM,StandardReferenceMaterialID,isotopedilutionRMP,referencemeasurementprocedureUPLC,ultraperformanceliquidchromatographyMr,relativemolecularmassLoQ,limitofquantificationLoD,limitofdetectionSN,signaltonoiseClinicalChemistry:–()AutomationandAnalyticalTechniquesolitesandsumuptheresultstothetotal(OH)DconcentrationHowever,Singhetal()showedthatcommonlyusedLCMSMSproceduresmayoverestimatethe(OH)DconcentrationwhentheCepimeraccountsforasubstantialproportionofthecirculating(OH)DconcentrationThisoccurrenceresultsfrominsufficientchromatographicresolutiontoseparatetheepimetaboliteAnothermajorlimitationthatappliesforallcurrent(OH)Dmethodsisthepoorcomparabilityofresults,asdemonstratedindedicatedproficiencytestingschemes(,)Thisisconfoundingfordiagnosisagainstexpertrecommendationstomaintaincirculating(OH)Dconcentrationsaboveacertainconcentrationforoptimalhealth(–)Thisconcernincreasinglycallsforstandardization(–)ThereisgeneralconsensusthatthisshouldbedonebyusingahigherorderreferencemeasurementsystemtoestablishtraceabilitytotheSyste`meInternationald’Unite´s(SI)()SItraceabilityortruenessisindeedthemostvalidbasisforgenerationoflaboratorydatathatservetheestablishmentofguidelinesandclinicaldiagnosis,longtermepidemiologicstudies,andprogramstoevaluatetheeffectandsafetyofdietarysupplements(–)ItwasinthisregardthattheOfficeofDietarySupplementsfromtheNationalInstitutesofHealthworkedtogetherwiththeNISTtodevelopstandardreferencematerials(SRMs)for(OH)DD,ie,theserumbasedSRM(levels)andtheSRMcalibrationsolutions(–)Thelatterwashelpfulindecreasinginterlaboratoryvariationofchromatographicprocedures()TheSRM,certifiedwithisotopedilution(ID)LCMSMSreferencemeasurementprocedures(RMPs)(),wasintendedforvalidationofthetruenessofhierarchicallylowermethods()However,somelevelsoftheSRMweredeemednoncommutablewhenmeasuredwithimmunoassaysandthereforenotfitfortruenessassessment()ThisproblemisnotexpectedforroutineMSMSproceduresbecausetheyarebasedonthesamemeasurementprincipleasthecertificationprocedureBecauseoftheissueofnoncommutabilityofreferencematerials,theapproachofdirectlyusingunadulteratedsera,assignedwithvaluesbyanRMP,isthepreferredalternative(,)Ofcourse,whenthelaboratorycommunityadvocatesthatinvitrodiagnosticcompaniesandIDLCMSMSlaboratoriesshouldmakeuseofthisapproach,thereisaneedforsufficientcapacityoflaboratoriesofferingRMPservicesHerewereportthedevelopmentofcandidateIDLCMSMSRMPsforquantificationofserum(OH)Dand(OH)D,thefirstwithsufficientchromatographicresolutiontoseparate(OH)DandtheepimetaboliteAspartoftheprocessofcriticallyevaluatingtheprocedures,wevalidatedtruenessandimprecisionagainstpreviouslyderivedspecificationsandassessedtheapplicabilityformeasurementofnativepatientsera()MaterialsandMethodsMATERIALS(OH)D,(OH)D,anddehydrocholesterolwerepurchasedfromSigmaAldrich,andhexadeuteratedanalogsd(OH)DDwereobtainedfromMedicalIsotopesSRMandSRMwereobtainedfromNIST,SephadexLHfromGEHealthcare,andhorseserumfromInvitrogenThecomponentstestedforinterferencewereagiftfromImmunotechandHDeLuca(UniversityofWisconsin,Madison)Chemicalsandsolventswereanalyticalgrade,withtheexceptionofmethanolandwaterofultraperformanceliquidchromatography(UPLC)grade(Biosolve)Fordetailsonthemeasuredhumanserumsamples,seeFileintheDataSupplementthataccompaniestheonlineversionofthisarticleathttp:wwwclinchemorgcontentvolissueCALIBRATIONThreeworkingsolutionsofapproximatelynmolL(OH)DDinethanolwerepreparedTheywereassignedwithexactvaluesfromacomparisonprotocolwiththeSRM,furtherreferredtoas“calibrationtransferprotocol”Thesolutionsofd(OH)DDwerepreparedsimilarlyForquantification,apointcalibrationprocedureatthe:isotoperatio(�)wasused()(seeSupplementalDataFile)SAMPLEPREPARATIONSamplepreparationisfullydescribedinonlineSupplementalDataFileTypically,�Lserum(maximum�L)wassampledanddilutedtomLwith(gg)sodiumchloridesolutionSubsequently,equalabsoluteamountsofeitherd(OH)Dord(OH)DwereaddedAfterequilibration,theserumwasalkalinized,extractedwithnhexane,andfractionatedbySephadexLHchromatographyLCMSMSANALYSISAdimensionalAcquity®UPLCsystemconnectedtoanAcquity®tandemquadrupolemassdetector(bothfromWaters),operatinginthepositiveelectrosprayionizationmode,wasusedThefirstdimensionusedanAcquity®BEHCcolumn(�mm,�m,Å)Theseconddimensioncolumnsweremethodspecific:the(OH)DcandidateRMPusedanAcquityBEHCcolumn(�mm,�m,Å),whilethe(OH)DusedaZorbaxSBCNcolumn(�mm,�m,Åheldat°C,AgilentTechnologies)Theselectedreactionmonitoringtransitionsweremz(OH)Dandepi(OH)Dandmzd(OH)D),ClinicalChemistry:()andmz(OH)Dandmzd(OH)DFurtherdetailsontheUPLCandMSconditionsaregiveninonlineSupplementalDataFileMETHODVALIDATIONCalibrationWeestimatedtheuncertaintyofthecalibrationtransferprotocolfromtheimprecisionoftheexperimentaldesignusedforvalueassignmentFurthermore,weassessedwhetheritwasjustifiedtodothecalibrationfromdirectanalysisofthecalibrators,ie,withoutsubmissiontothesamplepretreatmentprocedureTheexperimentalprotocolisdescribedinonlineSupplementalDataFileRecoveryandmatrixeffectonionizationTheabsoluterecoveryofthesamplepreparationprocedurefor(OH)DDwasinvestigatedbycomparisonoftheisotoperatiosobtainedforsetsofaliquotsfromthesameserumpoolTothefirstset,theisotopicallylabeledanalogwasaddedbeforesamplepreparationtothesecondset,itwasaddedonlyafterextractionandfractionation(seeonlineSupplementalDataFile)Matrixeffectonionizationwasinvestigatedbycomparisonoftheareaunderthepeakobtainedfromdirectinjectionofthelabeledanalogvsinjectionafteradditiontoaprocessedserummatrix(n�)InterferenceStructuralanalogsof(OH)DDweretestedforinterference(fortheamountsinjected,seeonlineSupplementalDataFile)Therelativemolecularmass(Mr)oftheM�H�andM�Na�ionswasafirstcriterionforpotentialinterferenceatthemzvaluesmonitoredfor(OH)DDandthedanalogsForthesubstanceswithahigherMr,afullscanspectrumwasregistered(mzrangeof–)toverifythepresenceofaninterferingMSsignalThesubstancestestingpositivewereevaluatedfortheirretentiontimerelativeto(OH)DDinthefirstdimensionoftheUPLCprocedureonlyInaddition,nativeserumsamples,notsupplementedwithd(OH)DD,wereanalyzedtoassessinterferenceLimitofquantification(LoQ)andlimitofdetection(LoD)TheLoQfor(OH)DwasevaluatedfromanalysisofSRMlevelcertifiedconcentration:�nmolL(expandeduncertainty)andfromananalyticalrecoveryexperimentwithahumanserumpoolsupplementedtoatotalconcentrationofnmolLNotethattheendogenousconcentrationwas(–)nmolL(sidedCI)For(OH)D,theLoQwasestimatedfromanalysisofhorseserumsupplementedwithnmolLAllsampleswerequantifiedinduplicateonindependentdays(n�),whichallowedtheestimationofthetotalCVandthemeansignaltonoise(SN)ratioNotethatthesameprotocolwasusedtodeterminetheendogenousconcentrationintheseraoftherecoveryexperimentOurspecificationsforLoQwereatotalCV�andamaximumabsolutedeviation�nmolL(seeDiscussion)TheestimationoftheLoDatanSNratioofwasbasedontheLoQsamplesImprecisionandtruenessThesecharacteristicswerevalidatedfromanalysisofthelevelsoftheSRMagainstspecificationsofamaximumtotalCVofandamaximumsystematicdeviationof()BecauseofthelimitedvolumesoftheNISTSRMmaterials,measurementsweredoneaccordingtoareducedCLSIEPprotocol,ie,duplicatesonindependentdays(n�)()Foreachlevel,thewithinrun,betweenrun,andtotalCVswerecalculatedbywayANOVAThemeantotalCVwascalculatedasthesquarerootofthemeanofthequadratictotalCVsForthedifferentinternalqualitycontrolsamples(seeonlineSupplementalDataFile),thetotalCVwassimilarlycalculatedusingwayANOVATheCVformeasurementofnativeserumsampleswasderivedfromthedifferencebetweentheduplicateshowever,itwasmeasuredassinglicateonindependentoccasionsThetruenesswasexpressedaspercentrecoveryoftheNISTcertifiedvaluesPerformanceofnativeserumsamplesThecandidateRMPswerechallengedwithanalysisofnativeserumsamplesAfterscreeningofthesamplesforthepresenceof(OH)Dandepi(OH)D,allmetabolitesweredeterminedinduplicatewithrigorousinternalqualitycontrol(seeonlineSupplementalDataFile)Inthereconstructedionchromatograms,wedeterminedthetypicalSNratio,verifiedthepresenceofnonidentifiedinterferingpeaks,andevaluatedtheresolvingpowertoseparateepi(OH)Datofthe(OH)DpeakheightThe(OH)Dand(OH)Dconcentrationswerequantifiedinabsoluteterms,whereasfortheepiform,thecontentrelativeto(OH)D()wasestimatedExpandeduncertaintyofmeasurementFirst,thecombineduncertaintywascalculatedfrompropagatingtheuncertaintyduetotheimprecision,thecalibrationtransferprotocol,theSRMcertification,andunspecificinterferencesToobtainarepresentativeimprecision,thetotalCVestimatedfromanalysisofthedifferentSRMlevelswascombinedwiththeCVscalculatedfortheinternalqualitycontrolandnativeserumsamplesThisstepwasdonebytakingthesquarerootofthemeanofeachquadraticCVvalueFinally,theexpandeduncertaintywasestimatedwithk�()StatisticaldataanalysisMicrosoftOfficeExcel®(version)wasusedtoperformtheGrubbsoutliertest,HydroxyvitaminDCandidateReferenceMeasurementProceduresClinicalChemistry:()thesidedFtest,orsided(dependingontheapplication)Student’sttestswithequalorunequalvariances(basedontheFtest),andawayANOVA,allatprobabilityResultsCALIBRATIONTheuncertaintyofthecalibrationtransferprotocolamountedtoTheisotoperatiosmeasuredinthesetsofcalibrators(directlyanalyzedandaftersubmissiontosamplepreparation)werenotsignificantlydifferent(OH)D:difference,Psidedttest�(OH)D:,Psidedttest�Thisresulttogetherwiththeevidenceforabsenceofmatrixeffectsonionization(seebelow)showedthatcalibrationwithdirectlyanalyzedcalibratorswasjustifiedRECOVERYANDMATRIXEFFECTONIONIZATIONTheabsoluterecovery�sidedCIvaluesofthesamplepreparationprocedureswere�(OH)Dand�(OH)DThematrixeffectonionizationwasfoundtobestatisticallynotsignificant(OH)D,observeddifference,Psidedttest�(OH)D,,Psidedttest�INTERFERENCETheresultsfortheinterferencestudyaresummarizedinTable(fortheretentiontimeoftheexaminedcomponents,seeonlineSupplementalTableS)Althoughtheregisteredfullscanspectraidentifiedpotentialinterferencebysomecompounds,therelativeretentiontimeallowedustoexcludeitforall,exceptepi(OH)DAsshowninFig,theepiformisseparatedatofthe(OH)DpeakheightontheZorbaxSBCNcolumnAnalysisofthesampleswithoutaddeddanalogsconfirmedabsenceofinterferenceonthetransitionmzvaluesandLoQANDLoDTheLoQfor(OH)D,estimatedfromanalysisofSRMlevel,was�nmolL(sidedCI)Theabsolutedifferencefromthetargetwas�nmolL,thetotalCVwas,andthemeanSNratiowasTheLoQ,estimatedfromtheanalyticalrecoveryexperiment,was�nmolL,withadeviationof�nmolLfromthetargetThemeasuredtotal(OH)Dconcentrationinthesupplementedhorseserumwas�nmolLThedeviationfromthetarget(nmolL,becauseofsupplementationoftheendogenousconcentrationat�nmolLwithnmolL)was�nmolL,thetotalCVwas,andthemeanSNratiowasTheLoD,estimatedfromtheabovedata,waspmol(pg)andpmol(pg)oncolumnsfor(OH)Dand(OH)D,respectivelyIMPRECISIONANDTRUENESSTablesandlistthedifferentimprecisionandtruenessdataThemeanwithinrunandtotalimprecision(expressedasCV)estimatedfromanalysisofthedifferentlevelsoftheSRMwereandfor(OH)Dandandfor(OH)DThemeantruenesswas�(sidedCI)for(OH)DandTableVitaminDanalogstestedforpotentialinterferenceVitaminDmetaboliteorstructuralanalogMrInterferingmzvalueaRelativeretentiontimebInterferenceM�HHO�M�H�M�Na�Cholecalciferol�r�NoErgocalciferol�r�NoDehydrocholesterol�NoepiHydroxyvitaminD�r�Yes�HydroxyvitaminD�r�No�,DihydroxyvitaminD�No�,DihydroxyvitaminD�No,DihydroxyvitaminD�No,DihydroxyvitaminD�No�,,TrihydroxyvitaminD�NoaCodesusedtodescribeinterferingmzvalue:�,nosignal,obtainedinthescanmode,atthemzvaluescorrespondingtotherelativemolecularmassofM�H�:(OH)D,d(OH)D,(OH)D,andd(OH)D�,becausethemoleculehadaninterferingmzvalue(note:mzvaluesareinitalics),wedeterminedtherelativeretentiontimeOnlythemoleculesthatelutedfromthefirstdimensioncolumnbetweenandmincorrespondingrelativeretentiontimes:–minfor(OH)Dand–minfor(OH)Daretransferredtotheseconddimensioncolumn)bRelativeretentiontimeincomparisonwith(OH)Dor(OH)D,respectivelyClinicalChemistry:()�for(OH)DNoneoftheobtainedpercentrecoveriesoftheNISTtargetvaluesexceededthedeviationlimit,asconfirmedbyasidedttestagainstthelimitsformaximumdeviation(ie,or)(seeTableforthePvalues)PERFORMANCEONNATIVESERUMSAMPLESTheconcentrationsof(OH)DinsamplesrangedfromtonmolL,withameanofnmol

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