生物样品中化学药物定量分析方法验证的进展与解读

*“”“”2012ZX09302001＊＊[e-mail][e-mail]*＊＊＊＊100176、、、。。、、。Ｒ917A0254－1793201311－2019－06Progressandinterpretationofquantitativeanalyticalmethodvalidationofchemicalmedicinesinbiologicalsamples*YUMinZHANGShuang－qing＊＊LIZuo－gang＊＊LaboratoryofDrugMetabolismandPharmacokineticsNationalCenterforSafetyEvaluationofDrugsNationalInstitutesforFoodandDrugControlBeijing100176ChinaAbstractThereliableandreproduciblequantitativebioanalysismethodisthebackboneofpharmacokinetictoxico-kineticbioequivalenceandbioavailabilitystudies．Itisnecessarytovalidatebioanalyticalmethodstoensurethere-liabilityoftheanalyticalmethods．ByretrievingguidelinespublicationsandmeetingpresentationsfromdifferentcountriesseveralimportantconferencesinthebioanalysisfieldandtheprocessofissueandimplementationofguidingprincipleswereoutlinedfundamentalparametersanddetailedprotocolsofbioanalyticalmethodvalidationformicromoleculemedicinesweredescribedthedifferencesbetweentheUSEMAandChineseregulationsweresummarizedandtheglobalharmonizationofbioanalyticalguidanceinthefuturewasdiscussed．Keywordsbioanalysismicromoleculemedicinesmethodvalidationpartialvalidationcrossvalidationguidingprinciples、、、、、。、、。HPLC、GCLC－MS、GC－MS、、90%。、、、。。FDA200151EMA200992012212。1。—9102—ChinJPharmAnal201333111.11990。1990AmericanAssociationofPharma-ceuticalScientistsAAPS、FDA、InternationalPharmaceuticalFederationFIP、HealthProtectionBranchHPB、AssociationofAnalyticalChemistsAOACThefirstBioanalyticalMethodValidationWorkshop。1999FDA31990。2000FDA。2001FDA4。2006、、、FDAchromatographicassaysligand－bindingassays2“4－6－20”。、、、。5。1.22007CalibratonandValidationGroupCVGGLPThefirstCalibratonandValidationGroupCVGWorkshoponＲecentIssuesinGLPBioanalysis。CVG。CVG、、、。2008GLP。、、、ContractＲe-searchOrganizationsCＲOs、、。IncurredSampleＲe－analysisISＲ、、6。2009。、、、7。2010。、、、FDA、EMA、HealthCanadaTherapeuticProductDirectorateTPDMedi-cinesandHealthcareproductsＲegulatoryAgencyMHＲAInternationalConferenceonHarmonizationICH、OrganisationforEconom-icCo－operatinandDevelopmentOECD8。2011WＲIB。。、、。。AMS、high－resolutionMS、ICP－MS9。2012WＲIB。、、6LC－MS、、QC、GLP10。10。1.3EMA200812conceptpa-per11—0202—ChinJPharmAnal20133311200911draftGuide-line12201053120116PKWP20117CHMP2012212。FDA200110。2015FDA。2FDA、EMA。2.1FullValidation2.1.1specificity、。、。。6、。LC－MS、LC－MS－MS、13。LLOQ20%5%。2.1.2carry－over。LLOQ20%5%。。2.1.3calibrationcurve。。range。/。6。。。3。%ＲErelativeerror%bias。LLOQULOQ±20%±15%75%6ULOQLLOQ。。ＲE%=－/×100%2.1.4lowlimitofquantitationLLOQ。LLOQ3～5Cmax1/10～1/20。±20%coefficientofvariation20%。5LLOQ。2.1.5accuracyQCQC。±15%LLOQ±20%。precision—1202—ChinJPharmAnal20133311。QCＲSD。ＲSD15%LLOQ20%。LLOQ、、3QC。LLOQ375%～85%ULOQ50%ULOQ。5。360。2.1.6extractionrecov-ery。14。、2ULOQLLOQ3515%。2.1.7matrixeffect6、。、2MF。MFMFMF。6MF15%15。2.1.8ULOQ。ULOQ10010005±15%15%。dilutionintegrity。2.1.9stability、、。、、、。、2QC3。。312h1。±15%15%。2.2partialvalidation。。、、、、、、、、、、。2.3crossvalidation。。2QC。QC2±15%67%±15%。2.4analysisofstudysamples。、QC。1。。1、、6、65%QC3。1—2202—ChinJPharmAnal20133311、1、、。±15%LLOQULOQ±20%。675%。LLOQULOQLLOQULOQ1QC。QC±15%15%。67%QC50%QC。±15%15%。UL-OQ。LLOQCmaxCmaxAUC。3FDA、EMAEMAFDA。2005《》、《》《》2010IIXIXB《》FDAEMA。1。1FDA、EMATab1SummaryofthemaindifferencesbetweentheUSEMAandChineseregulationsforbioanalyticalmethodvalidationrequirementFDAEMASFDAresidualeffectnotmentionedLLOQ20%5%carry－overintheblanksamplefollowingthehighconcentrationstand-ardshouldnotbegreaterthan20%oftheLLOQand5%fortheinternalstandardNotmentionedLLOQlowlimitofquantitationtheaccuracyandpreci-sionoftheLLOQarerecommendedbutbe-tween－runaccuracyandprecisionarenotrecommendedthewithin－runandbetween－runaccuracyandprecisionoftheLLOQarerecommendedtheaccuracyandpreci-sionoftheLLOQarerecommendedbutbe-tween－runaccuracyandprecisionarenotrecommendedQCselectionofQCsamplesLLOQ375%～85%ULOQ50%ULOQonewithin3×theLLOQlowQCsampleonenearthecentermiddleQCandoneneartheupperboundaryofthestandardcurvehighQCLLOQ375%～85%ULOQ50%ULOQwithinthreetimestheLLOQlowQCaround50%ofthecalibrationcurverangemediumQCandatleastat75%oftheuppercalibrationcurverangehighQC3onewithin3×theLLOQlowQCsam-pleonenearthecentermiddleQCandoneneartheupperboundaryofthestandardcurvehighQCintra－andinter－dayaccuracyandprecision3QC5。3accuracyandprecisionshouldbedeter-minedusingaminimumoffivedeterminationsperconcentrationlevel．Aminimumofthreeconcentrationsintherangeofexpectedcon-centrationsisrecommended．Between－runpre-cisionmeasurementsshouldbemadeacrossthreeconsecutivedays3QCLLOQ5。2d3within－runaccuracyandprecisionshouldbedeterminedbyanalysinginasinglerunaminimumof5samplesperlevelatamini-mumof4concentrationlevelswhicharetheLLOQlowmediumandhighQC．Between－runaccuracyandprecisionshouldbeevalua-tedLLOQlowmediumandhighQCsam-plesfromatleastthreerunsanalysedonatleasttwodifferentdays3QCaccuracyandprecisionshouldbeevaluatedlowmediumandhighQCsamples—3202—ChinJPharmAnal201333111requirementFDAEMASFDAcalibrationcurve64atleastfouroutofsixnon－zerostandardsshouldmeetthecri-teria75%6atleast75%ofthecalibrationstandardswithaminimumofsixcalibrationstanda