生物样品分析方法验证指南

DrugEvaluationResearch第35卷第5期2012年10月-396-•国外信息专栏•Guidelineonbioanalyticalmethodvalidation[编者按]：在创新药物研发过程中，生物基质（如血清、血浆、血液、尿液、唾液）中的药物浓度测定是一个重要的方面。其数据可用于支持新活性物质的应用和仿制药及已授权药品的变更申请。动物的毒代动力学研究和临床试验，包括生物等效性研究的结果为原料药或产品的安全性和有效性提供关键性的数据支持。因此，应用经过充分验证并记录到一个满意标准的生物分析方法以得到可靠的结果，这是非常重要的。2012年2月1日，欧洲药品管理局（EMA）开始实施昀新的《生物样品分析方法验证指南》（Guidelineonbioanalyticalmethodvalidation），本指南适用于动物的毒代动力学研究和所有阶段的临床试验中获得的生物样品中的药物浓度的生物分析方法的验证。本文摘录其方法学验证部分，抛砖引玉，供相关研究人员参考。4.Methodvalidation4.1.FullvalidationofananalyticalmethodAfullmethodvalidationshouldbeperformedforanyanalyticalmethodwhetherneworbaseduponliterature.Themainobjectiveofmethodvalidationistodemonstratethereliabilityofaparticularmethodforthedeterminationofananalyteconcentrationinaspecificbiologicalmatrix,suchasblood,serum,plasma,urine,orsaliva.Moreover,ifananticoagulantisused,validationshouldbeperformedusingthesameanticoagulantasforthestudysamples.Generallyafullvalidationshouldbeperformedforeachspeciesandmatrixconcerned.Insomecases,itmaybeproblematicforvalidationpurposestoobtainanidenticalmatrixcomparedtothematrixofthestudysamples.Asuitablealternativematrixmaybeused,e.g.syntheticallypreparedcerebrospinalfluid,ifjustified.Themaincharacteristicsofabioanalyticalmethodthatareessentialtoensuretheacceptabilityoftheperformanceandthereliabilityofanalyticalresultsare:selectivity,lowerlimitofquantification,theresponsefunctionandcalibrationrange(calibrationcurveperformance),accuracy,precision,matrixeffects,stabilityoftheanalyte(s)inthebiologicalmatrixandstabilityoftheanalyte(s)andoftheinternalstandardinthestockandworkingsolutionsandinextractsundertheentireperiodofstorageandprocessingconditions.Usuallyoneanalyteordrughastobedetermined,butonoccasionsitmaybeappropriatetomeasuremorethanoneanalyte.Thismayinvolvetwodifferentdrugs,butcanalsoinvolveaparentdrugwithitsmetabolites,ortheenantiomersorisomersofadrug.Inthesecasestheprinciplesofvalidationandanalysisapplytoallanalytesofinterest.ReferencestandardsDuringmethodvalidationandanalysisofstudysamples,ablankbiologicalmatrixwillbespikedwiththeanalyte(s)ofinterestusingsolutionsofreferencestandard(s)topreparecalibrationstandards,qualitycontrolsamplesandstabilitysamples.Inaddition,suitableinternalstandard(s)(IS)canbeaddedduringsampleprocessinginchromatographicmethods.ItisimportantthatthequalityofthereferencestandardandISisensured,asthequality(purity)mayaffecttheoutcomeoftheanalysis,andthereforetheoutcomeofthestudydata.Thereforethereferencestandardsusedduringthevalidationandstudysampleanalysisshouldbeobtainedfromanauthenticandtraceablesource.Suitablereferencestandards,includecertifiedstandardssuchascompendialstandards(EPCRS,USP,WHO),commerciallyavailablestandards,orsufficientlycharacterisedstandardspreparedin-houseorbyanexternalnon-commercialorganisation.Acertificateofanalysisisrequiredtoensurepurityandprovideinformationonstorageconditions,expirationdateandbatchnumberofthereferencestandard.TheuseofcertifiedstandardsisnotneededforIS,aslongasthesuitabilityforuseisdemonstrated,e.g.lackofanalyticalinterferenceisshownforthesubstanceitselforanyimpuritiesthereof.Acertificateofanalysisisnotrequired.Whenmass-spectrometry(MS)detectionisusedinthebioanalyticalmethod,astableisotope-labelledISisrecommendedtobeusedwheneverpossible.However,itisessentialthatthelabelledstandardofthehighestisotopepurityandthatnoisotopeexchangereactionoccurs.Thepresenceofanyunlabelledanalyteshouldbecheckedandifrelativeamountsofunlabelledanalytearedetectedthepotentialinfluencehastobeevaluatedduringmethodvalidation.4.1.1.SelectivityTheanalyticalmethodshouldbeabletodifferentiatetheanalyte(s)ofinterestandISfromendogenouscomponentsinthematrixorothercomponentsinthesample.Selectivityshouldbeprovedusingatleast6individualsourcesoftheappropriateblankmatrix,whichareindividuallyanalysedandevaluatedforinterference.Useoffewersourcesisacceptableincaseofrarematrices.Normally,absenceofinterferingcomponentsisacceptedwheretheresponseislessthan20%ofthelowerlimitofquantificationfortheanalyteand5%fortheinternalstandard.Itmayalsobenecessarytoinvestigatetheextentofanyinterferencecausedbymetabolitesofthedrug(s),interferencefromdegradationproductsformedduringsamplepreparation,andinterferencefrompossibleco-administeredmedications.Co-medicationsnormallyusedinthesubjectpopulationstudiedwhichmaypotentiallyinterfereshouldbetakenintoaccountatthestageofmethodvalidation,oronastudyspecificandcompoundspecificbase.Thepossibilityofback-conversionofametaboliteintoparentanalyteduringthesuccessivestepsoftheanalysis(includingextractionproceduresorintheMSsource)shouldalsobeevaluated,whenrelevant(i.e.potentiallyunstablemetabolitese.g.acidicmetabolitestoester,unstableN-oxidesorglucuronidemetabolites,lactone-ringstructures).Theextentofback-conversionshouldbeestablishedandtheimpactonthestudyresultsdiscussed.Itisacknowledgedthatthisevaluationwillnotbepossibleearlyduringdrugdevelopmentofanewchemicalentitywhenthemetabolismisnotyetevaluated.However,itisexpectedthatthisissueistakenintoaccountandapartialvalidationisperformedifrelevantasfurthe