24(2001)755–767
www.elsevier.com/locate/jpba
Validationinpharmaceuticalanalysis.
PartI:Anintegratedapproach
JoachimErmer*
A6entisPharmaAG,GlobalAnalyticalDe6elopment,QOTSS,Poseidonhaus,D-65926FrankfurtamMain,Frankfurt,Germany
Received16May2000;receivedinrevisedform16November2000;accepted17November2000
Abstract
TheICHguidelinesachievedagreatdealinharmonisingthedefinitionsoftherequiredvalidationcharacteristicsandtheirbasicrequirements.However,theyprovideonlyabasisforageneraldiscussionofthevalidationparameters,theircalculationandinterpretation.Itistheresponsibilityoftheanalysttoidentifyparameterswhicharerelevanttotheperformanceofthegivenanalyticalprocedureaswellastodesignpropervalidationprotocolsincludingacceptancecriteriaandtoperformanappropriateevaluation.Inordertofulfilthisresposibilityproperly,thebackgroundofthevalidationparametersandtheirconsequencesmustbeunderstood.Inthispart,thegeneralconceptofanintegratedvalidationisdiscussed.TheinterdependenciestootherICHguidelinesandtopicsduringdrugdevelopment(e.g.impuritiesanddegradants,stabilityandspecificationdesign)mustbetakenintoaccounttodefinetherequiredacceptancecriteria.Evaluationoftheresultsinordertoprovethesuitabilityoftheanalyticalproceduremustbebasedonthespecificationlimits.Importantparametersandaspectsarediscussedfortheindividualvalidationcharacteristics.Inthefollowingparts,theseparameterswillbediscussedindetail.Exampleswillbegivenfortheirinterpretationinordertofacilitatetheselectionofparameterswhicharerelevanttotheperformanceandsuitabilityofthegivenanalyticalprocedure.©2001ElsevierScienceB.V.Allrightsreserved.
Keywords:Pharmaceuticalanalysis;Validation;Qualityassurance;Acceptancelimits;Specificationlimits
1.Introduction
Thevalidationofanalyticalprocedures,i.e.theproofofitssuitabilityfortheintendedpurpose,isanimportantpartoftheregistrationapplicationforanewdrug[1–8].TheInternationalConfer-enceontheHarmonizationoftheTechnicalRe-*Tel.:+49-69-30584890;fax:+49-69-30525538.
E-mailaddress:joachim.ermer@aventis.com(J.Ermer).
quirementsforRegistrationofPharmaceuticalsforHumanUse(ICH)hasharmonisedthere-quirementsintwoguidelines[7,8].Thefirstonesummarizesanddefinesthevalidationcharacteris-ticsneededforvarioustypesoftestprocedures,thesecondoneextendstheprevioustexttoin-cludetheexperimentaldatarequiredandsomestatisticalinterpretation.Theseguidelinesserveasabasisworldwidebothforregulatoryauthoritiesandindustryandbringtheimportanceofa
0731-7085/01/$-seefrontmatter©2001ElsevierScienceB.V.Allrightsreserved.PII:S0731-7085(00)00530-6
756J.Ermer/J.Pharm.Biomed.Anal.24(2001)755–767propervalidationtotheattentionofallthoseinvolvedintheprocessofsubmission.
Nowadays,thevalidationcharacteristicsneededforthevarioustestproceduresandtheirgeneralrequirements(seeTable1)arewellunderstood.However,inspiteofrecommendationsnottodoso[8],thereisanincreasingtendencytomisusetheguidelinesasakindofchecklistwhichisautomaticallyapplied.Sucha‘checklistmentality’maybecausedbyanincorrectunderstandingofstandardisationandimprovingefficiency.Duetotheintegrationofanalyticsinallaspectsofdrugdevelopmentandqualitycontrol,inappropriateanalyticalproceduresmay,however,leadto,forexample,wrongdecisions,workwhichhastoberepeated(out-ofspecificationresults)anddelays.Consequently,boththedesignofthevalidationstudiesandtheevaluationoftheresultsmustbeadjustedtotheindividualanalyticalprocedureinordertoachieveanunderstandingofitsrealperformance.Onlyonthisbasiscanproofbeobtainedthattheprocedure‘issuitableforitsintendedpurpose’[14].
2.Integrationandinterdependenciesofanalyticalvalidation
Analyticalproceduresareusedthroughoutdrugdevelopmentandthemanufacturingofdrugsub-stancesanddrugproducts.Importantdecisionssuchastheestablishmentoftheshelf-lifefromstabilitystudies,theneedforadditionaltoxicolog-icaltrialsifnewimpuritiesappearorifknownimpuritiesexceedthequalifiedlevels,therework-ingofbatchesandbatchreleaseorrejectionarebasedonanalyticalresults.Inordertomaketherightdecisionsandtoavoidadditionalwork,anappropriateperformanceoftheanalyticalproce-duresisessential.
Butwhatdoes‘suitabilityforitsintendedpur-pose’mean?Forsomeapplications,therequire-mentsaredefinedintheICHguidelines,forexample,forimpuritytesting[9–11].Ifthereport-inglevelforunknownimpuritiesindrugsub-stancesissetto0.05or0.03%[9],thecorrespondingtestproceduremustbeabletoquantifyimpuritiesatthisconcentrationwithanappropriatelevelofprecisionandaccuracy.
Table1
Validationcharacteristicsnormallyevaluatedforthedifferenttypesoftestprocedures[7]andtheminimumnumberofdeterminationsrequired(ifapplicable)[8]Validationcharacteristics
Minimumnumber
TestprocedureIdentity
ImpuritiesQuantitative
SpecificitybLinearityRangeAccuracyPrecisionRepeatability
Intermediateprecision/reproducibilitycDetectionlimitQuantitationlimit
aAssayaLimitYesNoNoNo
YesYesYesYes
–
5concentrations–
9determinationsover3concentrationlevels(e.g.3×3)
6determinationsat100%or9determinationsover3concentrationlevels(e.g.3×3)2series––
YesNoNoNoYesYesYesYes
NoNoNoNo
YesYesNodYes
NoNoYesNo
YesYesNoNo
Includingdissolution,content/potency.
Lackofspecificityofoneanalyticalprocedurecouldbecompensatedbyothersupportinganalyticalprocedure(s).cIntermediateprecisionsufficientforsubmission.dMaybeneededinsomecases.
bJ.Ermer/J.Pharm.Biomed.Anal.24(2001)755–767757
Fig.1.Suitabilityoftheanalyticalprocedurewithrespecttospecificationlimits.
Withrespecttoassaydeterminations,thevari-abilityoftheanalyticalprocedureisoftenlargerthanthevariabilityofthemanufacturing.Thismustbetakenintoaccountintheestablishmentofspecificationlimits[12,13],i.e.thevariabilityoftheprocedureandtheacceptancelimitsmustbecompatible(Fig.1).Ofcourse,safetyrequire-mentsareofprimaryimportancebutiftheyaresatisfied,specificationlimitscanalsobedefinedbasedontheanalyticalvariability[15,16].
Theanalyticalstateoftheartshouldbetakenintoaccountalthoughitisnottheultimategoaltooptimiseanindividualanalyticalprocedureaswellaspossible.Itisalsoveryimportanttorecognisethatthereleaseofagivenbatchisbasedonawholesetoftestprocedureswhichcomple-mentandsupplementeachother.Theirselectioninthespecificationdesign[12,13]has,therefore,considerableinfluenceontherequiredperfor-manceoftheindividualcontroltestand,hence,onitsvalidation.
Besidesthis‘horizontal’integration,therearealso‘vertical’connections.Thereisastrongfeed-backbetweenmethoddevelopmentandvalidation[17].Duetotheimportantaspectoftime,itisadvisable(ifpossible)toperforma‘progressive’validationstartingfromabasicdatasetwhichissupplemented,forinstance,withrespectto(inter-mediate)precisionandrobustness.Thevalidationresultscanalsohaveafeedbackeffectondetailsoftheanalyticalproceduresuchasonthenumberofreplicatedeterminationsorthemodeofcali-bration(seenextchapter).
Duringmethoddevelopmentandvalidationthecriticalperformanceparametersoftheanalytical
procedureshouldbeidentifiedinordertodesigndiscriminatingsystemsuitabilitytests.
Iftheseinterdependenciesareignoredandifparametersaredeterminedduringthevalidationwhichdonotdescribethecriticalperformanceoftheanalyticalprocedure,severeconsequencescanbeexpected.Forexample,iftheprocedureisnotsufficientlyrobust,problemsarelikelytooccurinamethodtransferandrepeatedadjustmentsinthesystemsuitabilitytestwill,forexample,bere-quired.Iftheacceptancelimitsfor,forexample,assayaretoonarrow,out-ofspecificationresultswillrequireextensiveinvestigations[18].Conse-quently,thetimeandeffort(perhaps)savedina‘checklist’validationapproachwillmostlikelyresultinproblemsatalaterdatewhichcouldbemuchmoreexpensive.
3.AcceptancecriteriaforvalidationparametersUsingtheICHguidelinesasabasis[7,8],itistheresponsibilityoftheanalysttoselectforthegivenindividualtestprocedurerelevantparame-tersandappropriateacceptancecriteriaandtodesigntheexperimentalstudiesaccordingly.Theseacceptancecriteriacanoftenbederivedfromspe-cificationlimits.
Asageneralrule,thestandarddeviationoftheanalyticalprocedureshouldbelowerthan1/6ofthespecificationrange.Adetailedapproachtak-ingthenumberofrepeateddeterminationsintoaccountisbasedonconfidenceintervals[15,16](Fig.2).Basicspecificationlimits(BL)includethevariabilityofthemanufacturingprocessandrep-resentthefinallimits(SL)ifanerror-freeanalyti-calprocedureisused.Describingtheanalyticalvariabilityasanormaldistributionoftheexperi-mentalresults,confidenceintervalscanbecon-structedasarepresentationoftheresultprobabilityforagivennumberofreplicates.Thecombinationofthebasiclimitandthelimit(up-perorlower)ofthe95%confidenceinterval(one-sided)thenrepresentstheoverallspecificationlimit(Eq.(1)).Itmustbetakenintoaccountthatthiscalculationisbasedonthetruestandarddeviationwhereasthestandarddeviationobtainedinavalidationstudyisonlyarandomestimate
758J.Ermer/J.Pharm.Biomed.Anal.24(2001)755–767Fig.2.Constructionofspecificationlimitsfrom95%confi-denceintervals.Theprocedureisshownforthelowerlimits.BL,basiclimits,imposedbythevariabilityofthemanufactur-ingprocess;SL,overallspecificationlimit,combinationofBLandthe(lower)limitoftheone-sided95%confidenceintervaloftheanalyticalvariability.
whichalsodisplaysavariability.Areliableexper-imentaldeterminationofthetruevaluerequiresrepeatedintermediateprecisionstudies,butitcanbeestimatedfromthestatistical(2)distributionofstandarddeviations.Theupper95%confidencelimitofthisdistribution(UL)willrepresentthemaximumvalueforthetruestandarddeviation(Eq.(2)).Forsixexperimentalvalues,itcanbeapproximatedbytwicetheexperimentalstandarddeviation[16].SL=BL9tn−1,0.95×UL(strue)=sexp×
struen(1)
RearrangingEqs.(1)and(2)givesthemaxi-mumpermittedexperimentalstandarddeviationforthegivenspecificationlimits,i.e.theaccep-tancelimitforthevalidation.Forexample,adrugsubstanceLC-assay(performedwithfourrepeti-tions)withalowerspecificationlimitof98.0%andalimitforthesumofimpuritiesof0.5%(i.e.alowerbasiclimitof99.5%)wouldrequireanexperimentalstandarddeviationinvalidationbe-low0.64%.ItshouldbenotedthatBLandSLinEq.(3)refertothe‘critical’halfofthespecifica-tionrange.Fordrugsubstances,duetothepres-enceofimpurities,thesearethelowerlimits.Astherequiredstandarddeviationisdependentonthenumberofrepetitionsintheassay,adjust-mentsarepossible(Table2).Thus,thenumberofrepeateddeterminationscanalsobefine-tunedaccordingtotheresultsofthevalidation.Incaseofsufficientlywidespecificationlimitscomparedtotheanalyticalvariability,thisallowsaneffi-ciencyoptimisationoftheanalyticalprocedure.smax=
(BL−SL)×n2×tn−1,0.95(3)
'
(n−1)2n−1,0.95(2)
Whileinthecaseofadrugsubstanceassaythe
basiclimitsaredefinedmainlybythesumofimpurities,inthecaseofdrugproducts,oftenonlyanestimationispossible.Forsimpledosageforms,thesamevariabilitymaybeexpectedforanalyticsandmanufacturing.
Itshouldbenotedthattheestimationofthetruestandarddeviationwillresultinamaximumvalueand,therefore,reducetherequiredaccep-tancelimit.Consequently,incriticalcasesanex-
Table2
Maximumpermittedstandarddeviationforassaydeterminationsindependenceonthenumberofrepetitionsandthespecificationrange
Drugproduct(%)
SpecificationrangeBasiclimit(lower)Numberofrepetitions2346
95–105
97.5(estimated)
Drugproduct(%)95–105
99.0(estimated)
Drugsubstance(%)98–102
99.5(sumofimpurities)
Acceptancelimitforexperimentalstandarddeviationinvalidation(n=6)0.280.741.061.44
0.451.191.72.3
0.170.450.640.86
J.Ermer/J.Pharm.Biomed.Anal.24(2001)755–767759
perimentaldetermination(usingatleastfourtosixseries)mightbereasonabletoobtainanesti-mateforthetruestandarddeviation.Then,Eq.(1)canbedirectlyrearrangedandthefactoroftwoinEq.(3)canbeomitted.
Ifspecificationlimitsarenotyetdefinedorimpliedduetosafetyrequirements,Eq.(1)canbeuseddirectlytocalculatethelimits.Forthetruestandarddeviation,theupperlimitoftheexperi-mentaldetermination(Eq.(2))canbecalculatedortwicethisvalueasanapproximation.Alterna-tively,asoundlybasedintermediateprecisionmaybeusedasanestimationforthetruestandarddeviation.
Thevalidationacceptancelimitshouldthenbederivedfrompreviousexperiencesofcomparableanalyticalprocedures(analyticalstateoftheart).Forexample,fromawellbasedaswellasaccept-ableintermediateprecisionof1.0%andabasiclimitof99.0%,thespecificationrangeforadrugsubstancetobedeterminedwithfourrepetitionscanbecalculatedto97.8–101.2%.Iftheexperi-mentalrepeatabilitywasdeterminedwith1.0%,limitsfrom96.4–102.4%wouldresult.
Withrespecttoimpuritydeterminations,theICHreportingthresholdof0.05%[9]canberegardedastherequiredquantitationlimitforunknownimpuritieswhichwouldguaranteeareli-ablequantitationatthespecificationlimitof0.1%.Ifotherlimitsarerequireddue,forexam-ple,tosafetyconsiderations,theabove-mentionedapproachcanbeapplied.
StatisticaltestssuchastheStudent’st-testortheevaluationof95%confidenceintervalsshouldonlybecarefully(directly)appliedasacceptancecriteriabecausetheytestforstatisticaldifferences.Duetosometimesabnormallysmallvariabilitiesintheanalyticalseries,differencesareidentifiedassignificantwhichareofnopracticalrelevance[19].Inaddition,whencomparingindependentmethodsfortheproofofaccuracy,differentspe-cificitiescanbeexpectedwhichaddasystematicbias,thusincreasingtheriskoftheaforemen-tioneddanger.
Theanalystmustdecide,ifdetectedstatisticaldifferencesareofpracticalrelevance.Forexam-ple,if(e.g.duetoabnormalsmallvariabilityinoneseries)forameant-testorinlinearitya
Fig.3.Dependenceofthetestpoweronthenumberofdeterminations.Meant-testat95%levelofstatisticalsignifi-cance,assumingatruestandarddeviationof0.5andthesamenumberofdeterminationsforeachmean.
(statistical)significantdifferencetoanotherseriesoroftheintercepttozeroisdetected,theabsolutemagnitudeofthisdifferenceshouldbetakenintoaccount.Ontheotherhand,alargevariabilitycanalsoobscuredifferenceswhicharenotacceptable.
Inaddition,thepowerofthesetestsincreaseswiththenumberofdeterminations.AsshowninFig.3,withsixdeterminations,adifferenceof0.64betweentwomeanscanbedetected,whereasadifferenceof0.25canbedistinguishedwith32repetitions.Althoughthelatterdifferenceisstatis-ticallysignificant,inmostcases(suchasLC-assaydetermination)ithasnopracticalrelevance.
Forpracticalpurposes,asufficientagreementbetweentworesults(twomeansorameanandanominalvalue,e.g.recovery)iscompletelyade-quate.Theacceptancecriteriacanbederivedfrompreviousexperienceorcalculatedbasedonspecificationlimitsandstatisticalconsiderations(seenextpart).Forexample,assumingaLC-as-saywithspecificationlimitsfrom95to105%,arecoveryrangefrom98to102%wouldbeacceptable.
Theacceptancecriteriaandlimitsshouldbedefinedbeforestartingthevalidationandincludedinaprotocol.Afterthevalidationstudies,theywillserveasabasisfortheevaluation.
760J.Ermer/J.Pharm.Biomed.Anal.24(2001)755–7674.Validationcharacteristics
Whenperformingvalidationstudies,thewholeanalyticalprocedureincludingallthestepsofthesamplepreparationshouldbeapplied,asfaraspossible.Incontrast,theterm‘method’shouldberestrictedtothemodeofanalyticaldeterminationalone(e.g.capillaryelectrophoresis,(reversed-phase)chromatographyandspectrometry).
Allowedexceptionstothewrittenprocedureconcernthenumberofrepetitionsasthenumberofdeterminationsforthevariousvalidationchar-acteristicsisdescribedintheICHguideline(Table2)andtherepetitionsintheproceduremaybefine-tunedbasedonthevalidationresults(seepreviouschapter).Suchanadjustmentmayalsobeusedforthefinalcalibrationmode(Section4.2).
Asfaraspossible,theanalyticalprocedureshouldbeindependentoftheactualequipmentusedprovidedthattheequipmenthasbeenappro-priatelyqualified.Thismustbetakenintoaccountforthevalidationstudies.
4.1.Specificity
Therehasbeensomecontroversyregardingthetechnicaltermforthisvalidationcharacteristic,i.e.specificityvs.selectivity[20].Incontrasttoanisolatedtestprocedure,inpharmaceuticalanalysisthesumofvariouscontroltestsandhencetheircombinedspecificityisusedfortheoverallbatchevaluation.Averypragmaticdefinitiondescribesselectivityasthe(physical)separationofsub-stancemixtureswith,forexample,chromatogra-phyandelectrophoresis,i.e.thedeterminationoftheanalyteinadditiontoothersubstances.Theindividualdeterminationofananalyteinthepres-enceofothersubstances,(i.e.withoutsignificantinfluenceofothersubstancesorclassesofsub-stances)isdefinedasspecificby,forinstance,massspectrometry,NMR,infrared,fluorescenceorUVspectrometry,electrochemicaldetectionandtitration[21].
Inspiteofthisdiscussion,thereisabroadagreementthatthisvalidationcharacteristicisofcrucialimportanceandisthecriticalbasisforeachanalyticalprocedure.Asnoabsoluteand
quantitativemeasureexists(atleastfortheoverallspecificity),therequirementsdependontheindi-vidualanalyticalprocedureaswellasonitscom-binationwithothers.Forexample,theoverallspecificityofaqualitycontrolcanbeobtainedbysecuring(orcorrecting)apreciseandefficientassaytitrationwithaselectivechromatographicimpuritydetermination.Assumingatitrationfortheassayofabasicdrugsubstancewithamolec-ularweightof300.Threeimpuritiesarespecified,twoofwhicharealsobasiccompounds(A:200andB:100MW).Thethirdimpurity,aneutralmoleculewillhavenotitrationresponse,fortheotherimpuritiestitrationresponsefactorsof300/200=1.5and300/100=3.0canbecalculated.Therefore,theresultofthebatchtitrationmustbecorrectedwiththeamountsofthebasicimpuritiesobtainedbyaselectiveLC-procedure.Ofcourse,theamountofunknownimpuritiesmustbelim-itedinordertopreventnonpredictabletitrationresponses.
drug[%asis]=titration[%]×
100−1.5×A[%,LC]−3×B[%,LC]
100
(4)
Withrespecttochromatographictechniques,specificitycanbedemonstratedbyasufficientseparationofthesubstancespresent.Fortheas-say,appropriateseparationmeansanadequateresolutionbetweenthemainpeakandtheimpu-rityandplacebopeakswhichneednottobeseparatedfromeachother.Thesamecanbeappliedtoindividualimpurityordegradantdeter-mination.Incontrast,universalproceduresforthedeterminationofimpuritiesrequireasufficientseparationofallrelevantimpuritypeaks.Therequiredresolutionisstronglydependentonthedifferenceinthesizeofthecorrespondingpeaksaswellasontheirelutionorder[22].Inordertobeabletodetectthecoelutionofunknownsub-stances,peakhomogeneityinvestigationssuchasrechromatography,diodearraydetectionorLC-MScouplingshouldbeperformed[nextpart].Ifsamplesfromstresstestingareusedtodemonstrateappropriateseparationpower,care
J.Ermer/J.Pharm.Biomed.Anal.24(2001)755–767761
shouldbetakentoavoidoverdegradationasthiswouldresultinsecondary(orevenhigherorder)degradantswhichareofnopracticalrelevance.Therefore,degradationshouldberestrictedtoabout10%.Alternatively,samplesfromregularstabilitystudies(acceleratedstorageconditions)maybeused.
4.2.Linearity/range
Alineardependenceofthesignalandtheana-lyteconcentrationiscertainlythemostconvenientcaseandwidelyusedinpharmaceuticalanalysis.However,thereareanalyticalprocedureswitha
nonlinearresponsesuchasTLC,fluorescencede-tectionandatomabsorptionspectrometry.There-fore,theterm‘analyticalresponse’wouldhavebeenmoreappropriateforthisvalidationcharacteristic.
Theessentialquestiontobeansweredhereisonthesuitabilityofthecalibrationmodetobeusedinthetestprocedure.Therequirementsandrele-vantparametersforthevariouscalibrationsaregiveninTable3.Adetaileddiscussionwillfollowinthenextpart.
Itshouldbenotedthatinmostcasesonlyaqualitativestatementisneeded.Forexample,ifasingle-pointcalibration(externalstandard)is
Table3
RequirementsfordifferentcalibrationmodeswithrelevantparametersQuantitation
Single-pointcalibrationExternalstandard
Requirements
Relevantparameters
Linearfunction
Non-significantordinateinterceptHomogeneityofvariancesaMultiple-pointcalibrationLinear,unweighted
Linearfunction
Standarderrorofslope(residualstandarddeviation),sensitivities(relativestandarddeviation,graph),residualanalysis,statisticaltests(vs.quadraticregression)
Inclusionofzeroinconfidenceintervaloftheordinateintercept,magnitudeoftheintercept(aspercentofthesignalat100%testconcentration)
F-testofthevariancesatthelowerandupperlimitoftherange
Linear,weighted
Non-linear
100%-method(areanormalisationforimpurities):
Standarderrorofslope(residualstandarddeviation),sensitivities(relativestandarddeviation,graph),residualanalysis,statisticaltests(vs.quadraticregression)
HomogeneityofF-testofthevariancesatthelowerandupperlimitofthe
avariancesrangeLinearfunctionStandarderrorofslope(residualstandarddeviation),
sensitivities(relativestandarddeviation,graph),residualanalysis,statisticaltests(vs.quadraticregression)
ContinuousfunctionAppropriateequationFormainpeak:linearfunctionNon-significantordinateinterceptHomogeneityofvariancesaForimpurities:linearfunction
Standarderrorofslope(residualstandarddeviation),sensitivities(relativestandarddeviation,graph),residualanalysis,statisticaltests(vs.quadraticregression)
Inclusionofzeroinconfidenceintervaloftheordinateintercept,magnitudeoftheintercept(aspercentofthesignalat100%testconcentration)
F-testofthevariancesatthelowerandupperlimitoftherange
Standarderrorofslope(residualstandarddeviation),sensitivities(relativestandarddeviation,graph),residualanalysis,statisticaltests(vs.quadraticregression)
aMaybepresumedforalimitedrange(factor10–20).
762J.Ermer/J.Pharm.Biomed.Anal.24(2001)755–767Table4
QuantitativeapproachestodemonstrateaccuracyaccordingtoICH[8]
DrugsubstanceApplicationoftheanalyticalprocedureto
areferencematerial
Comparisonoftheresultswiththoseofasecond,wellcharacterisedprocedureDrugproduct
Applicationoftheanalyticalproceduretosyntheticmixturesofdrugproductcomponents
Spikingofanalyttodrugproduct
Comparisonoftheresultswiththoseofasecond,wellcharacterisedprocedure
ImpuritiesSpikingoftheimpuritytodrugsubstance(quantitative)orproduct
Comparisonoftheresultswiththoseofasecond,wellcharacterisedprocedure
aimedat,therequirementsarealinearresponsefunctionandthezerointercept.Iftheseprerequi-sitesarefulfilled,theactualfiguresforthestan-darderrorofslopeortheconfidenceintervaloftheintercept,forexample,arenotusedfurtherorreferredto.Therefore,itmakesnosensetorepeatlinearityinvestigationsonotherdaysorwithotheroperators[23].However,careshouldbetakentoremainwithinthelinearrangeoftheindividualdetectorusedbutthisinformationcanbeobtainedfromtheequipmentqualification.
4.3.Accuracy
TheICHguidelinerecommendsthedemonstra-tionofaccuracyoverthewholeworkingrange(seeTable1).However,ifonlyanarrowrangeisrequired(e.g.assayorimpuritieswithalowspe-cificationlimit),asix-folddeterminationata100%testconcentrationasdescribedforthepreci-sionstudiesmayalsobeused.
SeveralapproachesdiscussedintheICHguide-linearegiveninTable4.
Iftheanalyticaltesttobevalidatediscom-paredwithanotherprocedureorappliedtoareferencesubstance,theprobablydifferentspecifi-citiesmustbetakenintoaccount.Therefore,statisticaltestsshouldbeperformedonlyifthesystematicbiasbasedonthesedifferencescanbequantifiedandthuscorrectedorarenegligible.
Otherwise,thecomparisonshouldbeperformedasaqualitativeverificationofplausibilityoranacceptablemaximumdifferenceshouldbedefined(e.g.2.0%foranLCassay).
Spikingexperimentsforrecoveryinvestigationsshouldbeperformedascloselytotheauthenticconditionsaspossiblesothatpossibleinterfer-encesbetweentheanalyteandmatrixcanberecognised.Thisranges,forexample,fromthedirectpreparationofadrugproductwithvariouscontentsofactiveingredienttowhichthewholeanalyticalprocedureisappliedtotheadditionofadrugsubstancestocksolutiontoaplacebosolution.
Forthequantitationoftheanalyte,thesamecalibrationmodeasdescribedinthefinaltestproceduremustbeused.Again,statisticaltestsshouldbeusedcarefully,especiallywithcomplexmatricesandlowconcentrationsofimpurities.Alternatively,acceptabledeviationsfromthethe-oreticalrecoveryof100%canbedefinedbasedontheapplication,experiencesorgeneralstatisticalconsiderations(nextpart).
UsingUVdetection,responsefactorsforknownandavailableimpuritiesanddegradantscanbeobtainedfromlinearityorrecoverystudies(ratiooftheslopesorofspecificpeakareaofanalyteandactiveingredient).However,thisisnotpossibleforunknownimpuritiesorimpuritieswhicharenotavailable.Inthesecases,safetychecksshouldbeperformedtoidentifypossibleproblems.Asafirststep,thepeakareapercentcanbeobtainedadditionallyata‘check’wave-lengthinthelowerUVrange(e.g.210nm)[24]whereabsorbancecoefficientsoftendifferless.Comparableresultsforthetwowavelengthsindi-catesimilarresponsefactors,wheraslargediffer-encesinthepeakareamayindicateresponsefactorsdifferenttounity(Fig.4).However,thelatterbelongsrathertotheanalyticaldevelopmentandthedesignoftheanalyticalprocedure.
4.4.Precision
InadditiontotheICHprecisionlevels(Table1),itisadvisabletodeterminethesystempreci-sion(injectionrepeatability)eitherbyrepeated
J.Ermer/J.Pharm.Biomed.Anal.24(2001)755–767763
determinationsofthesametestsolutionorfromdoubledeterminationsofeachtestsolutionusedforrepeatability(Eq.(5)).s1md=
'
%(xi,1−xi,2)22m(5)
i=1Repeatability,alsotermedintra-assayprecision,referstotheprecisionobtainedunderthesameoperatingconditionsoverashortintervaloftimebyapplyingthewholeanalyticalproceduretothesample.Intermediateprecisionreferstowithin-laboratoryvariations.Theextentofinvestigationswilldependontheintendeduseoftheanalyticalprocedure.Atypicalinvestigationmightincludeanalysts,days,equipment,reagents,columns,etc.Preferably,theintermediateprecisionstudiesshouldbeextendedoveralongerperiodoftime,inordertoobtainameasureoftheanalyticalvariabilitywhichisrepresentativeforthelong-termroutineuse.Abasicvalidationstudycanalsobesupplementedbyincorporatinginvestiga-tionsintotheroutineapplicationoftheanalyticalprocedure.
Repeatabilityandintermediateprecisioncanbecalculatedbyananalysisofvariances[25,26].Theformerfigurerepresentstheoverallvariabilitywithintheperformedseries,thelatteralsoin-cludesthevariabilitybetweentheseries(Table5).Thedifferencebetweentheprecisionlevelsaswell
Fig.4.SafetycheckfordeviatingresponsefactorsofunknownimpuritiesinUVdetection.Inadditiontothetestprocedurewavelengthof240nm,relativepeakareaaredisplayedat220nm.Theratioforthelabelledpeaksisaboutseven.Infurtherinvestigations,aresponsefactorof14wasdetermined.
Table5
AnalysisofvariancesfortheinvestigationofintermediateprecisionaSeries1
Series2Numberofdeterminations6
7
Relativestandarddeviation0.32%0.63%Mean
10.0710.1495%confidenceinterval0.0340.060
Overallmean10.11Repeatability
0.52%Intermediateprecision
0.65%
aTwooperatorsperformedanassayofatabletformulation(10mg)withdifferentLC-systems,mobilephases,andcolumns.
astheirabsolutemagnitudeindicatetherobust-nessoftheanalyticalprocedure.Fortheevalua-tionofthesuitability(compatibilitywithspecificationlimits,seechapter‘AcceptanceCrite-riaforValidationParameters’),theintermediateprecisioncanberegardedastherelevantparame-ter,especiallyiftheanalyticalinvestigationsex-tendoverseveralyearssuchasduringstabilitystudies.
Fromthestandarddeviation,therepeatabilitylimitcanbecalculated(Eq.(6))whichrepresentsthemaximumpermitteddifferencebetweentworepeatedmeasurements.Inthisway,astraightfor-wardverificationispossibleifthedegreeofscat-teringundertheactualconditionsiscomparabletothevalidation.Bythisparameter,theprecisionobtainedduringthevalidationstudiesusingalargernumberofdata(thusincreasingthereliabil-ity)islinkedtotheroutineanalyseswithoutre-quiringalargernumberofexperimentaldata.r=tn−1,0.95×2×s:2.8×s
(6)
Ofcourse,areliableestimateofthestandarddeviationisrequiredtocalculateappropriatere-peatabilitylimits.
Moststatisticaltestsandcalculationsarebasedontheassumptionthattheexperimentalvaluesareonlyinfluencedbyrandomvariability(i.e.thattheyarenormallydistributed).Data,whichdonotfulfilltheseassumptions(e.g.duetosocalled‘grosserrors’,weighing,dilution,orbyproblemswiththeinstrument,etc.)willaffecttheresults.
764J.Ermer/J.Pharm.Biomed.Anal.24(2001)755–767Suchvaluescanbeidentifiedbystatisticaloutliertests(e.g.accordingtoDixonorGrubbs[25])inordertoeliminatethembeforeperformingfurthercalculations.However,theproblemis—espe-ciallywithasmallnumberofdatawheregroup-ingscouldeasilyoccur—avoidingtheincorrectrejectionofvaluesbelongingtothesamedistribu-tion.Whenanoutlierisidentified,theabsolutemagnitudeofthestandarddeviationmustalsobeconsideredforevaluation.Ifthisparameter(cal-culatedincludingthesuspected‘outlier’)liesinanormallyexpectedrange,preferablyallvaluesshouldberetained.Outliertestsshouldbeappliedcarefullyandonlyobviouslydeviatingvalues(‘grosserrors’)shouldbeeliminated.Itisprefer-abletoincreasethereliabilityoftheobtainedstandarddeviationbyrepeatedinvestigations,e.g.usingtheoverallrepeatabilityobtainedwithananalysisofvariancesofanintermediateprecisionstudy.
4.5.Detectionandquantitationlimit
SeveralapproachesaregivenintheICHguide-linetodeterminethedetectionandquantitationlimits(Table6).Generally,theyarebasedeitherontheanalysisofblanksoronthescattering(variability)oftheanalyticalsignalsinthelowconcentrationrange.
Usingtheblankprocedures,thecorrespondingcalculationvalueismultipliedbythefactorsof
Table6
Approachesfordeterminingthedetectionandquantitationlimit[8]aApproach
Detectionlimit
Quantitationlimit
VisualevaluationMinimumlevelMinimumleveldetectablequantifiableSignal-to-noise
3:1or2:110:1Standarddeviationof3.3×|/S
10×|/S
theresponse(|)bandtheslope(S)
aVerificationwithasuitablenumberofsamples.
bStandarddeviationoftheblank,residualstandarddevia-tionofthecalibrationline,orstandarddeviationoftheintercept.
3.3and10forthedetectionandquantitationlimits,respectively.Thecalculationvaluemayrepresentthesignaloftheblank,thestandarddeviationoftheblankoroftheinterceptofacalibrationline(correspondingtoanextrapolatedblank).Inthelattertwocases,theanalyticalsignalistransformedbytheslopeofthecalibra-tionlineintoaconcentration[8].Usingthecali-brationlinedirectly,theaforementionedfactors(3.3and10)canbemultipliedbytheratiofromtheresidualstandarddeviationandtheslope(cor-respondingtothestandarderrorofslope)[8].Limitscalculatedorextrapolatedbythesepro-ceduresshouldbeverifiedbytheanalysisofsam-plesinthecorrespondingconcentrationrange[8].Thisadditionalverificationisalreadyincludedinotherprocedureswhichmakedirectuseofthescatteringaroundthecalibrationlinebymeansofthe95%predictionintervalaroundtheregressionline[27,28].
Thequantitationlimitcanalsobeobtaineddirectlyfromprecisionstudies.Forthisapproach,decreasinganalyteconcentrationsareanalysedre-peatedly.Thecoefficientofvariation(relativestandarddeviation)isplottedagainstthecorre-spondingconcentration.Ifapredefinedlimitforthecoefficientofvariation(relativestandarddevi-ation)isexceeded(e.g.10or20%),thecorre-spondingconcentrationisestablishedasthequantitationlimit[6,29].However,asufficientlylargenumberofanalyteconcentrationsmustbeanalysedbecausealargescatteringofstandarddeviationsoccurinthelowconcentrationrange.Morethantheothervalidationcharacteristics,detectionandquantitationlimitsaredependentontheequipmentusedandtheactualconditionsaswellasonthemethodofcalculation.InFig.5,theresultsofarepeateddeterminationusingfiveLC-systemsoveraperiodofabout9monthsareshown.Acalibrationusingamodelcompoundwasperformedwithsixconcentrationsintherangefrom0.05–1mg/ml.Threeapproacheswereappliedtoobtainthequantitationlimit:thecalcu-lationsfromtheresidualstandarddeviationandfromthe95%predictionintervalwereperformedusingthelinearregressionparameters,thecon-centrationscorrespondingtoasignal-to-noisera-tioof10wereinterpolatedfromtheexperimental
J.Ermer/J.Pharm.Biomed.Anal.24(2001)755–767765
Fig.5.Reproducibilityofthequantitationlimit(‘intermediateQL’).Threedifferentprocedureswereusedtocalculatethequantitationlimit.1.1–1.6,sixdeterminationswereperformedonthesameLCsystem;2.1–2.3,threeserieswereperformedonasecondLC-system;3.1–5.1,threeotherLC-systemswereused.Theserieswereanalysedoveratimeperiodofabout9months.
signal-to-noiseratiooftheconcentrationsbelow0.5mg/ml.
Largedifferencescanbeseenforthevariouscalculationprocedures(factor2–3),butalsointhecaseofrepeatedanalysisforthesamecalcula-tion(factor2).
However,theselimitsareofspecialimportanceinthetransferofanalyticalproceduresandforthereportingofimpurities.Therefore,the(gen-eral)quantitationlimitoftheanalyticalprocedureshouldbedefinedtakingtherequirementsoftheanalyticalprocedureintoaccount,ratherthandeterminedexperimentally.Inordertoguaranteeareliablequantitation,itmustbeatleastthreestandarddeviationsawayfromthespecificationlimit.Fororientationpurposes—takingthehighervariabilityinthisconcentrationrangeintoaccount—thequantitationlimitmaybedefinedas50%ofthespecificationlimit.Forexample,withregardtoanimpurityspecifiedas0.1%,thequantitationlimitcanbeestablishedat0.05%.ThiscorrespondstothereportingthresholdsoftheICHimpurityguideline[9].Duringvalidation,oneofthedescribedmethodsisusedtoverify(generally)whetherthisquantitationlimitcanbereliablyachieved.Takingtherandomvariability
ofthedeterminationintoaccount,itdoesnotmatterwhetherthe‘actual’QLisdeterminedto0.02or0.04%,providedthattheupperlimitofthe‘general’QLof0.05%canbeachieved.Ifimpuritiesarepresentorcanbespikedatthequantitationlimitinbatchesusedforassaypreci-sionstudies,thestandarddeviationwithrespecttotheimpuritiescanbecalculatedfromthesameexperimentaldata.TheQLcanberegardedasverifiedifanacceptableprecision(e.g.below10–20%)isachieved.
5.Conclusions
Beyondtheregulatoryrequirements,theperfor-manceandreliabilityofthecontroltestprocedureareessentialtothequalitycontrolofdrugs.Re-sultswhichreflectthequalityoftheanalyticalproceduremorethanthequalityofthepharma-ceuticaltobetestedmayeasilyleadtodrasticfinancialconsequences.Therefore,validationshouldberegardedaspartofanintegratedcon-cepttoensurethequality,safety,andefficacyofpharmaceuticals.
766J.Ermer/J.Pharm.Biomed.Anal.24(2001)755–767Basedonthevalidationcharacteristicsandre-quirementsoftheICHguidelines,eachanalyticalproceduremustbevalidatedwithrespecttoparameterswhicharerelevanttoitsperformance.Itistheresponsibilityoftheanalysttoidentifythecriticalperformanceparametersanddesignthevalidationstudyaccordingly.Acceptancecriteriashouldbedefinedinthevalidationproto-col.Theycanbeestablishedfrompreviousexperi-ences(analyticalstateoftheart)orcalculatedfromspecificationlimits.Fortheintendeduseofthetestprocedureacceptableabsoluteacceptancelimitsarepreferred.Statisticaltestsshouldbeusedcarefullyandpreferablyfororientationpur-poses.Theevaluationofthevalidationresultsistheresponsibilityoftheanalystandmustnotbeleftorreducedtotheoutcomeofastatisticaltest!However,statisticalanalysisandconsiderationsareveryhelpfulinverifyingthecompatibilityofspecificationlimitsandanalyticalvariability,cal-culatingacceptancelimitsandperformingsimula-tionsinordertopredictfuturerisks.
Relevantparametersfortheevaluationoflin-earityaredependentontheintendedcalibrationmodeoftheanalyticalprocedure.Fortheevalua-tionandfurthercalculations,thedifferentlevelsofprecisionmustbetakenintoaccount.Duetothelargevariabilityofexperimentallyobtainedresults,the‘general’quantitationlimitshouldbedefinedaccordingtotherequirements,forexam-plethereportingthresholdforunknownimpuri-tiesof0.05%.
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