MPPA202

ANALYTICALMETHOD VALIDATIONFORSOLIDDOSAGE FORMS

Trainer:ChandramouliR

Raisond'etre

Testproceduresforassessmentofthequalityof pharmaceuticalproductssubjecttovarious requirements Usersofanalyticalmethodsdescribedinthe pharmacopeiasUSP/NF,IP,Phar.Eur.,arenot requiredtovalidateaccuracyandreliabilityof thesemethods,butmerelyverifytheirsuitability underactualconditionsofuse itisessentialthatproposalsforadoptionofnewor revisedcompendialanalyticalmethodoralternate inhousemethodsbeshownequivalentto,orbetter 2 than,thecurrentmethod

ProposedNewmethodistobesupportedby sufficientlaboratorydatatodocumenttheir validity Ifthereisnocompendialprocedure,the analyticalmethodmustbefullyvalidated priortouse

AspectsofAMVanditstestingquorum

VerificationofCompendialMethods

Acompendialprocedureisconsideredvalidatedifitis publishedasofficialtextinapharmacopeiaoran interimannouncement(addendums) Whenusingcompendialmethods,thefullvalidation isnotnecessary,butverificationoftheprocedureis veryimportant Verificationensuresthattheprocedureissuitablefor usewithaspecificingredientorproduct,inaspecific laboratory,withspecificlaboratorypersonnel, equipment,andreagents
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Examples...

titrimetricmethodsforwaterdeterminationshouldbe verifiedforaccuracy(andabsenceofpossible interference)whenusedforanewproductorraw material Forimpuritytesting,thesuitabilityofacompendial proceduremaybeanissueforseveralreasons(e.g., impurityprofilechangefromdifferentroutesof synthesis,compositionofformulation,orinterference fromexcipients). Itisrecommendedthattheprocedureforcertification ofsuitabilityofthemonographsofthepharmacopeia beused

CharacterizationofReference Standard

Duringmethodvalidation,awellcharacterized standardshouldbeused wellcharacterizedreferencestandardisacritical factorformethodvalidation Forpotencyassay,thepurityofthestandardmustbe assigned thereferencestandard(usedasprimarystandard) shouldalwaysbeacquiredfromarecognizedauthority, suchastheNationalInstituteforStandardand Technology(NIST),USP,EP,etc.
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Fornewdrugsaninhousereferencestandardmust besynthesizedandcharacterized Thereferencestandardshouldbeminimally characterizedbythefollowingtests:physical appearance,identification,andpurityassignment. Thestructureshouldbeconfirmedusingmultiple analyticaltechniques,suchaselementalanalysis,IR &UVvisiblespectroscopicanalysis,MS,1HNMR, and13CNMR

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Purityassignmentcanbeestablishedbytests forthefollowingitems:organicimpurity, inorganicimpurity,moisture,andresidual solvents. TotalorganicimpuritiesdeterminedbyHPLC, and/orotherchromatographicmethods. Moistureandresidualsolventsgravimetric analysis,suchasTGAorcombinedKarl Fischertitration,andGCmethod.

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inorganicresidueresidueonignition KarlFischermethodforwatercontentandthe GCmethodforresidualsolventsareusedthese shouldbevalidatedusingacompendial method)priortouse. Theinhousestandardqualifiedagainsta primarystandard,followingawelldefined qualificationprotocol.

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StabilityIndicatingMethod

requiredpertheICHguidelines Tochecktheprocedureisabletodetectthechanges withtimeinthepertinentproperties(e.g.,active ingredient,preservativelevel)ofthedrugsubstance anddrugproduct,andshouldaccuratelymeasurethe activeingredientswithoutinterferencefrom degradationproducts,processimpurities,excipientsor otherpotentialsystemcomponents. comprehensiveforceddegradationstudyandHPLC coelutionevaluationconducted,inorderto demonstratethesuitabilityoftheproceduretodetect 11 anychangesthatareattributabletodegradation.

ForcedDegradationStudies(Stress Studies)

maintoolusedtopredictstabilityissues,develop analyticalmethods,andidentifydegradationproducts orpathways performedpriortotheothervalidationparameters (e.g.,accuracy,repeatability,intermediateprecision, specificity,DL,QL,linearityandrange,solution stability,androbustness) multiplestrengthsofdrugproductswiththesame excipientcomposition(includingdifferentformulation ratios),forceddegradationstudiescanbeperformed withonlyoneformulation 12

Formultiplestrengthsoraformulationofdrugproduct withdifferentexcipients,eachdifferentformulation compositionshouldbeevaluatedusingforced degradationstudies. ForINDphase1andINDphase2applications,the forceddegradationstudiesforthestabilityindicating natureoftheassaymethodismethodspecific, therefore,iftheassaymethodsfordrugsubstanceand drugproducthavedifferentconditionswhichcan causechangesinselectivity
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ForINDphase3andlaterphaseapplications, forceddegradationstudiesshouldbedonefor bothdrugsubstancesanddrugproducts. Additionally,massbalanceshouldbeevaluated atphase3,byaddingtherelatedsubstances detectedtotheassayresultsobtainedforforced degradationsamples. 520%degradationisoptimalforforced degradationstudies

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moreasampleisdegraded,themorelikelya lossinmassbalancewillbeobserveddueto secondarydegradation,lossofimpuritiesinthe solventfront,andlossofabsorptionduetoring openingorotherdegradationpathways Forceddegradationstudiesfordrugproduct shouldbeperformedbeforecommencing stabilitystudiesofregistrationbatches Duringdataacquisition,DADshouldbeused forHPLCandallspectraofpeaksshouldbe
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MethodValidationParameters

TheprotocolsorgeneralSOPstodescribethe detailedparametersasperTable Executionoftheprotocolinthelaboratory. Redevelopmentandvalidationifdeviationor failureisobservedduringvalidationthatis attributedtotheanalyticalmethod. Validationreport.

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Dataelementsrequiredfor analyticalmethodvalidation

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SpecialconsiderationsforAMVifdoneby chromatographicmethods...

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FilterBias

theanalysisistypicallycarriedoutbyspectroscopy(UVVis) utilizingachromatographicmethod(HPLC,GC) Filterorcentrifugeisutilizedtoremoveparticulatesthatmay clogthecolumnoraffectabsorbancereadings differenttypesofsyringefilters,suchasnylonorPTFEwitha sizeof0.45or0.2m,shouldbeinvestigateddependenton thesamples. Thefiltershouldbevalidatedbyfilteringaportionofworking standardsolutionthrougheachsyringefilter,discardingthefirst 23mL,andcollectingthefiltrateforanalysis. resultfromthefilteredsolutionshouldbecomparabletothatof unfilteredsolution. 20

SystemSuitability

demonstratesthatthesystemisworking properlyatthetimeofanalysis injectionrepeatability,expressedasRSDof peakresponsesobtainedfromfiveorsix consecutiveinjectionsofworkingstandard solution,tailingfactor,theoreticalplatenumber, resolution,etc

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HPLCSystemSuitabilityParameters

injectionrepeatability checkstandard USPtailing theoreticalplatenumber systemdrift resolution

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Injectionrepeatability

workingstandardsolutionwillbeinjectedfiveor sixtimesontotheHPLCcolumn.Themeanand RSDforconcernedpeakresponsesuchas areawillbecalculatedas:

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acceptancecriteriaareasfollows,theRSDfor peakareaofinterestfromfiveorsixinjections ofworkingstandardsolutionshouldbe2.0%for potencyassay,10%forimpuritytestingand residualcleaningtesting,and3.0%dissolution testing. IftheproducthaslowstrengthorS/Nofthe activepeakislessthan50,theRSDofthe peakareaoftheactivefromthesixconsecutive injectionsof3%maybeacceptableforpotency 24 assay

Checkstandard

partofintegratedsystemsuitabilityforpotency assayordissolutionassay. The%recoveryoftheactivefromthecheck standardiscalculatedasfollows:

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percentagerecoveryforthecheckstandard solutionisintherangeof100.02.0. Forlowdose/strengthofdrugproducts,3.0% differencemaybeacceptable.

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Tailingfactor(T)

Thetailingfactor(T)ofactivepeakfromthe workingstandardsolutioniscalculatedas follows:

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Thepeakissymmetricaliftailingfactoris1.0. generallyconsideredtobereasonableiftailing factorisnomorethan2.0 theacceptabletailingfactorshouldbe confirmedduringrobustnessexperimentssince excessivetailingorfrontingcouldimpact resolutionbetweenpeaks.

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Theoreticalplatenumber(N)

theoreticalplatenumberpercolumn(N)forthe peakcanbecalculatedfromthefirstinjectionof theworkingstandardsolutionasfollows:

Where t is the retention time of active peak and w is the peak width of the peak, obtained by extrapolating the relatively straight sides of the peak to the baseline. Appropriate requirements for this parameter should be derived from validation data

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Systemdrift

Periodicinjectionsoftheworkingstandard solutionshouldbemadeafteracertainnumber ofsampleinjectionsandattheendoftherun. Thepercentagerecoveryofsystemdrift injectioncanbecalculatedasfollows:

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percentagerecoveryofsystemdriftinjectionthroughout therunshouldbewithin98102%. Ifthesystemdriftmeetsthisrequirement,theaverage peakresponsefromthefirstconsecutiveinjectionscanbe usedforthecalculationofthesamples,otherwisea bracketingprocedureshouldbeusedforcalculationofthe samples. Inadditiontothepeakarea,theretentiontimeshouldalso beevaluated. Foridentificationtheretentiontimesshouldnotvaryby morethan2%.
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Resolution(R)

calculatedtodemonstratethatcriticalpairsof peaksareadequatelyseparated,andtherefore independentlyintegrated Theresolutionfactorfromthefirstinjectionof theworkingstandardsolutioncanbecalculated asfollows:

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Theminimumresolutionbetweeneach identifiedcriticalpairofadjacentpeaksshould be1.5,sincethisrepresentsbaseline separationoftwoneighboringGaussianpeaks. Resolutioncanalsohaveanupperlimittomake surethatpeakshavenotdriftedtoofaraway, minimizingtheriskofcoelutiondueto significantchangesintheretentionpropertiesof acolumn.

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ExplanationsforDataelementsrequiredfor analyticalmethodvalidation...

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Accuracy

determinedbyspikingknownamountsofthe activeatsuitablelevelsofthelabelclaimed amounttothecorrespondingplacebopowder (oranamountofplacebomixturecontainingall theingredientsfortheformulationexcept active),andthencalculatingthepercent recoveryoftheactive Forphase3accuracyexperiments,triplicate samplepreparationsarerequiredateach spikinglevel,andaminimumofthreelevels shouldbeassessed.

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Percentagerecoveryiscalculatedbytheassayed amountdividedbyaknownamountofanalytespiked inthesample Percentagerecovery,averagerecoveryfromeach levelandoveralllevels,andconfidenceintervals shouldbeevaluated potencyassays,RSDofrecoveriesforeachspiked levelNMT2.0%. Forlowstrengthofdrugproducts,e.g.,1mg,wider ranges(3.0%forpotency,5.0%fordissolution)maybe 36 applied.

Accuracyandacceptancecriteria

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Precision

evaluatedthroughrepeatability,intermediateprecisionor reproducibility Repeatability

Forrelatedsubstancesandresidualsolventstests, repeatabilityevaluatedbyspikingimpuritiesor solventsatthespecificationlimitfortheproducts potencyassay,usingdrugproducts(tabletorcapsules), withaminimumofsixdeterminations,at100%ofthe testconcentration. TheRSDforthepercentagelabelclaimoftheactive shouldbe3.0%forthefiveorsixsamples.

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Intermediateprecision

measureofthemethodssensitivitytominor changesinequipmentperformance,and/orto variationintheoperatorstechniqueonany givenday secondanalystshouldperformtheassay,using differentequipment,andonadifferentdayto confirmthatacceptableresultscanbeobtained Absolutedifferencebetweenthemean percentagelabelclaimsoftheactivegenerated 39 bythetwoanalystsshouldbe<3.0%

theexactcriteriaisbasedonthetypeoftest andultimatespecification e.g.,drugsubstanceassayspecificationis98 102%,thenthedifferencebetweenlaboratories shouldbe1.5%, butforadrugproductwithaspecificationof 90110%awidercriteriacouldbeused

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Reproducibility

measureofthemethodssensitivityto laboratorychangesduetomoderatechanges inequipmentperformance,and/orvariationin theoperatorstechnique,andthelaboratory environment generatedbytwoseparatelaboratoriesrunning thetest,andisthereforealsocalled interlaboratoryprecision.

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absolutedifferencebetweenthemean percentagelabelclaimsoftheactivegenerated bythetwoanalystsshouldbe<3.0% exactcriteriaisbasedonthetypeoftest,and theultimatespecification e.g.,drugsubstanceassayspecificationis98 102%,thenthedifferencebetweenlaboratories shouldbe1.5% butforadrugproductwithaspecificationof 90110%awidercriteriacouldbeused
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resultssuchasmean,standarddeviation, relativestandarddeviation,andconfidence intervalshouldbeevaluatedandreportedfor eachtypeofprecision

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Linearity

Alinearrelationshipbetweentheconcentration andrespectiveresponsecanbeobtainedby analyzingaseriesofstandardsolutions Atleastfivestandardsolutionswithaspecific rangeshouldbepreparedOneinjectionof eachofthelinearitystandardsolutions sufficient Thepeakareaoftheactivewillbemeasuredat differentconcentrationlevels,andplotted 44 againstthecorrespondingconcentrations

correlationcoefficient(r),yintercept,slopeof theregressionline,andresidualsumofsquares calculatedbythemethodofleastsquares,and aplotofthedatarecorded correlationcoefficientNLT0.999forpotency assay,&NLT0.99forothertests. veryusefultoevaluatethedifferencebetween estimatedvaluefromregressionlineandactual value

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Therangeintheregressionline

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somecases,acombinedmethodforpotency andimpurityassayisused Areapercentageoramountofimpurity calculatedfromstandardat100%leveloflabel claim(LC)arereported,ifitislinearfrom reportingortheQLlevelto120%oflabel claimedlevelofstrength ICHQ3B(R2)

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Ifimpuritiesareavailable,impuritystandards containingknownimpuritiesatrespective specificationlevelsprepared Comparingtheslopeofeachimpuritytothe slopeofthemainstandard,responsefactorsor normalizationfactorscanbeestablished singlepointstandardusedtoquantitate accuratelytheknownimpurities

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Specificity

toconfirmthatananalyticalprocedureisspecificfor theanalyteofinterestinthepresenceofcomponents suchasimpurities,degradants,andmatrix components(excipients) HPLCspecificitydemonstratedbyseparationof criticalpairsofthetwocomponents(theactiveand impurityortwoimpurities)thateluteclosesttoeach other bymakingindividualinjectionofdiluent,eachimpurity, theactive,andtheplacebo(oranalyticalprepared placebo) 49

diodearraydetectorisusefulindetectingco elutedpeaksinthesamplesspikedwithan impuritywhenimpurityisavailable,and/orin stressedsamples.

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StabilityofStandardandSample Solutions

essentialthatsampleandstandardsolutions arestablethroughoutsamplepreparationand analysis. Provingstabilityofthestandardspartofthe validationprocess standardsolutionsfreshlyprepared,andthe concentrationofthestandardsolutionusedas theinitialvalue

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potency assay, the standard & sample solutions considered to be stable if the % difference between initial values of standard and samples, and those at specific times, is NMT2.0%,butanydownwardtrendinthedata shouldalsobeevaluatedforpossibleimpacton theanalysis.

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dissolutionsamples,thestabilityofthesamples inthedissolutionvesselsalsopartofthe validationprocess Thesampleinthedissolutionvesselshouldbe stableatleastuptothefinalsamplingtime. Thesamplesolutioncanbeobtainedbyspiking analyteofinterest(eithersolutioninmediumor APItobedissolvedinmediumofvesselwithin averyshorttime),andplaceboat37.00.5C.

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impurityassay,thesamplesolutionconsidered stableifthefollowingconditionsaremet:

If0.10%individualrelatedsubstance<0.50%, theabsolutedifferencebetweentheinitialand specifictimepointvaluesshouldbe0.10%. Ifpercentageindividualrelatedsubstance 0.50%,thepercentagedifferencebetween theinitialandttimepointvaluesshouldbe 20.0%. NonewdegradationproductQLofanalyteof 54 interestshouldbedetected.

DetectionLimit(DL),and QuantitationLimit(QL)

ICHQ2(R1) thedetectionlimitofanindividualanalytical procedureisthelowestamountofanalyteina samplethatcanbedetected,butnot necessarilyquantitatedasanexactvalue. quantitationlimitisthelowestamountofanalyte inasamplethatcanbequantitatively determinedwithsuitableprecisionandaccuracy

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TheDLandQLcriticalparametersofanalytical procedurevalidationforresidualsolventand impurityassay. SeveralapproachesfordeterminingDLandQL

Visualevaluation Signaltonoiseratioapproach Standarddeviationoftheresponseandslope

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Visualevaluation

mostlikelytobeusedfornoninstrumental methods. determinedbytheanalysisofsampleswith knownamountsofanalyte,andbyestablishing theminimumlevelatwhichtheanalytescan bedetected(DL)orquantifiedwithacceptable accuracyandprecision(QL). ForQLdetermination,sixreplicatesamples mayberequiredtobepreparedandtested.

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Signaltonoiseratioapproach

signaltonoiseratioofpeakofanalyteofinterestin thesampleshouldbeatleast3:1fromDLsolution, and10:1fromtheQLsolution Chromatographictechniques,thesignalofthepeak andthebaselinenoisecanbemeasuredmanuallyor usingbuiltinsoftware. ThedetectionlimitandQLofanalytemaybe determinedbyserialdilutionofastandardsolution withdiluent,andinjectingontotheHPLCsystemfor assay.ThenQLandDLwillbedeterminedbysignalto noiseratio. 58

Standarddeviationoftheresponse andslope

is the standard deviation of the response; S is the slope of the calibration curve. may be estimated based on standard deviation of blank (measurement of the magnitude of analytical background response using six replicate blank samples) or residual standard deviation of regression line or the standard deviation of Y(gamma) -intercepts.
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Thereportingthresholdforimpuritytestingis 0.05%level reportinglevelsaredefinedasperICH Q3B(R2),basedondailyintakeanddose. itisprudentthattheconcentrationofthe analyteinsamplesolutionispreparedatQL level Thesignaltonoiseratioforthepeakofanalyte shouldbemorethan10:1

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QLsolutionshouldbeinjectedassixreplicates ontotheHPLCsystemforanalysis. TheRSDofthepeakareaofanalyteshouldbe 10.0%. IftheQLlevelcannotmeetanyoftheabove criteria,the0.10%levelwillbeevaluatedthe methodshouldbemodifiedtomeetallofthe aboveQLcriteria

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Robustness

Therobustnessofananalyticalprocedureis criticalforeffectivemethodvalidationandcost effectivenesslateroninroutineassay. Robustnessofchromatographicconditionswill beperformedonasamplesolution,suchasa repeatabilitysamplesolutionwithtriplicate injections,byvaryingtheparametersspecified Onlyoneparameteratatimeisalteredwhile therestoftheparametersremainunchanged.

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RobustnessExperimentaldesignfor chromatographicparameters

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Thedesignofexperiment(DOE)couldalsobe usedtoallowmultipleparameterstobevaried ineachexperiment,andthusreducethe numberofexperiments. Theinstrumentsystemmustbeequilibrated undereachtargetandrobustnesscondition. Thesystemsuitabilityrequirementsshouldbe evaluatedineachexperimenttoensurethe appropriatesystemsuitabilitycriteriaaresetfor 64 themethod.

Triplicateinjectionsofthesamplesolutionare thenmadeundereachcondition. Theindividualdeterminedmeanvaluesof analyteandRSDofthreeinjectionsforeach robustnessconditionarereported. Thepercentageoftargetvalueiscalculatedfor eachrobustnesstestconditionasfollows:

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Thepercentageoftargetshouldbewithin98% to102%. Ifanyoftherobustnessparametersfailsto meettheacceptancecriteria,thena precautionarystatementshouldbeincludedin themethodspecifyingthelimitations.

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Samplepreparationforpotencyassay

Robustnessofthesamplepreparationfor potencyassaywillbeperformedonduplicate samplepreparationsbyvaryingtheparameters Robustnessofthedissolutionmethodwillbe performedonthreetablets(orcapsules)by varyingtheparameters,suchasmediumpH value(0.1oftarget),orconcentrationof surfactant(5%oftarget).

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NonchromatographicMethodValidation

AdditionalQCDrugsubstances,excipients,and/or drugproductsparticlesizedistribution,opticalrotation methodologiessuchasDSC,PXRD,Raman spectroscopy,andnearinfraredspectroscopyshould bevalidatedpriortouse validationparameterslessextensivethan chromatographicmethods includerepeatability,intermediateprecision,and robustness,butspecificityandaccuracymayalsobe applicable
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FailureandRevalidation

failuretomeetvalidationacceptancecriteria maybeobserved investigationsshouldbeconductedtoreveal rootcauses attributedtotheprocedure,thevalidationwillbe terminatedatthatstage,andnewmethod developmentexperimentsinitiated Ifmethodappropriatelyrevised,andchanges aredocumentedpriortorevalidation

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Incaseofafailureofcomparisonduring repeatabilityandintermediateprecision,all aspectsshouldbetakenintoconsideration(i.e. chemistskills,labequipmentvariation,and samplevariation) Foranyfailuresduringvalidationexperiments,a thoroughevaluationisrequiredtoensurethat thefailureistrulyduetothemethod,andnot duetolaboratoryerrororotherunexpected issuessuchassamplehomogeneity 70

Theneedtorevalidatethemethodwillbe evaluatediftherearechanges,suchascolumn vendor,drugsubstancesrouteofsynthesis,and drugproductscomposition Somechangesmaynotrequirerevalidationor mayonlyrequirepartialrevalidation(e.g.,new excipientswouldrequirespecificexperiments), buttheevaluationshouldalwaysbemade,and thejustificationfornotrevalidatingshouldbe documented. 71