Validasi metode

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  • Analytical Methods ValidationM. Hatta PrabowoDepartment of Pharmaceuticals ChemistryUniversitas Islam Indonesia

  • Chemical analysisThe bioanalytical part of bioequivalence trials should be conducted according to the applicable principle of Good Laboratory Practice (GLP)

  • Good Laboratory Practice (GLP)Test plan (Analytical protocol)Sample traceability Documentation, possible to reconstruct the studyAnalytical method validation reportAnalytical report signed by responsible investigator

  • Pre-study phaseThe method used must be well characterisedStabilitySpecificityAccuracyPrecisionLimit of quantitationResponse function

  • Validation objectiveTo demonstrate that the analytical procedure is suitable for its intended purpose

  • Analytical method validationAnalytical ProcedureSelectivityAccuracy Precision- within-run - between-run RecoveryLimit of Quantitation LOQCalibration curveRobustnessValidationStability

  • Sample preparationSeparation DetectionAnalytical procedure

  • Specificity (selectivity)Ability of an analytical method to measure only what it is intended to measure

    Blank samples from six different subjectsWill other drugs, metabolites or endogenous components interfere in the measurements?

  • AccuracyThe closeness of mean test results obtained by the analytical method to the true value (concentration) of the analyte.

  • AccuracyAccuracy should be measured at minimum 3 levelsAt least 5 determinations per concentrationThe mean value should be within 15% of the actual value At the lower limit of quantitation level within 20% is accepted


  • PrecisionThe closeness of individual measurements of an analyte when the procedure is applied repeatedly to multiple aliquotes of a single homogenous volume of biological matrix

  • PrecisionPrecision should be measured at minimum 3 levelsAt least 5 determinations per concentrationThe calculated CV should not exceed 15% At the lower limit of quantitation level, CV should not exceed 20%Subdivided into within-run and between-run

  • Precision and AccuracyConc.nmol/lAccuracy(%)Precision%%n0.760.65.16.118233.61.71.8181223.91.31.318Within-runBetween-run

  • RecoveryThe extraction efficiency of an analytical method

    Recovery of an analyte need not be 100%

  • Lower limit of quantitation The lowest standard on the calibration curve should be accepted as the lower limit of quantitation (LLOQ)


  • Lower limit of quantificationThe analyte responce at LLOQ is at least 5 times the blank responseThe peak should be identifiable and discretePrecision within 20% CVAccuracy of 80-120%

  • LLOQ (1.50 nmol/l) for morphine

  • Calibration/Standard curve

    A calibration curve is the relation between instrument response and known concentrations of the analyteShould be prepared in the same biological matrix as the samplesShould consist of 6-8 samples covering the expected rangeShould include LLOQ and a blank sampleShould include a zero sample (with internal standard)Same curve fitting, weighting in prestudy and studyAny changes should be documented

  • Calibration curve

  • Sample dilutionAny required sample dilutions should use like matrixDilution QC sample should be used

  • RobustnessHow many samples can be analysed in one run?

  • 8590951001051101150102030405060708090100110Sample No.Found concentration %Robustness

  • Stability of your substanceIn the automatic injector

    In plasma during storage In room temperature (4 h)In Freeze/Thaw testsIn stock solutions

  • Analytical method validationAnalytical ProcedureSelectivityAccuracy Precision- within-run - between-run RecoveryLimit of Quantitation LOQCalibration curveRobustnessValidationStability

  • ReferencesGuidance for Industry Bioanalytical Method Validation FDA, May 2001Workshop Report: Shah, V.P. Et al., Pharmaceutical Research: 1992; 9:588-592.Workshop Report: Shah, V.P. et al., Pharmaceutical Research: 2000; 17:1551-1557

  • CostsValidation = 130-180.000 SEKStability = 15-20.000 SEK for each time pointQA = 11.000 SEK/study

  • The study phase (1) which the validated bioanalytical method is applied to the actual analysis of samples from the biostudy mainly in order to confirm the stability, accuracy and precision.

  • The study phase (2)Calibration curve in each runSix Quality Control samples in each runPre-stablished SOPs for procedures (method)Acceptance criteria for a run - accuracy and precision of the calibration curve - accuracy and precision of the QC samples - repeat analysisIt is preferable to analyse all study samples from a subject in a single run

  • The study phase (3)The QC samples should be used to accept or reject the run (2 samples at 3 levels)Four QC samples out of six should be within 15% of their nominal valueTwo QC samples can be outside 15% but not both at the same concentration

  • System suitability test

    Signal to noise ratio is above 5 for the substance.The peak shape is acceptable after visual inspection of the chromatogramThe retention times are within 10% of the previous run.The lowest calibration sample is injected before each run.

    The system is accepted if:

  • The analytical report should includeResults for all calibration curvesResults for all quality control samplesRepresentative number of chromatogramsShould include data from subjects who eventually dropped-outReanalysed samples and the reason for reanalyses The analytical validation reportThe responsible investigator(s) should sign for their respective section of the report

  • Chiral active substancesThe bio-analytical method should be enantiomericUnlessBoth products contain the same stable singel enantiomerBoth products contain the racemate and both enantiomers show linear pharmacokinetics

  • Also guidance forReference and test productData analysisIn vitro dissolution comlementary to a bioequivalence studyReporting of resultsApplication for products containing new active substancesApplication for products containing approved active substancesis given