Method Validation on Drug Substances

8/2/2019 Method Validation on Drug Substances

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Drug Substances

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Method Validation

ObjectiveThe objective of validation of an analyticalprocedure is to demonstrate that it is

suitable for its intended purpose.


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Methods to Validate

IdentificationAssay / DissolutionImpurities

Quantitative andLimit tests

Specific Tests


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ICH Parameters


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Parameters

SpecificityPrecisionLinearity and RangeRobustnessAccuracy

Limit of DetectionLimit of Quantitation

Assay method

Related

Substances


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Specificity

Specificity is the ability to assessunequivocally the analyte in the presence ofcomponents which may be expected to bepresent. Typically these might includeimpurities, degradants, matrix, etc.This is applicable for following Porcedures

IdentificationAssay andImpurities


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SpecificityFor Assay and Impurities

Impurities are availableFor Assay

Spike the pure substances with appropriate levelsof impurities and verify that the assay result is noteffected by the presence of impurities.

Compare the results of assay with spiked andunspiked samples.

For example inject matrix containing(spiked)

Voriconazole and Impurity B(at 0.15%) andidentify the RTs Inject test sample of Voriconazole and identify the

RT of Voriconazole and Impurity B(at 0.15%). Compare both spiked and test samples


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Specificity

For ImpuritiesSpiking the drug substance with appropriatelevels of impurities and demonstrate the

seperation of these impurities individually orother components from the sample matrix.

Specificity can be demonstrated by theresolution of the two componenetswhich elute closest to each other.


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Specificity

Impurities are not availableDemonstrate the specificity by comparingthe test results of sample containing

impurities to a second well characterizedprocedure Eg: Pharmacopoiel method orother validated independent procedure.This should include samples stored underrevelent stress conditions


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Specificity

Stress testingAcidic/SolutionBasic/Solution

OxidationThermalThermal / Humidity

Photo(UV Light)Photo(Fluorecent Light)


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Specificityfor the assay, the two results should becompared;for the impurity tests, the impurity profiles

should be compared.Peak purity tests may be useful to showthat the analyte chromatographic peak isnot attributable to more than one

component (e.g., photodiode array(PDA),mass spectroscopy).Peak purity > 99.9999%Purity angle < purity threshold


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Specificity

Acceptance crieteriaPeak purity test must be passed by theactive component and related substances

There is no interference at the elution zoneof the active component and relatedsubstancesThe resolution between the closest elutedpeaks must be > 2


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Specificity

In cases where a non-specific assay isused, other supporting analyticalprocedures should be used to demonstrateoverall specificity.For example, where a titration is adoptedto assay the drug substance for release, the

combination of the assay and a suitable testfor impurities can be used.


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PrecisionThe precision of an analytical procedureexpresses the closeness of agreement(degree of scatter) between a series ofmeasurements obtained from multiplesampling of the same homogeneoussample under the prescribed conditions.Precision may be considered at threelevels:

repeatability,intermediate precision andreproducibility.


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Precision

RepeatabilityA minimum 6 determinations at 100% of thetest concentration.

Intermediate Precision(ruggedness)The applicant should establish the effects ofrandom events on the precision of theanalytical procedure.Typical variations to be studied include days,analysts, equipment


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Precision

ReproducibiliyReproducibility is assessed by means of aninter-laboratory trial..

These data are not part of the marketingauthorization dossier .The standard deviation, relative standarddeviation (coefficient of variation) andconfidence interval should be reported foreach type of precision investigated.


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LinearityThe linearity of an analytical procedure is itsability (within a given range) to obtain testresults which are directly proportional to theconcentration (amount) of analyte in thesample.Linearity should be evaluated by visualinspection of a plot of signals as a function ofanalyte concentration or content. If there is a

linear relationship, test results should beevaluated by appropriate statistical methods,for example, by calculation of a regressionline by the method of least squares


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Linearity

The correlation coefficient, y-intercept, slopeof the regression line and residual sum ofsquares should be submitted. A plot of the

data should be included.For the establishment of linearity, a minimumof 5 concentrations is recommended.

For Assay method: 80-120%For Related Subsance: from the reporting level ofan impurity 1 to 120% of the specification


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Detection Limit

The detection limit of an individual analyticalprocedure is the lowest amount of analyte ina sample which can be detected but not

necessarily quantitated as an exact value.Several approaches for estabilishing LOD


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LOD

Visual InspectionSignal-to-Noise ratioStandard Deviation of the responseand the slope.

Based on the standard deviation of the blank

Based on the calibration curve


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LODBased on Signal-to-NoiseThis approach can only be applied to analyticalprocedures which exhibit baseline noise.Determination of the signal-to-noise ratio isperformed by comparing measured signals fromsamples with known low concentrations of analytewith those of blank samples and establishing theminimum concentration at which the analyte canbe reliably detected.

A signal-to-noise ratio between 3 or 2:1 isgenerally considered acceptable for estimating thedetection limit.


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LODBased on the Standard Deviation of theResponse and the SlopeDL =3.3 / S

= the standard deviation of the responseS = the slope of the calibration curveBased on the Standard Deviation of the Blank

Measurement of the magnitude of analyticalbackground response is performed by analyzing anappropriate number of blank samples and calculatingthe standard deviation of these responses.


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LODBased on the Calibration Curve

A specific calibration curve should be studied usingsamples containing an analyte in the range of DL.The residual standard deviation of a regression line

or the standard deviation of y-intercepts ofregression lines may be used as the standarddeviation.The detection limit and the method used fordetermining the detection limit should be presented.

If DL is determined based on visual based on signalto noise ratio, the presentation of the relevantchromatograms is considered acceptable for

justification.


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Quantitation Limit

The quantitation limit of an individual analyticalprocedure is the lowest amount of analyte in asample which can be quantitatively determined

with suitable precision and accuracy.The quantitation limit is generally determined bythe analysis of samples with knownconcentrations of analyte and by establishingthe minimum level at which the analyte can bequantified with acceptable accuracy andprecision.


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LOQ

Visual InspectionSignal-to-Noise ratioStandard Deviation of the responseand the slope.

Based on the standard deviation of the blank

Based on the calibration curve


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LOQBased on Signal-to-Noise ApproachThis approach can only be applied toanalytical procedures that exhibit baselinenoise.Determination of the signal-to-noise ratio isperformed by comparing measured signalsfrom samples with known lowconcentrations of analyte with those of

blank samples and by establishing theminimum concentration at which theanalyte can be reliably quantified. A typicalsignal-to-noise ratio is 10:1.


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LOQBased on the Standard Deviation of theResponse and the SlopeThe quantitation limit (QL) may be expressed as:

QL = 10 /S = the standard deviation of the responseS = the slope of the calibration curve

The slope S may be estimated from the

calibration curve of the analyte. The estimate ofmay be carried out in a variety of ways forexample:


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Accuracy

The accuracy of an analytical procedureexpresses the closeness of agreementbetween the value which is accepted

either as a conventional true value or anaccepted reference value and the valuefound.

This should be performed for both Assayand Related Substances


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Accuracy

Accuracy should be assessed on samplespiked with known amounts of impuritiesNine determinations over a minimum 3concentration levels (80, 100 and 120 %)Report the results of recovery as %Recovery


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Accuracy

Formula for recovery[(D-A) (C-A)/(B-A)] x 2 for 50%concentration[(D-A) (C-A)/(B-A)] x 2/3 for 150%concentration

A=Blank

B=Standard at 100%C=SampleD=Sample+standard(50% or 100% or 150%)


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Robustness

The robustness of an analyticalprocedure is a measure of its capacity toremain unaffected by small, but

deliberate variations in methodparameters and provides an indicationof its reliability during normal usage

Parameters of robustnessVariabilitySolution stability


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RobustnessVariabilityVariations in method parameters

LC PH of a Mobile Phase Mobile Phase Composition Different column Temperature Flow RateGC Different column Temperature Flow rate


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RobustnessParameter Variability Permitted

Flow rate 0.1 ml/min

Mobile Phase Composition 2 % major and Minor component

Temperature 2C

Column Different manufacturer

PH of Mobile Phase 1.0 Units


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Robustness(example)Parameter Low Actual Higher

Flow rate 0.4 ml/min 0.5 ml/min 0.6 ml/min

Temparature 23C 25C 27C

Column Exp Col-1 Main Column Exp Col-2

Mobile Phasecomposition

58:42 60:40 62:38

pH of mobilephase

5.0 6.0 7.0

Mobile Phase Composition50:30:20(A:B:C)Low: ----


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RobustnessParameter Variability Permitted

Flow rate 0.1 ml/min

Mobile Phase Composition 2 % major and Minor component

Temperature 2C

Column Different manufacturerPH of Mobile Phase 1.0 Units


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Robustness(example)Parameter Low Actual Higher

Flow rate 0.4 ml/min 0.5 ml/min 0.6 ml/min

Temparature 23C 25C 27C

Column Exp Col-1 Main Column Exp Col-2

Mobile Phasecomposition

58:42 60:40 62:38

pH of mobilephase

5.0 6.0 7.0

Mobile Phase Composition50:30:20(A:B:C)Low: ----


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Robustness

Solution stability:Sample, Standard solutions and MobilePhase solutions are prepared during

chromatographic analysis.Leftover solutions are stored after theanaysis. But we need to verify that thesolution does not loose its properties.

For the above reason, there is a need toperfrom the stability of analytical solutions atthe time of validation itself.


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RobustnessSolution stability .

Standard and sample solutions stored in acapped volumetric flask on a lab bench undernormal lighting conditions for 24 hours to 48hours based on the stability.

Perform the analysis by injecting same samplesolution at different time intervals. SampleSolution Stability should be monitor up to 24 hrswith minimum 4-5 time intervals or 48 hours withminimum 8-10 intervals. Perform more timeintervals after 12 Hrs. Based on Injection runtime and sequence time establish the samplesolution stability for about 24 Hrs or 48 hours.


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System Suitability Testing

System suitability testing is an integralpart of many analytical procedures.Parameters

Repeatability(RSD)Capacity factor ( k )Relative retention( )

Resolution(Rs)Tailing factor(T) at W0.05Theoretical plates ( N )


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Method Validation on Drug Substances