Validation of Analytical Method

Dr. Wael Abu Dayyih

• Introduction

• Method validation is the process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Results from method validation can be used to judge the quality, reliability and consistency of analytical results; it is an integral part of any good analytical practice.

•  Analytical methods need to be validated or revalidated:

-Before their introduction into routine use;

-Whenever the conditions change for which the method has been validated (e.g., an instrument with different characteristics or samples with a different matrix); and

-Whenever the method is changed and the change is outside the original scope of the method.

2

Method Characteristics to Be Considered for Validation

• Specificity (Selectivity)• Linearity• Range• Accuracy• Precision– Repeatability– Intermediate Precision– Reproducibility

(Ruggedness)

• Detection Limit

• Quantitation Limit

• Robustness

• System Suitability Testing

• Specificity and stability

3

Validation CharacteristicsValidation Characteristics

IdentificationIdentification ImpuritiesImpurities AssayAssay

QuantitativeQuantitative limitlimit

Accuracy Accuracy -- ++ -- ++

PrecisionPrecision -- ++ -- ++

SpecificitySpecificity ++ ++ ++ ++

Detection LimitDetection Limit -- -- ++ --

Quantitation LimitQuantitation Limit -- ++ -- --

LinearityLinearity -- ++ -- ++

RangeRange -- ++ -- ++

RobustnessRobustness ++ ++ ++ ++

SpecificitySpecificity Is the ability to assess unequivocally the analyte in the presence of components Is the ability to assess unequivocally the analyte in the presence of components

which may be expected to be present (impurities, degradants, matrix…).which may be expected to be present (impurities, degradants, matrix…).

Identity testingIdentity testingTo ensure the identity of an analyte.To ensure the identity of an analyte.

Purity testingPurity testingTo ensure accurate statement on the content of impurities of an analyte.To ensure accurate statement on the content of impurities of an analyte.

AssayAssayTo allow an accurate statement on the content of an analyte in a sampleTo allow an accurate statement on the content of an analyte in a sample

Specificity:Specificity: Overlay chromatogram of an impurity solution with Overlay chromatogram of an impurity solution with a sample solutiona sample solution

LinearityLinearity

It is the ability (It is the ability (within a givenwithin a given rangerange) to obtain test ) to obtain test resultsresults which are directly which are directly proportional to the concentrationproportional to the concentration (amount) of analyte in the sample. (amount) of analyte in the sample.

If there is a If there is a linear linear relationship relationship test results should be evaluatedtest results should be evaluated by appropriateby appropriate statistical methods:statistical methods:

Correlation coefficient (r)Correlation coefficient (r)Y-interceptY-interceptSlopeSlope

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.01 0.015 0.02 0.025 0.03 0.035 0.04

Calc

ula

ted a

nal

yte in

mg/m

L

Reference material mg/ml

Linearity of an analyte in a material

Table of values (x,y)

x y # Reference

material mg/mlCalculated

mg/ml

1 0.0100 0.0101

2 0.0150 0.0145

3 0.0200 0.0210

4 0.0250 0.0260

5 0.0300 0.0294

6 0.0400 0.0410

Linearity Statistics • Intercept -0.0002

• Limit of Linearity and Range

0.010 – 0.040 mg/mL

• Slope 1.0237• Correlation coefficient 0.9978

AAccuracy and precisionccuracy and precision

Accurate & Accurate & preciseprecise Inaccurate & Inaccurate &

impreciseimpreciseInaccurateInaccurate & &

preciseprecise Accurate & impreciseAccurate & imprecise

AccuracyAccuracy: : Application of the method to synthetic mixtures of the Application of the method to synthetic mixtures of the drug product components to which known quantities of the analyte drug product components to which known quantities of the analyte have been added.have been added.

RecoveryRecovery reduced reducedby by ~10 – 15%~10 – 15%

PrecisionPrecisionExpresses the Expresses the closeness of agreementcloseness of agreement between a series of between a series of

measurements obtained from multiple sampling of the same measurements obtained from multiple sampling of the same homogenous samplehomogenous sample

Is usually expressed as the Is usually expressed as the standard deviation (S)standard deviation (S) or or coefficient of variationcoefficient of variation (RSD)(RSD) of a series of measurements. of a series of measurements.

Precision may be considered at three levels:Precision may be considered at three levels:RepeatabilityRepeatability (intra-assay precision)(intra-assay precision)Intermediate PrecisionIntermediate Precision (variability within a laboratory)(variability within a laboratory)ReproducibilityReproducibility (precision between laboratories)(precision between laboratories)

RepeatabilityRepeatabilitySix replicate sample preparation steps from a homogenously prepared tablet Six replicate sample preparation steps from a homogenously prepared tablet

mixture (nominal value of API 150 mg)mixture (nominal value of API 150 mg)

InjectionInjection Peak areaPeak area AssayAssay

11 173865173865 98.06%98.06%

22 174926174926 98.66%98.66%

33 172933172933 97.54%97.54%

44 175011175011 98.72%98.72%

55 179557179557 101.30%101.30%

66 176425176425 99.52%99.52%

MeanMean 175453175453 98.96%98.96%

RSDRSD 1.32%1.32% 1.32%1.32%

Intermediate precisionIntermediate precisionExpresses within-laboratories variations (different days, Expresses within-laboratories variations (different days,

different analysts, different equipment etc.)different analysts, different equipment etc.)

InjectionInjection Peak area analyst 1Peak area analyst 1 Peak area analyst 2Peak area analyst 2 Peak area analyst 3Peak area analyst 3

11 173865173865 175656175656 177965177965

22 174926174926 175878175878 178556178556

33 172933172933 176004176004 177342177342

44 175011175011 176344176344 178011178011

55 179557179557 175332175332 179466179466

66 176425176425 174959174959 179688179688

MeanMean 175453175453 175695175695 178504178504

RSDRSD 1.32%1.32% 0.28%0.28% 0.51%0.51%

ReproducibilityReproducibility

Expresses the precision between laboratories:Expresses the precision between laboratories:Collaborative studies, usually applied to standardisation of Collaborative studies, usually applied to standardisation of

methodologymethodology

Transfer of technologyTransfer of technology

Compendial methodsCompendial methodsInjectionInjection Peak area analyst lab-1Peak area analyst lab-1 Peak area analyst lab-2Peak area analyst lab-2

11 175656175656 177965177965

22 175878175878 178556178556

33 176004176004 177342177342

44 176344176344 178011178011

55 175332175332 179466179466

66 174959174959 179688179688

MeanMean 1756917569 178504178504

RSDRSD 0.28%0.28% 0.51%0.51%

RangeRangeThe The rangerange of an analytical procedure is the interval between the upper and of an analytical procedure is the interval between the upper and

lower concentrations of analyte in the sample for which it has been lower concentrations of analyte in the sample for which it has been demonstrated that the analytical procedure has a demonstrated that the analytical procedure has a suitable level of precision, suitable level of precision, accuracy and linearityaccuracy and linearity

AssayAssay80 to 120% 80 to 120% of test concentration.of test concentration.

Content uniformityContent uniformity70 to 130% 70 to 130% of test concentration.of test concentration.

DissolutionDissolutionQ-20% to 120%.Q-20% to 120%.

ImpuritiesImpuritiesReporting level – 120% of specification limit (with respect to test Reporting level – 120% of specification limit (with respect to test

concentration of API).concentration of API).Assay & ImpuritiesAssay & Impurities

Reporting level to 120% of assay specificationReporting level to 120% of assay specification

Limit of Detection (LOD, DL):Limit of Detection (LOD, DL):The LOD of an analytical procedure is the lowest amount The LOD of an analytical procedure is the lowest amount

of analyte in sample which can be of analyte in sample which can be detected but not detected but not necessarily quantitated as an exact value.necessarily quantitated as an exact value.

Determination is usually based onDetermination is usually based on Signal to noise ratio (~3:1)Signal to noise ratio (~3:1) ((baseline noisebaseline noise).).

noise

Peak ALOD

Peak BLOQ

Baseline

Limit of Quantitation (LOQ, QL)Limit of Quantitation (LOQ, QL)The LOQ is the lowest amount of analyte in a sample which can The LOQ is the lowest amount of analyte in a sample which can

be quantitatively be quantitatively determined with suitable precision and accuracydetermined with suitable precision and accuracyThe quantitation limit is used particularly for the The quantitation limit is used particularly for the

determination of impurities and/or degradation productsdetermination of impurities and/or degradation productsDetermination is usually based onDetermination is usually based on

Signal to noise ratio (~10:1) Signal to noise ratio (~10:1)

((baseline noisebaseline noise))

noise

Peak ALOD

Peak BLOQ

Baseline

Specificity and Specificity and stability:stability:Stress stability testing to ensure the Stress stability testing to ensure the stability indicating potentialstability indicating potential of an of an

analytical methodanalytical method

Assure that the API can be assessed specifically in the presence of known Assure that the API can be assessed specifically in the presence of known and unknown (generated by stress) impurities.and unknown (generated by stress) impurities.

Assure that known impurities/degradants can be specifically assessed in Assure that known impurities/degradants can be specifically assessed in the presence of further degradants.the presence of further degradants.By By peak purity assessmentpeak purity assessment and (overlay of) and (overlay of) chromatogramschromatograms

Stress Stress stability studies versus stability studies versus forced forced degradationdegradation studies studies

Stress Stress parameterparameter

Forced degradationForced degradation Stress stabilityStress stability(5 – 15% decomposition)(5 – 15% decomposition)

AcidAcid 0.2 ml 0.2 ml 1N HCl1N HCl / 5 ml API-solution / / 5 ml API-solution / 3h, 6h3h, 6h, 12h, 24h…7d (RT & 60°C), 12h, 24h…7d (RT & 60°C)

pHpH ± 2 (2 weeks)± 2 (2 weeks)

BaseBase 0.2 ml 0.2 ml 1N NaOH1N NaOH / 5 ml API-solution / / 5 ml API-solution / 3h, 6h3h, 6h, 12h, 24h…7d (RT & 60°C), 12h, 24h…7d (RT & 60°C)

pHpH ± 10 (2 weeks)± 10 (2 weeks)

HH22OO22 / Oxygen / Oxygen 0.2 ml 0.2 ml 5%5% or or 35%35% HH22OO22 / 5 ml API-/ 5 ml API-

solution (RT, to 7d & 60°C, solution (RT, to 7d & 60°C, 3h3h))1 g/ml 1 g/ml oxygenoxygen bubbled through (8 hours) bubbled through (8 hours)

0.1 – 2% 0.1 – 2% HH22OO22 (24 hours) (24 hours)

HeatHeat 60°C60°C / 5 ml solution ( / 5 ml solution (3h,3h, 6h6h…7d)…7d) --

HeatHeat 105° C105° C / solid API ( / solid API (1d1d and 7d) and 7d) 60°C (4 weeks)60°C (4 weeks)

UV or LightUV or Light 365 nm or white fluorescent light / 365 nm or white fluorescent light / solid API (solid API (1d1d and 7d) and 7d)

--

HumidityHumidity -- 50°C / 80% RH (4 weeks)50°C / 80% RH (4 weeks)

RobustnessRobustness

Robustness of an analytical procedure should show the Robustness of an analytical procedure should show the reliabilityreliability of of an analysisan analysis with respect to with respect to deliberate variations in method deliberate variations in method parameters.parameters.

The evaluation of robustness should be considered during the The evaluation of robustness should be considered during the development phase.development phase.

If measurements are If measurements are susceptiblesusceptible to variations in analytical conditions to variations in analytical conditions the the analytical conditions should be suitably controlledanalytical conditions should be suitably controlled or a or a precautionary statementprecautionary statement should be included in the procedure. should be included in the procedure.

Influence of buffer pH and buffer concentration in mobile Influence of buffer pH and buffer concentration in mobile phase on retention times of API and impurities:phase on retention times of API and impurities:

System suitability testingSystem suitability testing

Based on the concept that equipment, electronics, analytical Based on the concept that equipment, electronics, analytical operations and samples to be analysed operations and samples to be analysed constitute an integral constitute an integral systemsystem that can be evaluated as such that can be evaluated as such

Suitability parameters are established for each analytical Suitability parameters are established for each analytical procedure procedure individuallyindividuallyDepend on the Depend on the type of analytical procedure type of analytical procedure

SummarySummaryAnalytical procedures play a critical role in pharmaceutical Analytical procedures play a critical role in pharmaceutical

equivalence and risk assessment/managementequivalence and risk assessment/management

Establishment of Establishment of product-specificproduct-specific acceptance criteriaacceptance criteria

Assessment of Assessment of stabilitystability of APIs of APIs

Validation of analytical procedures should demonstrate that they Validation of analytical procedures should demonstrate that they are are suitable for their intended usesuitable for their intended use

Validation of analytical procedures deserves Validation of analytical procedures deserves special attention special attention during assessmentduring assessment of dossiersof dossiers for prequalification for prequalification

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