Method validation :

It is the process which is used to confirm that the analytical procedure used for a specific test is suitable for its intended use. Results obtained 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 Method Validation :

• Validation should be performed in accordance with the validation protocol. The protocol should include procedures and acceptance criteria for all characteristics. The results should be documented in the validation report.
• Justification should be provided when non-pharmacopoeial methods are used. If pharmacopoeial methods are available, justification should include data such as comparisons with the pharmacopoeial or other methods.
• Standard test methods should be described in detail and should provide sufficient information to allow properly trained analysts to perform the analysis in a reliable manner.
• As a minimum, the description should include the chromatographic conditions (in the case of chromatographic tests), reagents needed, reference standards, the formulae for the calculation of results and system suitability tests.

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The below are the characteristics that should be considered during Validation of Analytical Methods (but not limited to)

Specificity:

Ability to assess unequivocally the analyte in the presence of components which may be expected to be present (impurities, degradants, matrix). It is a measure of the degree of interference from such things as other active ingredients, excipients, impurities, and degradation products, ensuring that a peak response  is due to a single component only.

Precision:

The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility.

Repeatability (Method Precision):

Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision.

Intermediate precision (Ruggedness):

Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment, etc.

Accuracy:

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found.

Linearity:

The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.

Range:

The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.

Detection Limit (DL):

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value.

Quantitation Limit (QL):

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities and/or degradation products.

Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

PROCEDURE

The typical process that is followed in an analytical method validation is chronologically listed below,

• Planning and deciding on the method validation experiments
• Preparation and approval of method validation protocol
• Execution of the method validation activity
• Reporting the analytical method
• Finalizing the analytical

Analytical methods to be revalidated in following circumstances;

• Changes in the composition of a finished product (Drug product).
• Changes in the Analytical
• During the optimization of the drug product process, significant changes were introduced into the process. To ensure that the analytical method will still be able to analyze the potentially different profile of the drug product, revalidation may be
• The degree of revalidation required depends on the nature of the changes. Certain other changes may require validation as

Types of analytical procedure to be validated,

• Category I : Limit test of the active moiety in samples of drug substance or drug product or other selected component (s) in the
• Category II: Quantitative tests for impurities content and Limit tests for the control of impurities;
• Category III: Determination of performance characteristic (e.g. Dissolution, drug release)
• Category IV: Identification Test

SPECIFICITY/SELECTIVITY

For identification test

Analyze the finished product sample along with reference standard or certified working standard or reference material and analyze the finished product sample which do not containing the analyte (Placebo), compare the results.

For assay and impurity tests

• For chromatographic procedure the specificity shall be demonstrated by resolution of the two closest eluting compounds and their peak purity and blank
• For non-chromatographic procedure (e.g. titration) combination of assay and a suitable test for impurities can be
• Spike the finished product or drug substance with impurities and/or excipients as per specification level and analyze it along with unspiked finished product or drug
• Check that the all the spiked components are resolved from each other according to the acceptance

Acceptance criteria:

• System suitability should
• No interfering peaks shall be eluted at the retention time of
• The resolution between analyte peak and any closely eluting peak should be more than 0.
• The peak purity due to analyte should not be less then 990 or 0.99 whichever is applicable.

PRECISION

Precision is measured as the percent relative standard deviation (% RSD) for significant number of samples.

System precision:

Carry out minimum 5 determinations of standard /reference solution at 100% of test concentration (concentration of compound of interest given in analytical method).

Acceptance criteria: The relative standard deviation of five replicate injections of standard/reference solution should not be more than 2.0%.

Repeatability (Method precision):

For Assay/Related substances: Prepare 6 different sample preparations as per concentration of  compound of interest given in analytical method from a sample of one batch and determine the results from these six-sample preparation.

Acceptance criteria:

For Dissolution: Prepare 2 sets of 6 units as per concentration of compound of interest given in analytical method from a sample of one batch and determine the results from 12 units preparation

Acceptance criteria: Over all % RSD of % release of two sets should not be more than 6.0 %. Intermediate precision:

For Assay / Related substances: Analyze the sample of single batch six times by different analysts on different days using different instrument and where applicable use different column or electrode.

Acceptance criteria: The % RSD of results of two different sets (method precision and intermediate precision) should be as per shown in below table.

For Dissolution: Analyze the sample of single batch (6 units) two times by different analysts on different days using different instrument.

Acceptance criteria: % RSD of results of two different sets (method precision and intermediate precision) should not be more than 6.0 %.

ACCURACY :

For the assay of the finished product:

Analyze the synthetic mixtures of finished product components (placebo) spiked with known quantities of drug substance to be analyzed.

Prepare the sample in the range of 80,100,120% of label claim and analyze it in   triplicate      at     each concentration.

If it is not possible to prepare placebo due to non-availability of other components then add known quantities of analyte to the finished product. (4.1.2 b of ICH Q2 (R1))

Accuracy may be inferred once precision, linearity and specificity have been established.

For impurities test (quantitation):

Analyze the finished product sample spiked with known amounts of impurities at the specification    level.

Prepare the sample in the range of 80%, 100%, and 120% of specification level and analyze it in triplicate at each concentration.

For dissolution:  Add known amount of standard to that of placebo (above and below the nominal   level) at 3 different levels i.e. 70%, 100% and 130% to cover both above and below the nominal levels. Calculate the data as % of label claim, mean and % RSD at each concentration.

Report the data  as the percent recovery by the assay of the known added amount of analyte in the  sample or as the difference between the mean and the accepted true value together with confidence intervals.

Acceptance criteria: For Assay / Related substances

• System suitability should
• Recovery should not less than 90.0 %.
• For % RSD of recovery for all levels,

Acceptance criteria: Dissolution

• Recovery at each level should in between 95.0% to 0%.
• % RSD for the recovery of all the levels should not be more than 5.0%.

LINEARITY AND RANGE:

Linearity should be evaluated by visual inspection of a plot of signals as a function of analyte concentration or content.

The below table details the methodology to perform linearity and range parameter.

Demonstrate the linearity by the use of correlation coefficient, y- intercept, and slope of the regression line.

Acceptance criteria:

The correlation coefficient should not be less than 0.99 for Assay method and for  impurity  quantification method the correlation coefficient should not be less than 0.98.

LIMIT OF DETECTION (applicable only for impurity methods):

It is a limit test that specifies whether or not an analyte is above or below certain value  which can  not  be quantified as an exact value.

Based on Visual Evaluation

Measurement by visual evaluation for non-instrumental methods (e.g. TLC/titration);  Determined the detection limit by the analysis of samples with known concentrations of analyte.

Prepare a sample at lowest concentration of analyte and establish the minimum level at which the analyte can be consistently detected.

Based on Signal-to-Noise:

Measurement by signal to noise ratio for instruments which exhibit baseline noise;

Determine the signal-to-noise ratio by comparing measured signals from samples with known lowest concentrations of analyte with those of blank samples and establish the minimum concentration at which the analyte can be consistently detected. And measure the signal-to-noise ratio.

Acceptance Criteria: A signal-to-noise ratio between 2:1 or 3:1 is required.

Based on the Standard Deviation of the Response and the Slope:

Determine the slope of calibration curve by analyzing the samples at different concentration  in  the  range of detection limit.

Detection Limit (DL) = 3.3 x Standard deviation (SD) of response/Slope of calibration curve

LIMIT OF QUANTITATION (applicable only for impurity methods): Based on Visual Evaluation:

Measurement by visual evaluation for non-instrumental methods (e.g. TLC/titration);

Determined the quantitation limit by the analysis of samples with known concentrations of analyte.

Prepare a sample at lowest concentration of analyte and establish minimum level at which analyte can be quantified with acceptable accuracy and precision.

Based on Signal-to-Noise:

Measurement by signal to noise ratio for instruments which exhibit baseline noise;

Analyze minimum 6-sample solution at decreasing concentration in the expected range of quantitation limit.

Determine the signal-to-noise ratio by comparing measured signals from samples with known lowest concentrations of analyte with those of blank samples and  establish the minimum concentration at which the analyte can be consistently quantified. And measure the signal-to-noise ratio.

Acceptance criteria: A signal to noise ratio 10:1 is required.

Based on the Standard Deviation of the Response and the Slope:

Determine the slope of calibration curve by analyzing the samples at different     concentration in the range of quantitation limit.

Quantitation Limit (QL) = 10 x Standard deviation (SD) of response/Slope of calibration curve

Precision at Quantitation Limit (QL):

Prepare a sample solution at the QL concentration determined by adapting any of above method and analyze it for six times and measured the precision (%RSD) at this concentration.