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Standard Operating Procedure and Guideline for Analytical Method Validation (AMV) for Oral solid dosage form as per ICH – Q2 (R1) and USP general chapter 1225.

Analytical Method Validation (AMV) – Validation is the proof needed to ensure that an analytical method can
produce results that are reliable and reproducible and which are fit for the purpose intended. 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.

SOP – Analytical Method Validation (AMV)

1.0    OBJECTIVE :

• • This SOP defines the characteristics for consideration during the validation of an analytical procedure in line with the ICH Q2 (R1) guideline for method validation. It describes the typical characteristics to be selected for validation of an analytical procedure and also demonstrates that it is suitable for its intended purpose.

2.0    SCOPE :

• • This procedure is applicable to all analytical method validations for drug substances and drug products carried out in the Quality control department / Analytical Development department.

3.0    RESPONSIBILITY :

• • Officer, Executive QC — For analytical method validation activity.

• • Head of Department QC— Compliance of SOP

• • Head of Department QA— Compliance of SOP

4.0    DEFINITION (S) :

• • Certain terms used in this SOP are defined as follows:

• • Validation: Validation of an analytical method is the process that establishes, by laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical applications.

• • Partial validation: The nature of the validation experiments selected should be based on the likely nature of the change to the response in the existing validated method.

For example Change in the specification of impurity limit (for example, 0.2% to 0.1% or vice versa) then linearity, precision, and accuracy shall be performed to cover the updated specification concentration.

• • Specificity: Specificity is the ability of the analytical method to assess unequivocally the analyte in the presence of components that may be expected to be present in the sample.

• • Limit of detection: 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.

• • Limit of quantitation: 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.

• • Linearity: The linearity of an analytical procedure is its ability (within a given range) to obtain test results that are directly proportional to the concentration 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.

• • Precision:

  • The precision of an analytical procedure expresses the closeness of agreement (degree of scattering) between a series of measurements obtained from multiple samplings of the same homogeneous sample under the prescribed conditions.

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

• • Intermediate Precision: Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment /instruments, etc.

• • Reproducibility: Reproducibility expresses the precision between laboratories (collaborative studies usually applied to standardization of methodology)

• • 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.

• • 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.

5.0    PROCEDURE – ANALYTICAL METHOD VALIDATION (AMV) :

• • The objective of the analytical procedure should be clearly understood since this will govern validation characteristics that need to be evaluated. Typical Analytical Method validation (AMV) characteristics that should be considered are listed below:

Accuracy

Precision

Repeatability

Intermediate Precision

Specificity

Detection Limit

Quantitation Limit Linearity

Range

• • The table lists those validation characteristics regarded as the most important for the validation of different types of analytical procedures.

• • This list should be considered typical for the analytical procedures cited but occasional exceptions should be dealt with on a case-by-case basis.

• • It should be noted that robustness, filtration study, and forced degradation are not listed in the table but should be considered at an appropriate stage in the development/validation of the analytical procedure.

• • Re validation / partial validation of analytical methodology:

  • Re validation / Partial validation of analytical methodology shall be performed in the following circumstances:
    • Changes in method of analysis (partial validation).

For Example: If additional system suitability is added in methodology like resolution, tailing factor, Theoretical plates, etc, then perform the robustness parameters.

• • • Changes in the formulation of drug products (Based on formulation changes, parameter to be )

For example, Additional excipients are added/processed modified or removed then perform the partial Analytical Method validation (AMV) as specificity, precision, intermediate precision, and accuracy.

• • • Changes in specification limits above the level, which was covered under validation. (Linearity and accuracy)

• • • All queries related to drug substances and drug products shall be performed.

• • • Partial Analytical Method validation (AMV) shall be performed for residual solvents of drug substances and drug products if the commonly validated method is available for the determination of residual solvents for other drug substances and drug

Changes in drug substance source. Following parameters to be performed based on the route of synthesis and impurity profile comparison between sources.

Test Parameter	If the route of synthesis / Impurity profile same of drug substances	If the route of synthesis change / Impurity profile same	When the route of synthesis change / Impurity profile change
			Process Impurity	Degradation Impurity
# Assay
1)     Specificity	×	×	×	√
2)     Forced Degradation	×	×	×	×
3)     Solution Stability	×	×	×	×
4)     Linearity and Range	×	×	×	×
5)     Precision	×	×	×	√
6)     Accuracy	×	×	×	×
7)     Robustness	×	×	×	×
8)     System Suitability	×	×	×	×

 

# Related Substances (HPLC Method)
1)     Specificity	×	√	√	√
2)     Forced Degradation	×	×	×	√
3)     LOD/LOQ	×	×	×	√
4)     Solution Stability	×	×	×	√
5)     Linearity and Range	×	×	×	√
6)     Precision	×	√	×	√
7)     Accuracy	×	×	×	√
8)     Robustness	×	×	×	√
9)     RRF	×	×	×	√
10)   System Suitability	×	×	×	√

 

# Related Substances (TLC Method)
1)    Specificity	×	×	√	√
2)    Linearity and Range	×	×	×	√
3)    Precision	×	×	×	√

 

# Dissolution
1)     Specificity	×	×	×	√
2)     Solution Stability	×	×	×	×
3)     Linearity and Range	×	×	×	×
4)     Precision	×	×	×	√
5)     Accuracy	×	×	×	×
6)     Robustness	×	×	×	×
7)     System Suitability	×	×	×	×

Note: –  √ – Need to be validated., X — Not required.

• General Notes – Analytical Method validation (AMV):

• • Pharmacopoeial methods are recognized as validated analytical methods and therefore validation for the same is not required. However, verification of the same is required to demonstrate that it is suitable for its intended purpose for drug substances and drug products.

• • For Analytical Method validation (AMV) of excipients and KSM/intermediate, only minimum parameter shall be evaluated which will be defined in the Analytical Method validation (AMV) protocol for in house method, and no need to perform the analytical method validation (AMV) for pharmacopoeial

• • Any analytical method validation (AMV) for drug product drug substance, excipients, and residual substances like leachable, extractable, etc. shall be carried out by referring to the above Analytical Method validation (AMV) parameter and as defined in the protocol for execution.

• • Numbering and preparation of protocol and report shall be done as per sop. Before initiation of protocol training for the Analytical Method validation (AMV) team shall be conducted for methods to be validated and development data of the same methods also to be

• • If any other tests are performed other than the above-mentioned validations then those particular tests will be executed as per the Analytical Method validation (AMV) protocol.

• • If any incidents (deviation) from the analytical methodology or related to the approved protocol shall be reported in the final report.

• • Qualitative tests such as description, average weight, pH, disintegration, hardness solubility, water content, LOD, color test, etc, but not limited to, need not be validated by the analytical method. If required based on criticality shall be performed.

• • Analytical Method Validation (AMV) recommends performing on single injection for the sample, if the recommendation is given by STP in the method, shall be performed as per the recommendation of the respective method.

For example

• • • Duplicate preparation of the sample and a single injection of each.

• • • Single preparation of sample with duplicate

• • Selection of Suitable Strength for analytical method validation (AMV)

• • If the method is applicable for single strength, method validation is to be performed on the specified strength.

• • In case of multiple strengths, based on the manufacturing formula, suitable strength shall be selected as below:

• • • If the manufacturing formula is a dose-proportional dosage form, then method validation activity shall be performed using the lowest and the highest strength of the dosage form.

• • • Ensure that the precision, accuracy, and linearity should cover the bracketing of the lowest and highest test concentration.

• • Specification level: Analytical method validation shall be performed by referring to the regulatory / shelf-life specification limit for all test procedures.

• • Spiking of impurity in sample preparation:

• • Recommend to spike known degradant impurities/ impurities (as per specification) in the sample preparation for the specificity of assay method validation.

• • In related substance method validation, recommend spiking all process impurities and degrading impurities for specificity study.

• • Based on impurity level and specification, the sample solution shall be prepared as per the below recommendations:

• • • If the known impurity is not listed in the specification, then prepare the sample solution and use it as is.

• • • If the known impurity is above the LOQ level, then prepare the sample solution and use it as is.

• • • If the known impurity is below or equal to the LOQ level, then spike the impurity at the specification limit in the sample solution. (Wherever applicable).

• • Preparation of a placebo solution is applicable for only drug product method validation.

• • Filtration study applicable for drug product only, if required for drug substance shall be performed based on protocol.

• • Forced degradation study (For HPLC and GC methods):

Note: For the preservative content (antioxidant) test, a forced degradation study is not applicable, only an Interference study shall be performed. Forced degradation studies are performed to demonstrate that the analytical method is stability-indicating. Refer to the forced degradation guideline for further details. Carry out the forced degradation studies using the following degradation conditions, but not limited to-

• • Solid degradation :

• • • Thermal Degradation

• • • Photolytic Degradation

• • • Humidity Degradation

• • Liquid Degradation :

• • • Acid Degradation

• • • Base Degradation

• • • Oxidation Degradation

Analytical Method Validation of Assay (Active) and Preservative (antioxidant) content

• • Specificity – Analytical Method Validation (AMV):

  • Interference study: Determination:

• • Prepare and analyze the blank (diluents), placebo, system suitability, standard, sample, and individual specified impurity/impurities at specification limit, to check the interference.

• • Spike the impurities at the specification limit with respect to sample concentration for HPLC and GC.

• • Determine the % assay or preservative (antioxidant) content for the sample and spiked sample. (Wherever applicable).

• • If the method is isocratic, then the specificity studies shall be performed by extending the run time for that particular method to be validated.

• • For UV- Spectrophotometry methods, scan the blank, placebo, standard, and sample to 200 to 400nm for UV range, in case of UV-visible range scan 200 to 700nm, and also take the absorbance at working wavelength.

• • For titrimetry methods, analyze the blank, placebo, and sample as per methodology.

• • If in case, different colors are used in the drug product then inject either an individual or mixed placebo and sample, measure the absorbance/peak response.

Note: Peak purity for the saturated peak shall be evaluated on the same sample by diluting the solution.

• Acceptance criteria for Specificity:

• • For HPLC and GC method

• • No peak should be observed due to blank, placebo, and specified impurity/impurities at the same retention time of the analyte peak/s as observed in the standard and sample.

• • The peak/s of interest should be well resolved from each other and the closest interfering peak/s (impurity, excipients, degradation, internal standard, etc.).

• • Generally, the resolution value is more than 1.5. If not, then make the appropriate recommendation with scientific justification.

• • The main analyte peak should be spectrally pure for the Purity index value shall be not less than 950 (Not applicable to GC methods as well as preservative content).

• • For UV – Spectrophotometry method

• • No Interference due to blank and placebo solution is allowed. (At the specified wavelength as per methodology). If any interference is observed due to the placebo make an appropriate recommendation with scientific justification.

• • For Titrimetry method

• • No interference should be observed due to blank and placebo. If interference is observed make appropriate recommendations such as subtraction of blank or placebo from the sample value.

• Solution stability – Analytical Method Validation (AMV):

• • Analytical Solutions stability: Determination:

• • Prepare blank, placebo, system suitability, standard, and the such sample as described in the methodology.

• • Keep the prepared solutions tightly closed and store them at either controlled room temperature (RT) and/or at a temperature below the controlled RT (as per recommended by the test procedure).

• • Analyze the system suitability, standard, and sample at pre-defined periodic intervals (interval shall not exceed more than 12hrs).

for example: Initial 00hrs, 6hrs, 12hrs, 18hrs and so on……., up to 48hrs, but not limited to.

• • Report the results of initial and actual time interval conditions.

• • Determine the % RSD of standard and absolute difference in assay/preservative content (antioxidant result at a particular time point with respect to the initial for standard and sample.

• Acceptance criteria for Analytical Solution Stability : 

• • System suitability shall meet the acceptance criteria at each time.

• • % RSD of the peak area/absorbance in standard obtained from initial and at each time interval should not be more than 2.0% HPLC/UV- spectrophotometer/GC for in DP and 1.0% for DS for HPLC/UV- spectrophotometer and 2.0% for GC methods or as per recommended in system suitability criteria for the same sequence.

• • In case HPLC/GC/UV-Spectrophotometer system changed, the absolute difference in assay results with respect to initial at each time interval should be not more than 2.0% for HPLC/UV-spectrophotometer / GC foF DP and 1.0% for DS for HPLC/UV- spectrophotometer and 2.0% for GC methods and 5.0% for preservative (antioxidant) content.

• • The absolute difference in assay result with respect to initial and at each time. interval ( for the sample should not be more than 2.0% for DP and 5 0% for preservative (antioxidant) content.

• • The absolute difference in assay result with respect to initial and at each time interval for the sample should not be more than 1.0% for DS for HPLC/UV- spectrophotometer and 2.0% for GC methods and 5.0 % for preservative (antioxidant) content.

Note: If absolute difference fails at any interval, then the previous interval is recommended as stability of the analytical solution. A minimum of two successive failure time interval results shall be considered to define the solution stability.

• Mobile Phase solution Stability:

Determination:

• • Prepare the mobile phase as per methodology and evaluate the system suitability criteria as per methodology at pre-defined time intervals. Mobile phase stability shall be evaluated for minimum of 72 hours or may extend.

• • System suitability criteria shall meet the acceptance criteria and also record the physical observation of the mobile phase at each time interval.

• • Acceptance Criteria

• • System suitability shall meet the acceptance criteria mentioned in the methodology.

• • The mobile phase shall be free from any particulate No turbidity or no opalescence is seen.

• Filtration study – Analytical Method Validation (AMV) :

Determination:

• • Prepare a sample as described in the methodology to be validated.

• • The sample shall be filtered as sequentially as per methodology and as –

• • • -1.0mL of specified saturated volume in methodology

• • • Specified volume in methodology

• • • + 1.0mL of specified.

• • • + 2.0mL of specified.

For example, The discard volume is mentioned as 5.0mL, then evaluate the discarded volume as 4.0mL, 5.0mL. 6.0mL and 7.0mL.

• • Analyze the obtained solutions as described in the method to be validated and calculate the assay/ preservative (antioxidant) content results.

Acceptance criteria:

• • The absolute difference in the assay result obtained from specified discard volume filtered solution and respective discard volume filtered solutions should not be more than 2% for active and 5% for preservative (antioxidant)

OR

• • Prepare sample solutions as described in the methodology to be validated.

• • Select the filter listed in the test method. Set up seven clean and dry test tubes marked with 1, 2, 3, 4, 5, 6, and 7 for the collection of filter solution.

• • Take about 10mL sample solution and filter 1mL in each test tube from 1 to 7 numbered marked test tubes. Analyze each filtrate solution.

Acceptance criteria:

• • The absolute difference in the assay result obtained from 7 test tube filtrate solution and other filtrate test tube 1, 2, 3, 4, 5, and 6 results should not differ more than 2% for active and 5% for preservative (antioxidant) content.

• Linearity Test – Analytical Method Validation (AMV):

Determination:

• • Prepare and analyze the series of linearity solutions from the stock solution of standard, to obtain solutions from 80% to 120% of the test concentration by preparing minimum 5 concentrations.

• • If the method is the same for the content uniformity and blend analysis test then perform the linearity in the specified range from 70 to 130% of test concentration (desired concentration 1X-3X to cover the content uniformity and blend analysis).

• • If possible, recommended to perform extended concentration from 50 to 150% of test concentration (wherever possible).

• • Plot a graph of corrected (actual) concentration (ppm) vs. peak area/ absorbance and report.

• • The correlation coefficient, deviation of Y-intercept, slope of the regression line, and residual sum of squares should be reported.

• • For Titrimetry methods

• • For the establishment of linearity, a minimum of 5 concentrations are to be prepared between 80 and 120% of the working concentration.

Acceptance criteria for Linearity Test :

• • The correlation coefficient should not be less than 0.999 for HPLC/UV- Spectrophotometry, GC, and Titrimetry methods.

• • % deviation from Y-intercept value shall be within + 2%

• Range – Analytical Method Validation (AMV) :

Determination:

• • The specified range is normally derived from linearity studies and it is established by confirming that the analytical procedure provides an acceptable degree of linearity, accuracy, and precision of extremes of the specified range.

Acceptance Criteria:

• • For DS: % RSD should not be more than 1.0% for HPLC/UV- spectrophotometer and 2.0% for GC methods for six replicate injection areas.

• • For DP: % RSD should not be more than 2.0% for HPLC/GC/UV- spectrophotometer methods for six replicate injection areas.

• • For preservative content: % RSD should not be more than 2.0% for HPLC/GC/UV- spectrophotometer methods for six replicate injection areas.

• Precision Test – Analytical Method Validation (AMV)

• • Repeatability :

Determination:

• • Analyze the sample (minimum three independent sample preparation at each concentration) for assay/preservative (antioxidant) content at three different concentrations covering the specified range from 80 to 120% of test concentration.

OR

• • Alternatively, analyze the sample for assay/preservative (antioxidant) content at 100% of the test concentration with six independent sample preparations.

• • Recommended number of sample preparations: 3 at 80%, 6 at 100%, and 3 at 120% test concentrations.

• • In case of multiple strengths in the drug product, bracketing of lowest and highest strengths should be covered the range from 80% – 120% of test concentration.

• • If the content uniformity and blend uniformity test procedure is the same as the assay, then the accuracy of the specified range should cover from 70 to 130% of the test concentration.

• • Recommended number of sample preparations: 3 at 70%, 6 at 100%, and 3 at 130% test concentrations.

• • If possible, recommended to perform extended concentration from 50 to 150% of test concentration (wherever possible).

• • Calculate the assay/preservative (antioxidant) content results. Determine the mean, standard deviation, relative standard deviation, and 95% confidence interval of the assay/preservative (antioxidant) content results.

Acceptance criteria :

• • Drug Product (DP): % RSD of the assay/preservative (antioxidant) content results obtained at each concentration should not be more than 2.0% and 5.0% respectively and cumulative % RSD of 9 determinations should not be more than 2.0% and 5.0% respectively.

• • For DP: % RSD of six assay results at 100% concentration should not be more than 2.0% for active and 5.0% for preservative (antioxidant) content.

• • Drug Substances (DS) : % RSD of assay results obtained at each concentration should not be more than 1.0% for HPLC / UV spectrophotometer methods and 2.0% for GC methods and cumulative % RSD of 9 determinations should not be more than 1.0% for HPLC/UV-Spectrophotometer and 2.0% for GC methods.

• • For DS: % RSD of six assay results at 100% concentration should not be more than 1.0% for HPLC / UV spectrophotometer methods and 2.0% for GC

• • Intermediate precision:

• • Determination:

• • Perform experiments same as performed under repeatability, but with following typical variables compared to repeatability experiment.

• • Typical variations to be studied include – day, analysts, equipment and column (different lot or different make) etc. It is not considered necessary to study these effects individually.

• • In case multiple strengths dosage forms in DP, perform the intermediate precision with both lowest and highest strengths.
  • Calculate the assay/preservative (antioxidant) content results.

• • Determine the mean, standard deviation, relative standard deviation and 95% confidence interval of the assay/ preservative (antioxidant) content results.

• • Calculate the % difference in the assay/ preservative (antioxidant) content results obtained in repeatability and Intermediate precision.

Acceptance criteria :

• • Drug Product (DP), % RSD of the assay/preservative (antioxidant) content results obtained at each concentration should not be more than 0% and 5.0^o/ respectively and cumulative % RSD of 9 determinations should not be more than 2.0% and 5.0% respectively.

• • For DP, % RSD of assay/preservative (antioxidant) content at 100% concentration should not be more than 2.0% and 5.0% respectively.

• • For DP, absolute difference between average results value obtained in the repeatability and intermediate precision, should not be more than 2.0% for assay and 5.0% for preservative (antioxidant) content.

• • Drug Substances (DS), % RSD of assay results obtained at each concentration should not be more than 1.0% for HPLC / UV-spectrophotometer methods and 2.0% for GC methods and cumulative % RSD of 9 determinations should not be more than 1.0% for HPLC/UV-spectrophotometer and 2.0% for GC methods.

• • For DS, % RSD of the assay results obtained from six sample preparations should not be more than 1.0% for DS for HPLC and UV-spectrophotometer methods and 2.0% for GC/Titrimetry method.

• • For DS, absolute difference between average result value obtained in repeatability and intermediate precision, should not be more than 1.0% for DS for HPLC and UV-spectrophotometer, and 2.0% for GC/Titrimetry methods.

• Reproducibility  – Analytical Method Validation (AMV):

Determination :

• • Perform the experiment, when the use of analytical procedure is in different lab, as in collaborative study. lt shall be perform same as repeatability study.

• • In case multiple strengths dosage forms in DP, perform the reproducibility with both lowest and highest strengths.

• • Calculate the assay/preservative (antioxidant) content results. Determine the mean, standard deviation, relative standard deviation and 95% confidence interval of the assay/ preservative (antioxidant) content results.

• • Calculate the % difference in the assay/preservative (antioxidant) content results obtained in repeatability and reproducibility.

Acceptance criteria :

• • Drug Product (DP), % RSD of the assay/preservative (antioxidant) content results obtained at each concentration should not be more than 2.0% and 5.0% respectively and cumulative % RSD of 9 determinations should not be more than 0% and 5.0 % respectively.

• • For DP, % RSD of assay/preservative (antioxidant) content at 100% concentration should not be more than 2.0% and 5.0 % respectively.

• • For DP, absolute difference between average results value obtained in the repeatability and reproducibility, should not be more than 2.0% for assay and 5.0 % or preservative (antioxidant) content.

• • Drug Substances (DS) : % RSD of assay results obtained at each concentration should not be more than 0% for HPLC / UV-spectrophotometer methods and 2.0% for GC methods and cumulative % RSD of 9 determinations should not be more than 1.0% for HPLC/UV-spectrophotometer and 2.0% for GC methods.

• • For DS, % RSD of the assay results obtained from six sample preparations should not be more than 0% for DS for HPLC and UV-spectrophotometer methods, and 2.0 % for GC/Titrimetry method.

• • For DS, absolute difference between average result value obtained in repeatability and reproducibility, should not be more than 1.0% for DS for HPLC and UV-spectrophotometer, and 2.0% for GC/Titrimetry.

• Accuracy (Recovery) – Analytical Method Validation (AMV):

Determination :

• • Recovery solutions shall be prepared by adding known quantities of the analyte into the volumetric flask, in case of DP flask containing placebo powder.

• • Analyse the sample (minimum three sample preparations at each concentration) for assay/preservative (antioxidant) content at three different concentrations covering the specified range from 80 to 120% of test concentration with considering the listed points as…

• • • In case of multiple strengths in DP, bracketing of lowest and highest strengths should cover the range from 80% to 120% of test concentration.

• • • For DP, if content uniformity and blend uniformity test procedure is same as assay, the accuracy of specified range should cover from 70 to 130% of test concentration, also perform the accuracy on additional concentration levels to cover the content uniformity and blend analysis desired concentration (1X- 3X)).

• • • If possible, recommended to perform extended concentration from 50 to 150% of test concentration (wherever possible).

• • • If in case different colors are used for multiple strength dosage forms then accuracy shall be performed either individually or on mixed placebo.

• • • For DP, the quantity of placebo in recovery solutions at all levels shall remain constant as in sample solution described in the method to be validated.

• • • For DP, in case of very low strength, then prepare a stock solution of the drug substance/preservative (antioxidant) content with suitable solvent for spiking to prepare the recovery solutions (wherever applicable).

• • • Recovery study shall be performed by covering first stock sample concentration (i.e. spiking API standards or preservative (antioxidant) content in placebo matrix) and further diluted as per methodology.

• • • Calculate the quantity recovered in mg and the % recovery, for each level and the mean recovery for all solutions.