Analytical Method Validation SOP

SOP for analytical method validation – AMV | ICH guidelines for analytical method validation | Analytical method validation acceptance criteria |

Analytical Method Validation SOP covers below points:
- Principle purpose of the analytical method validation
- Analytical method validation definitions
- Accuracy
- Precision
- Specificity
- Detection limit
- Quantitation limit
- Linearity and Range
- Robustness
- Test and selection of validation parameter for Drug substances (API)
- Test and selection of validation parameter for Finished drug product
- Analytical method validation for Assay (by HPLC/UV)- for drug substance (API)
- Analytical method validation for Assay (by potentiometric/manual titration) for drug substances(API)
- Analytical method validation for Chromatographic purity/related substances (by HPLC) where known impurities are available – for drug substance (API).
- Analytical method validation for Chromatographic purity/related substances (by HPLC) where known impurities are not available – for drug substance (API).
- Analytical method validation for Chromatographic purity/ related substances (by TLC) – for drug substances (API)
- Analytical method validation for Cleaning validation – for drug substances (API)
- Analytical method validation for Assay/content uniformity (by HPLC/UV/GC)-for finished drug product.
- Analytical method validation for Assay (by potentiometric/manual titration)-for drug product.
- Analytical method validation for Dissolution (by HPLC/GC/UV)-for drug product
- Analytical method validation for Chromatographic purity/related substances (by HPLC) where known impurities are available for drug product
- Analytical method validation for Chromatographic purity / related substances (by HPLC) where known impurities are not available – for drug product.
- Analytical method validation for Chromatographic purity/related substances (by TLC) for drug product.
- Analytical method validation for Residual solvent (by GC with head space)-for drug product.
- Analytical method Verification of standard and compendial methods
- Analytical method validation protocol format
- Analytical method validation report format

1.0 OBJECTIVE :

To lay down the procedure for validation of an analytical method.

2.0 SCOPE :

This SOP is applicable for validation of an analytical method of following categories;
- Category I: Analytical method for quantitation of major components of bulk drugs substances or active ingredients (including preservatives) in finished drug products.
- Category II: Analytical method for determination of impurities in bulk drug substances or in finished drug products.
- Category III: Analytical method for determination of performance characteristics (e.g. dissolution, drug release).
- Category IV: Identification tests.

3.0 RESPONSIBILITY :

QC Officer: To prepare the protocol, performing validation as per protocol and preparing the report.
QC Sr. Officer/ Executive: To review the protocol, monitoring the entire validation, reviewing raw data and report.
Head-QC: To review the protocol and report.
Head-QA: To Approve the protocol and report.

4.0 ACCOUNTABILITY:

Quality Control Head

5.0 PROCEDURE :

5.1 Analytical monitoring of a pharmaceutical product is necessary to ensure its safety & efficacy throughout all phases of its shelf life, including storage, distribution & use.

5.2 The principle purpose of the analytical method validation is to ensure that the selected analytical procedures will give reproducible & reliable results that are adequate for the intended purpose.

5.3 DEFINATIONS:

Accuracy: The accuracy of an analytical method is the closeness of test results obtained by that method to the true value. The accuracy of an analytical method should be established across its range.
Precision: The precision of an analytical method is the closeness of agreement (degree of scatter) between series of measurements obtained from multiple samplings of the same homogeneous sample under the prescribed conditions.
Specificity: Specificity is defined as the ability to assess unequivocally the analyte in the presence of components that may be expected to be present, such as impurities, degradation products and matrix components.
Detection limit: The detection limit of an analytical procedure is the lowest concentration of analyte in a sample that can be detected but not necessarily quantitated as an exact value.
Quantitation limit: The quantitation limit of an analytical procedure is the lowest concentration of analyte in a sample that can be determined with suitable precision and accuracy under the stated experimental conditions.
Linearity and Range: The linearity of an analytical method is its ability to elicit test results that are directly, or by a well-defined mathematical transformation, proportional to the concentration of analyte in samples within a given range where as the range of an analytical method is the interval between the upper and lower levels of analyte (including these levels) that have been demonstrated to be determined with a suitable level of precision, accuracy, and linearity using the method as written.
Robustness: The robustness of analytical method is a measure of its capacity to remain unaffected by small, but deliberate variations in the method parameters and provides an indication of its reliability during normal usage.

5.4 Validation of analytical method will be necessary if method is new.

5.5 Revalidation may be necessary if any of the following changes are made:

Changes in synthesis of drug substance.
Changes in the composition of finished drug product.
Changes in analytical procedure.

5.6 Test and selection of validation parameter for Drug substances (API).

*Carry out Accuracy only when impurities are known and available.

** Carry out Specificity by stress study (forced degradation) only by HPLC.

5.7 Test and selection of validation parameter for Finished drug product.

*Carry out Accuracy only when impurities are known and available.

** Carry out Specificity by stress study (forced degradation) only by HPLC.

5.8 Not all validation parameter and criteria are applicable to all method as outlined in 5.4 and 5.5. A decision to omit or include test is the responsibility of department head/designee.

5.9 Prepare an analytical method validation protocol (Refer Experimental Design/matrix for specific method mentioned).

5.10 Protocol should be prepared by QC person, checked by Senior QC person, reviewed by Head-QC and approved by Head-QA

5.11 Carry out analytical method validation as per protocol.

5.12 On completion of validation prepare validation report and conclusion to summarize the results.

5.13 Validation report and raw data should be checked by senior QC person, reviewed by senior QA person and Head-QC and approved by Head-QA.

5.14 EXPERIMENTAL Design/ MATRIX:

Drug substances (API):
- Assay (by HPLC/UV).
- Assay (by Potentiometric/Manual Titration).
- Chromatographic Purity/Related Substances (by HPLC) where known impurities are available.
- Chromatographic Purity/Related Substances (by HPLC) where known impurities are not available.
- Chromatographic Purity/Related Substances (by TLC).
- Cleaning Validation.
Finished Drug Product
- Assay/Content Uniformity (by HPLC/UV/GC).
- Assay (by Potentiometric/Manual Titration).
- Dissolution.
- Chromatographic Purity/Related Substances (by HPLC) where known impurities are available.
- Chromatographic Purity/Related Substances (by HPLC) where known impurities are not available.
- Chromatographic Purity/Related Substances (by TLC).
- Cleaning Validation.
Verification of standard and Compendial methods.

5.15 ASSAY (By HPLC/UV)- FOR DRUG SUBSTANCE (API)

5.15.1 Specificity
5.15.1.1 Specificity –I

Experimental Design	Acceptance criteria
By UV: Check interference of diluent.	There should be no interference of any peaks due to diluent with drug substance. The observed should not be more than 1% with respect to standard solution.
By HPLC: Inject individually diluent as a blank, system suitability solution if applicable, standard solution and test solution. In case of HPLC carry out the test using PDA detector.	There should be no interference of any peaks due to diluent with standard and test. The standard and test should be comparable with respect to retention time. Peak purity of standard should pass, Peak purity index should be greater than single point threshold.

5.15.1.2 Specificity –II (By stress study / forced degradation)

5.15.2 Precision
5.15.2.1 System precision (System suitability)

Experimental Design	Acceptance criteria
By HPLC: Inject blank and system suitability solution as applicable and determine system suitability.Inject five replicate injection of standard solution (in case of GC six replicate). Determine % RSD of peak area or peak area ratio with internal standard.	For chromatographic method, system suitability parameters such as resolution, column efficiency, tailing factor should be determines. The RSD for peak area or peak area ratio with internal standard should not be more than 2.0%.
By UV: Check absorbance of standard five times and determine % RSD of absorbance.	The RSD of absorbance should be not more than 2.0%.

5.15.2.2 Method precision (Repeatability)

5.15.2.3 Intermediate precision (Ruggedness)

Experimental Design	Acceptance criteria
Perform same exercise as that of Method precision (Repeatability) by a different analyst, different make of column, different day and on different instrument.	1. The similarity factor between standard and check standard should be 0.98 to 1.02. 2. Meet requirement of system suitability under system precision. 3. The RSD of % assay from six results should not be more than 2.0%. 4. The overall RSD of % assay from method precision study and intermediate precision together should not be more than 2.0%.

5.15.3 Linearity and Range

Experimental Design

Acceptance criteria

Perform the linearity of the standard from 50% to 150% of the target concentration at 50%, 60%, 80%, 100%, 120%, 140%, 150% level. Six replicates injection/absorbance at 50% level and 150% level. At other levels in duplicate. Plot a graph of concentration against the peak area / absorbance.
Calculate correlation coefficient, y-intercept, and %RSD of 50% level and 150% level for peak area responses.

1. The similarity factor between standard and check standard should be 0.98 to 1.02.
2. Meet requirement of system suitability under system precision.
3. Correlation coefficient should be ≥0.999.
4. The y-intercept should be ± 3.
5. RSD of peak area/absorbance of 6 replicates at 50% and 150% levels should not be more than 2.0%.

5.15.4 Accuracy

Experimental Design	Acceptance criteria
Prepare three samples of drug substances at each of the three concentrations, 80% 100%, 120% of the working test concentrations and analyze against standard solution. Determine the % recovery for each sample and RSD of overall recovery.	1. The similarity factor between standard and check standard should be 0.98 to 1.02. 2. Meet requirement of system suitability under system precision. 3. Recovery should be between 98.0% and 102.0%. 4. The relative standard deviation of overall recovery should not be more than 2.0%.

5.15.5 Robustness

5.15.5.1 Solution Stability

Experimental Design	Acceptance criteria
Analyze sample solution and standard solution at interval between initial and predetermine time interval (e.g. 0, 12, 24, 36, 48, 60 and 72 hrs) against freshly prepared standard solution. Note: Time interval can be reduced for sensitive drug substances. If solution stability is specified in method, no need to perform solution stability.	1. The similarity factor between standard and check standard should be 0.98 to 1.02. 2. Meet requirement of system suitability under system precision. 3. Assay at predetermine interval using freshly prepared standard solution should be ± 2.0% from initial result. 4. Overall RSD of Area/absorbance of standard solution including initial replicate injections should not be more than 2.0%.

5.15.5.2 Variation in method parameter

Experimental Design

Acceptance criteria

Vary one parameter at a time and carry out assay as per method. Compare result with result obtained by method precision.
By HPLC:
a. pH of mobile phase/Buffer (±0.2)
b. Composition of Mobile phase (±5%)
c. Column temperature (± 5°C)
d. Detection wavelength (± 2 nm)
e. Flow rate (±10%)
f. Different type and make of column
By UV: a. Change make of chemical
b. Wavelength (± 2 nm)
Note: Following parameter also consider if mention in method.
a. Extraction time (±10%).
Parameter for variation to be decided by department head / designee.

1. The similarity factor between standard and check standard should be 0.98 to 1.02.
2. Meet the requirement of system suitability under system precision.
3. Assay value should be ± 2% of actual assay result obtained by method precision.

5.16 ASSAY (BY POTENTIOMETRIC/MANUAL TITRATION) FOR DRUG SUBSTANCES(API)

5.16.1 Specificity

Experimental Design	Acceptance criteria
Check interference of diluent.	The burette reading for diluents should be negligible(About 0.1ml)

5.16.2 Precision

5.16.2.1 Method precision (Repeatability)

Experimental Design	Acceptance criteria
Perform six individual Assay determinations.	The RSD of % assay from six results should not be more than 2.0%.

5.16.2.2 Intermediate precision (Ruggedness)

Experimental Design	Acceptance criteria
Perform same exercise as that of Method precision (Repeatability) by a different analyst, different day and on different instrument.	1. The RSD of % assay from six results should not be more than 2.0%. 2. The overall RSD of %assay from method precision study and intermediate precision study together should not be more than 2.0%.

5.16.3 Linearity and Range

Experimental Design	Acceptance criteria
Perform the linearity of the standard from 50% to 150% of the target concentration at 50%, 60%, 80%, 100%, 120%, 140%, 150% level. Perform six replicate titrations at 50% level and 150% level. At other levels in duplicate. Plot a graph of concentration against the volume. Calculate correlation coefficient, y-intercept, and %RSD of 50% level and 150% level.	1. Correlation coefficient should be ≥0.999. 2. The y-intercept should be ± 3. 3. RSD of volume of titrant of 6 replicates at 50% and 150% levels should not be more than 2.0%.

5.16.4 Accuracy

Experimental Design	Acceptance criteria
Titrate samples of drug substance at concentrations 80% 100%, 120% of the working test concentrations in triplicate at each level. Determine the % recovery for each sample and RSD of overall recovery.	1. Recovery should be between 98.0% and 102.0%. 2. The relative standard deviation of overall recovery should not be more than 2.0 %.

5.16.5 Robustness

Experimental Design	Acceptance criteria
Vary one parameter at a time and carry out assay as per method. Compare result with result obtained by method precision. 1. Change in volume of masking reagent (e.g. mercuric acetate, acetic anhydride etc. as applicable) 2. Change in make of chemicals. Parameter for variation to be decided by department head /designee.	Assay value should be ±2 % of actual assay result obtained by method precision.

5.17 CHROMATOGRAPHIC PURITY/RELATED SUBSTANCES (BY HPLC) WHERE KNOWN IMPURITIES ARE AVAILABLE – FOR DRUG SUBSTANCE (API).

5.17.1 Specificity
5.17.1.1 Specificity –I

Experimental Design	Acceptance criteria
Inject individual diluent, system suitability solution if applicable, standard solution, known impurities (at limit level concentration) and test solution. Carry out the test using PDA detector.	1. There should be no interference of any peaks due to diluent with known impurities, standard and test. 2. The standard and test should be comparable with respect to retention time. 3. Peak purity of analyte should pass, Peak purity index should be greater than single point threshold.

5.17.1.2 Specificity –II (By stress study/ forced degradation)

5.17.2 Precision
5.17.2.1 System suitability (system suitability)

Experimental Design	Acceptance criteria
1. Inject diluent and system suitability solution as applicable and determine system suitability. 2. Perform six replicate injection of standard solution at limit level concentration.	1. System suitability parameters such as resolution, column efficiency, tailing factor should be determined. 2. The RSD for peak area standard solution should not be more than 5.0%.

5.17.2.2 Method precision (Repeatability)

5.17.2.3 Intermediate precision (Ruggedness)

Experimental Design

Acceptance criteria

Perform same exercise as that of Method precision (Repeatability) by a different analyst, different column, different day and on different instrument.

1. The similarity factor between standard and check standard should be 0.95 to 1.05.
2. Meet requirement of system suitability under system precision.
3. The % RSD for content of known impurities, single maximum unknown impurity and total impurities should not be more than limit specified below as per result observed.,
Result observed        % RSD
≤ 0.01%                     20%
0.01% to 0.10%         10%
≥ 0.10%                       5%
4. The overall % RSD for content of known impurities, single maximum unknown impurity, total impurities from method precision study and intermediate precision study together should not be more than limit specified below as per result observed.,
Result observed        % RSD
≤ 0.01%                      20%
0.01% to 0.10%          10%
≥ 0.10%                        5%

5.17.3 Limit of Detection (LOD) and Limit of Quantitation (LOQ)

5.17.4 Linearity and Range

Experimental Design	Acceptance criteria
Perform the linearity of the standard from LOQ to 150% of limit level concentration (LOQ, 50%, 60%, 80%, 100%, 120%, 140%, 150% level). Six replicates injection at LOQ and 150% level. At other levels in duplicate. Plot a graph of concentration against the average peak area. Calculate correlation coefficient, y-intercept, and % RSD of LOQ level and 150% level.	1. The similarity factor between standard and check standard should be 0.95 to 1.05. 2. Meet requirement of system suitability under system precision 3. Correlation coefficient should be ≥0.990. 4. The y-intercept should be ± 3 4. The % RSD of peak area should not be more than limit specified below. Level % RSD LOQ 20% 150% 5%

5.17.5 Accuracy

Experimental Design	Acceptance criteria
Prepare triplicate samples of drug substances spiked with impurities at LOQ, 50%, 100% and 150% of limit level concentration. Determine the %recovery for each impurity.	1. The similarity factor between standard and check standard should be 0.95 to 1.05. 2. Meet requirement of system suitability under system precision 3. % Recovery of each impurity should be not more than limit specified below. Level % Recovery LOQ 80% to 120% 50%,100%,150% 85% to 115%

5.17.6 Robustness
5.17.6.1 Solution stability

Experimental Design

Acceptance criteria

Analyze sample solution and standard solution at interval between initial and predetermine time interval (e.g. 0, 12, 24,36,48,60 and 72 hrs) against freshly prepared standard solution.
Note: Time interval can be reduced for sensitive drug substances. If solution stability is specified in method, no need to perform solution stability.

1. The similarity factor between standard and check standard should be 0.95 to 1.05.
2. Meet requirement of system suitability under system precision.
3. Overall RSD of Area of standard solution including initial replicate injections should not be more than 5.0%.
4. The overall RSD for content of known impurities, single maximum unknown impurity, total impurities at predetermine intervals should not be more than limit specified below as per result observed.
Result observed        % RSD
≤ 0.01%                      20%
0.01% to 0.10%          10%
≥ 0.10%                       5%

5.17.6.2 Variation in method parameters

Experimental Design

Acceptance criteria

Vary one parameter (as mention below) at a time and perform analysis of three samples spiked with all known impurities at limit level concentration. Also perform analysis of unspiked sample.
1. Determine the % RSD for content of known impurities, single maximum unknown impurity and total impurities.
2. Determine the overall % RSD for content of known impurities, single maximum unknown impurity and total impurities along with method precision under precision study.
Parameter:
a. pH of mobile phase/Buffer (± 0.2)
b. Composition of Mobile phase (± 5%)
c. Column temperature (± 5°C)
d. Detection wavelength (± 2nm)
e. Flow rate (± 10%)
f. Different type and make of column

Parameter for variation to be decided by department head /designee.

5.18 CHROMATOGRAPHIC PURITY/RELATED SUBSTANCES (BY HPLC) WHERE KNOWN IMPURITIES ARE NOTAVAILABLE – FOR DRUG SUBSTANCE (API).

5.18.1 Specificity
5.18.1.1 Specificity –I

Experimental Design	Acceptance criteria
Inject individual diluent, system suitability solution if applicable, standard solution, and drug substance. Carry out the test using PDA detector.	1. There should be no interference of any peaks due to diluent with known impurities, standard and test solution. 2. The standard and test should be comparable with respect to retention time. 3. Peak purity of analyte should pass, Peak purity index should be greater than single point threshold.

5.18.1.2 Specificity –II (By stress study/forced degradation)

5.18.2 Precision
5.18.2.1 System precision (System suitability)

Experimental Design	Acceptance criteria
Inject diluent and system suitability solution as applicable and determine system suitability. Perform six replicate injection of standard solution at limit level concentration).	1. System suitability parameters such as resolution, column efficiency, tailing factor should be determines. 2. The RSD for peak area should not be more than 5.0%.

5.18.2.2 Method precision (Repeatability)

5.18.2.3 Intermediate precision (Ruggedness)

Experimental Design

Acceptance criteria

Perform same exercise as that of Method precision (Repeatability) by a different analyst, different column, different day and on different instrument.

1. The similarity factor between standard and check standard should be 0.95 to 1.05.
2. Meet requirement of system suitability under system precision.
3. The % RSD for content of known impurities, single maximum unknown impurity and total impurities should not be more than limit specified below as per result observed.
Result observed        % RSD
≤ 0.01%                     20%
0.01% to 0.10%         10%
≥ 0.10%                       5%
4. The overall % RSD for content of known impurities, single maximum unknown impurity, total impurities from method precision study and intermediate precision study together should not be more than limit specified below as per result observed.
Result observed        % RSD ≤
0.01%                      20%
0.01% to 0.10%          10%
≥ 0.10%                        5%

5.18.3 Limit of Detection (LOD) and Limit of Quantitation (LOQ)

5.18.4 Linearity and Range

Experimental Design	Acceptance criteria
Perform the linearity of the standard from LOQ to 150% of limit level concentration (LOQ, 50%, 60%, 80%, 100%, 120%, 140%, 150% level). Six replicates injection at LOQ and 150% level. At other levels in duplicate. Plot a graph of concentration against the average peak area. Calculate correlation coefficient, y-intercept, and % RSD of LOQ level and 150% level.	1. The similarity factor between standard and check standard should be 0.95 to 1.05. 2. Meet requirement of system suitability under system precision 3. Correlation coefficient should be ≥0.990. 4. The y-intercept should be ± 3. 5. The % RSD of peak area should not be more than limit specified below. Level % RSD LOQ 20% 150% 5%

5.18.5 Robustness
5.18.5.1 Solution stability

Experimental Design

Acceptance criteria

Analyze sample solution and standard solution at interval between initial and predetermine time interval (e.g. 0, 12, 24, 36, 48, 60 and 72 hrs) against freshly prepared standard solution.

Note: Time interval can be reduced for sensitive drug substances. If solution stability is specified in method, no need to perform solution stability.

1. The similarity factor between standard and check standard should be 0.95 to 1.05.
2. Meet requirement of system suitability under system precision
3. Overall RSD of Area of standard solution including initial replicate injections should not be more than 5.0%.
4. The overall RSD for content of known impurities, single maximum unknown impurity, total impurities at predetermine intervals should not be more than limit specified below as per result observed.
Result observed        % RSD
≤ 0.01%                       20%
0.01% to 0.10%           10%
≥ 0.10%                        5%

5.18.5.2 Variation in method parameters

Experimental Design

Acceptance criteria

Vary one parameter (as mention below) at a time and perform analysis of three samples.
1. Determine the % RSD for content of single maximum unknown impurity and total impurities.
2. Determine the overall % RSD for content of single maximum unknown impurity and total impurities along with method precision under precision study.
Parameter:
a. pH of mobile phase/Buffer (± 0.2)
b. Composition of mobile phase (± 5%)
c. Column temperature (± 5°C)
d. Detection wavelength (± 2 nm)
e. Flow rate (± 10%)
f. Different type and make of column
Parameter for variation to be decided by department head /designee.

1. The similarity factor between standard and check standard should be 0.95 to 1.05.
2. Meet requirement of system suitability under system precision.
3. The overall RSD for content of single maximum unknown impurity and total impurities with method precision should not be more than limit specified below as per result observed.
Result observed        % RSD
≤ 0.01%                       20%
0.01% to 0.10%           10%
≥ 0.10%                        5%

5.19 CHROMATOGRAPHIC PURITY/ RELATED SUBSTANCES (BY TLC) – FOR DRUG SUSBTANCES (API)

5.19.1 Specificity

Experimental Design	Acceptance criteria
Spot diluent, individual impurity and drug substance.	1. There should be no interference of diluent at the Rf value of known impurity and drug substance. 2. All impurities should be well separated from drug substance.

5.19.2 Precision
5.19.2.1 System precision (System suitability)

Experimental Design	Acceptance criteria
Spot diluent, system suitability solution (If applicable) and determine system suitability.	Meet system suitability parameters.

5.19.2.2 Method precision (Repeatability)

Experimental Design	Acceptance criteria
Perform analysis of six samples spiked with all known impurities at limit level concentration. Also perform analysis of unspiked sample. Compare intensities of impurity spots visually.	1. The intensities of all impurities of same Rf value in six samples should be comparable. 2. The % RSD of Rf value of six samples solution should not be more than 10%.

5.19.2.3 Intermediate precision (Ruggedness)

Experimental Design	Acceptance criteria
Perform same exercise as that of Method precision (Repeatability) by a different analyst, different day.	1. The intensities of all impurities of same Rf value in six samples should be comparable. 2. The RSD of Rf value of six samples solution should not be more than 10% 3. The overall RSD of Rf value along with method precision study should not be more than 10%.

5.19.3 Limit of Detection (LOD)

Experimental Design	Acceptance criteria
Prepare solutions of different concentration of impurity and test solution. Check the lowest detection level where the spot clearly visible.	The concentration is acceptable as LOD, if the spot is clearly visible.

5.19.4 Robustness- solution stability

Experimental Design	Acceptance criteria
Spot individual impurity solution and drug solution at predetermine time interval (e.g. 0,4,8,12,16,20 and 24 hrs) Note: 1. Time interval can be reduced for sensitive drug substances. 2. If solution stability is specified in method, no need to perform solution stability.	The intensities of all impurities of same Rf values should be comparable.

5.20 CLEANING VALIDATION – FOR DRUG SUBSTANCES (API)

5.20.1 Specificity

Experimental Design

Acceptance criteria

Check interference of analytical diluent, cleaning agent and swab.
1. Cleaning agent solution: Prepare 1 ppm solution of cleaning agent in analytical diluent if cleaning agent is different than analytical diluent.
2. Swab solution: Wet the swab with diluent. Rub on the clean SS surface as per procedure and transfer into a volumetric flask. Add diluent and swirl.
3. Standard solution: Prepare as per method.

Preferably there should be no interference of diluent, cleaning agent and swab.
If observed it should be less than limit of detection of standard.

5.20.2 Precision
5.20.2.1 System precision (System suitability)

Experimental Design	Acceptance criteria
By HPLC: Inject diluent, system suitability solution as applicable and determine system suitability. Perform six replicate injection of standard solution at working level.	1. Parameters such as resolution, column efficiency, tailing factor should be determined. 2. The RSD for peak area should not be more than 5.0%.
By UV: Check six replicate absorbance of standard solution at working level.	The RSD for absorbance should not be more than 2.0%.

5.20.2.2 Precision

5.20.2.3 Intermediate precision (Ruggedness)

Experimental Design

Acceptance criteria

Perform same exercise as that of Method precision (Repeatability) by a different analyst, different column, different day and on different instrument.

1. The similarity factor between standard and check standard should be 0.95 to 1.05.
2. Meet requirement of system suitability under system precision
3. The RSD of % recovery for six replicate determinations should not be more than 10% and individual % recovery should be between 85% and 115%.
4. The overall RSD of % recovery from method precision study and intermediate precision study together should not be more than 10%.

5.20.3 Limit of Detection (LOD) and Limit of Quantitation (LOQ)

5.20.4 Linearity and Range

Experimental Design	Acceptance criteria
Perform the linearity of the standard from LOQ to 150% of working level concentration (LOQ, 50%, 60%, 80%, 100%, 120%, 140%,150% level). Six replicates injection/absorbance at LOQ and 150% level. At other levels in duplicate. Plot a graph of concentration against the average peak area/absorbance. Calculate correlation coefficient, y-intercept and % RSD of LOQ level and 150% level.	1. The similarity factor between standard and check standard should be 0.95 to 1.05. 2. Meet requirement of system suitability under system precision. 3. Correlation coefficient should be ≥0.990. 4. The y-intercept should be ± 3. 5. The % RSD of peak area should not be more than limit specified below. Level % RSD LOQ 20% 150% 5%

5.20.5 Accuracy

Experimental Design

Acceptance criteria

Carry out recovery at LOQ, 50%, 150% of working level concentration in triplicate and 100% of working level concentration in six replicates.
Apply known quantity of standard stock solution of drug substance and apply onto a clean stainless steel cleaning validation pan which has an area of about 100 cm². The solution will be left to dry at room temperature. Wet a swab by dipping it into swabbing solvent (usually IPA or water) and squeeze out the excess solvent. Wipe the pan twice using the above wet swab (use one side of the swab to wipe the whole area in one direction, and then use the other side of the swab to wipe the whole area in a perpendicular direction). Transfer this swab into volumetric flask. Repeat the above procedure using three other swabs. Put all wiped swabs into the same volumetric flask. Add known amount of diluting solvent to volumetric flask, swirl and carry out determination.

% Recovery should be within below specified limit.
Level %Recovery LOQ 80% to 120% 50%,100%, 85% to 115% & 150%

5.21 ASSAY/CONTENT UNIFORMITY (BY HPLC/UV/GC)-FOR FINISHED DRUG PRODUCT

5.21.1 SPECIFICITY
5.21.1.1 Specificity –I

Experimental Design	Acceptance criteria
By HPLC: Inject individually diluent as a blank, Placebo, system suitability solution if applicable, standard solution and test solution. In case of HPLC carry out the test using PDA detector.	There should be no interference of any peaks due to diluent and placebo with standard and test. The standard and test should be comparable with respect to retention time. Peak purity of standard should pass, Peak purity index should be greater than single point threshold.
By UV: Check interference of diluent and placebo. Placebo interference may be determined by weighing placebo and dissolving or dispersing them in dissolution medium as per dissolution method.	There should be no interference of diluent and placebo with standard and test. If observed should not be more than 1% with respect to standard solution.

5.21.1.2 Specificity-II (By stress study/ forced degradation)

5.21.2 Precision
5.21.2.1 System precision (System suitability)

Experimental Design	Acceptance criteria
By HPLC/GC: Inject system suitability solution as applicable and determine system suitability. Inject five replicate injection of standard solution (in case of GC six replicate). Determine % RSD of peak area or peak area ratio with internal standard.	1. System suitability parameters such as resolution, column efficiency, tailing factor should be determined. 2. The RSD for peak area or peak area ratio with internal standard should not be more than 2.0%.
By UV: Check absorbance of standard five times and determine % RSD of absorbance.	The RSD of absorbance should not be more than 2.0%.

5.21.2.2 Method precision (Repeatability)

Experimental Design	Acceptance criteria
Perform six individual Assay determinations and ten determination of content uniformity.	1. Similarity factor of standard solution and check standard solution should be between 0.98 and 1.02. 2. Meet requirement of system suitability under system precision 3. The RSD of % assay values and content uniformity values should not be more than 2.0% and 6.0% respectively.

5.21.2.3 Intermediate precision (Ruggedness)

Experimental Design

Acceptance criteria

Perform same exercise as that of Method precision (Repeatability) by a different analyst, different column, different day and on different instrument.

1. Similarity factor of standard solution and check standard solution should be between 0.98 and 1.02.
2. Meet requirement of system suitability under system precision
3. The RSD of % assay values and content uniformity values should not be more than 2.0% and 6.0% respectively.
4. The overall RSD of % assay values and content uniformity values from method precision study and intermediate precision study together should not be more than 2.0% and 6.0% respectively.

5.21.3 Linearity and Range

Experimental Design

Acceptance criteria

Perform the linearity of the standard from 50% to 150% of the target concentration at 50%, 60%, 80%, 100%, 120%, 140%,150% level. Six replicates injection/absorbance at 50% level and 150% level.
At other levels in duplicate.
Plot a graph of concentration against the peak area / absorbance. Calculate correlation coefficient, y-intercept, slope and %RSD of 50% level and 150% level for peak area responses.

1. Similarity factor of standard solution and check standard solution should be between 0.98 and 1.02
2. Meet requirement of system suitability under system precision
3. Correlation coefficient should be ≥0.999.
4. The y-intercept should be ± 3
5. RSD of peak area/absorbance of 6 replicates at 50% and 150% levels should not be more than 2.0%.

5.21.4 Accuracy

Experimental Design	Acceptance criteria
For assay: To placebo, add standard at 80% 100%, 120% of the working test concentrations in triplicate at each level and analyze against standard solution. Placebo concentration should be same as that of working concentration. Determine the %recovery for each sample and RSD of overall recovery.	1. Similarity factor of standard solution and check standard solution should be between 0.98 and 1.02. 2. Meet requirement of system suitability under system precision 3. Recovery should be between 98.0% and 102.0%. 4. The relative standard deviation of overall recovery should not be more than 2.0%.
For content uniformity: To placebo, add standard at 70% 100%, 130% of the working test concentrations in triplicate at each level and analyze against standard solution. Placebo concentration should be same as that of working concentration. Determine the %recovery for each sample and RSD of overall recovery.	1. Similarity factor of standard solution and check standard solution should be between 0.98 and 1.02. 2. Meet requirement of system suitability under system precision 3. Recovery should be between 98.0% and 102.0%. 4. The relative standard deviation of overall recovery should not be more than 2.0%.

5.21.5 Robustness
5.21.5.1 Solution stability

Experimental Design

Acceptance criteria

Analyze sample solution and standard solution at interval between initial and predetermine time interval (e.g. 0, 12, 24, 36, 48, 60 and 72hrs) against freshly prepared standard solution.
Note:
1. Time interval can be reduced for sensitive drug substances.
2. If solution stability is specified in method, no need to perform solution stability.

1. Similarity factor of standard solution and check standard solution should be between 0.98 and 1.02.
2. Meet requirement of system suitability under system precision
3. % Assay at predetermine interval using freshly prepared standard solution should be ± 2.0% from initial result.
4. Overall RSD of Area/absorbance of standard solution including initial replicate injections should not be more than 2.0%.

5.21.5.1 Variation in method parameter

Experimental Design

Acceptance criteria

Vary one parameter at a time and carry out assay as per method. Compare result with result obtained by method precision.
By HPLC:
a. pH of mobile phase/Buffer(±0.2)
b. Mobile phase composition (±5%)
c. Column temperature(±5°C)
d. Detection wavelength(±2nm)
e. Flow rate (±10%)
f. Different type and make of column
By UV:
a. Change make of chemical
b. Wavelength(±2nm)
Following parameter also consider if mention in method.
c. Extraction time (±10%)
Parameter for variation to be decided by department head /designee.

1. Similarity factor of standard solution and check standard solution should be between 0.98 and 1.02.
2. Meet requirement of system suitability under system precision.
3. % assay values and content uniformity values should be ±2% and ±6% of actual average assay result and average content uniformity obtained by method precision respectively.

5.22 ASSAY (BY POTENTIOMETRIC/MANUAL TITRATION)-FOR DRUG PRODUCT

5.22.1 Specificity

Experimental Design	Acceptance criteria
Check interference of placebo and blank.	There should be no interference of Placebo and blank.

5.22.2 Precision
5.22.2.1 Method precision (Repeatability)

Experimental Design	Acceptance criteria
Perform six individual Assay determinations.	The RSD of %assay from six results should not be more than 2.0%.

5.22.2.2 Intermediate precision (Ruggedness)

Experimental Design	Acceptance criteria
Perform same exercise as that of Method precision (Repeatability) by a different analyst, different day and on different instrument.	1. The RSD of % assay from six results should not be more than 2.0%. 2. The overall RSD of % assay from method precision study and intermediate precision study together should not be more than 2.0%.

5.22.3 Linearity and Range

Experimental Design	Acceptance criteria
Perform the linearity of the standard from 50% to 150% of the target concentration by taking minimum five concentration levels. Perform six replicate titrations at 50% level and 150% level. At other levels in duplicate. Plot a graph of concentration against the volume. Calculate correlation coefficient, y-intercept and %RSD of 50% level and 150% level.	1. Correlation coefficient should be ≥0.999. 2. The y-intercept should be ± 3. 3. RSD of peak area/absorbance of 6 replicates at 50% and 150% levels should not be more than 2.0%.

5.22.4 Accuracy

Experimental Design	Acceptance criteria
To placebo, add standard at 80% 100%, 120% of the working test concentrations in triplicate at each level and titrate. Placebo concentration should be same as that of working concentration. Determine the % recovery for each sample and RSD of overall recovery.	Recovery should be between 98.0% and 102.0%. The relative standard deviation of overall recovery should not be more than 2.0%.

5.22.5 Robustness

Experimental Design	Acceptance criteria
Vary one parameter at a time and carry out assay as per method. Compare result with result obtained by method precision. 1. Change in volume of masking reagent (e.g. mercuric acetate, acetic anhydride etc. as applicable). 2. Change in make of chemicals. Parameter for variation to be decided by department head /designee.	Assay value should be ±2% of actual assay result obtained by method precision.

5.23 DISSOLUTION (BY HPLC/GC/UV)-FOR DRUG PRODUCT

5.23.1 Specificity
5.23.1.1 Specificity –I

Experimental Design	Acceptance criteria
By HPLC: Inject individually dissolution medium, diluent, placebo, standard and test solution. Placebo interference may be determined by weighing placebo and dissolving or dispersing them in dissolution medium as per dissolution method. In case of HPLC carry out the analysis using PDA detector.	There should be no interference of peaks due to dissolution medium, diluent and placebo with standard and test. The standard and test should be comparable with respect to retention time Peak purity of standard should pass, Peak purity index should be greater than single point threshold.
By UV: Check interference of dissolution medium, diluent and placebo. Placebo interference may be determined by weighing placebo and dissolving or dispersing them in dissolution medium as per dissolution method.	There should be no interference of diluent and placebo with standard and test. If observed should not be more than 1% with respect to standard solution.

5.23.1.2 Specificity-II (By stress study/forced degradation)

5.23.2 Precision
5.23.2.1 System precision (System suitability)

Experimental Design	Acceptance criteria
By HPLC/GC: Inject system suitability solution as applicable and determine system suitability.Inject five replicate injection of standard solution (in case of GC six replicate). Determine % RSD of peak area or peak area ratio with internal standard.	1. System suitability parameters such as resolution, column efficiency, tailing factor should be determines. 2. The RSD for peak area or peak area ratio with internal standard should not be more than 2.0%.
By UV: Check absorbance of standard five times and determine % RSD of absorbance.	The RSD of absorbance should be not more than 2.0%.

5.23.2.2 Method precision (Repeatability)

Experimental Design	Acceptance criteria
Perform six individual dissolutions test (each of having six units) as per method and determine % RSD of average % releases.	1. Similarity factor of standard solution and check standard solution should be between 0.98 and 1.02. 2. Meet requirement of system suitability under system precision 3. The RSD of average % release should not be more than 5%.

5.23.2.3 Intermediate precision (Ruggedness)

Experimental Design	Acceptance criteria
Perform same exercise as that of Method precision (Repeatability) by a different analyst, different column, different day and on different instrument.	1. Similarity factor of standard solution and check standard solution should be between 0.98 and 1.02. 2. Meet requirement of system suitability under system precision 3. The RSD of average % release should not be more than 5%. 4. The overall RSD of average % releases values from method precision study and intermediate precision study together should not be more than 5%.

5.23.3 Linearity and Range

Experimental Design	Acceptance criteria
Perform the linearity of the standard from 10% to 150% of working level at 10%,30%, 50%, 80%, 100%, 120%, 150% level. Six replicates injection/absorbance at 10% level and 150% level. At other levels in duplicate. Plot a graph of concentration against the peak area / absorbance. Calculate correlation coefficient, y-intercept and %RSD of 10% level and 150% level for peak area responses.	1. Similarity factor of standard solution and check standard solution should be between 0.98 and 1.02. 2. Meet requirement of system suitability under system precision 3. Correlation coefficient should be ≥0.999. 4. The y-intercept should be ± 3RSD of peak area/absorbance of 6 replicates at 10% and 150% levels should not be more than 5.0% and 2.0% respectively.

5.23.4 Accuracy

Experimental Design

Acceptance criteria

To the vessel add placebo and standard stock solution at 10% ,100%, 150% of the working level (Triplicate at each level) and carryout dissolution.
Placebo concentration should be same as that of working level.
Organic solvents may be used to enhance drug solubility for preparation of standard stock solution; however, no more than 5 %( v/v) of organic solvent in final solution should be used.
Determine the %recovery and RSD of overall recovery.

1. Similarity factor of standard solution and check standard solution should be between 0.98 and 1.02.
2. Meet requirement of system suitability under system precision
3. Recovery should be between 95.0% and 105.0%.
4. The relative standard deviation of overall recovery should not be more than 2.0%.

5.23.5 Robustness
5.21.5.1 Solution stability

Experimental Design

Acceptance criteria

Analyze sample solution and standard solution at interval between initial and predetermine time interval (e.g. 0, 12, 24, 36, 48 hrs) against freshly prepared standard solution.
Note:
1. Time interval can be reduced for sensitive drug substances.
2. If solution stability is specified in method, no need to perform solution stability.

1. Similarity factor of standard solution and check standard solution should be between 0.98 and 1.02.
2. Meet requirement of system suitability under system precision
3. % release at predetermine interval using freshly prepared standard solution should be ± 2.0% from initial result.
4. Overall RSD of Area/absorbance of standard solution including initial replicate injections should not be more than 2.0%.

5.23.5.2 Filter study

Experimental Design

Acceptance criteria

Analyze sample solution and standard solution after using different filters (e.g. Whatman filter, SS filter, Nylon filter paper).
For standard solution, compare the results for filtered solutions (after discarding the appropriate volume) to those for the unfiltered solution.
For sample solutions, compare the results for filtered solutions (after discarding the appropriate volume) to those for centrifuged, unfiltered solutions.
Prepare each solution in triplicate.
Evaluate alternative type of filter.

1. Similarity factor of standard solution and check standard solution should be between 0.98 and 1.02.
2. Meet requirement of system suitability under system precision
3. Area/absorbance of standard solution (filtered) should ± 2.0% of standard solution (unfiltered).
4. Area/absorbance of sample solution (filtered) should ±2.0% of sample solution (centrifuged)
5. For alternative type filter, both above acceptance criteria should meet.

5.23.5.3 Variation in method parameter

Experimental Design

Acceptance criteria

Vary one parameter at a time and carry out dissolution as per method. Compare result with result obtained by method precision.
By HPLC:
a. pH of mobile phase/Buffer (±0.2)
b. Composition of Mobile phase (± 5%)
c. Column temperature (± 5°C)
d. Detection wavelength (± 2nm)
e. Flow rate (± 10%)
f. Different lots and suppliers of column
By UV:
a. Change make of chemical
b. Wavelength (±2 nm)
Following parameter also consider if mention in method.
a. Extraction time (±10%)
Parameter for variation to be decided by department head /designee.

1. Similarity factor of standard solution and check standard solution should be between 0.98 and 1.02.
2. Meet requirement of system suitability under system precision.
3. Average % release values should be ±5% of actual average result obtained by method precision.

5.24 CHROMATOGRAPHIC PURITY/RELATED SUBSTANCES (BY HPLC) WHERE KNOWN IMPURITIES ARE AVAILABLE FOR DRUG PRODUCT

5.24.1 Specificity
5.24.1.1 Specificity-I

Experimental Design	Acceptance criteria
Inject individual diluent, placebo, system suitability solution if applicable, standard solution, known impurities (at limit level concentration) and test solution. Carry out the test using PDA detector.	There should be no interference of any peaks due to diluent and placebo with known impurities, standard and test. The standard and test should be comparable with respect to retention time. Peak purity of analyte should pass, Peak purity index should be greater than single point threshold.

5.24.1.2 Specificity-II (By stress study/forced degradation)

5.24.2 Precision
5.24.2.1 System precision (System suitability)

Experimental Design	Acceptance criteria
1. Inject diluent, placebo and system suitability solution as applicable and determine system suitability. 2. Perform six replicate injection of standard solution at limit level concentration.	1. System suitability parameters such as resolution, column efficiency, tailing factor should be determined. 2. The RSD for peak area standard solution should not be more than 5.0%.

5.24.2.2 Method precision (Repeatability)

Experimental Design

Acceptance criteria

Perform analysis of six samples spiked with all known impurities at limit level concentration. Also perform analysis of unspiked sample.
Determine the % RSD for content of known impurities, single maximum unknown impurity and total impurities.

1. Similarity factor of standard solution and check standard solution should be between 0.95 and 1.05.
2. Meet requirement of system suitability under system precision
3. The % RSD for content of known impurities, single maximum unknown impurity and total impurities should not be more than limit specified below as per result observed.
Result observed        % RSD
≤ 0.01%                      20%
0.01% to 0.10%         10%
≥ 0.10%                       5%

5.24.2.3 Intermediate precision (Ruggedness)

Experimental Design

Acceptance criteria

Perform same exercise as that of Method precision (Repeatability) by a different analyst, different column, different day and on different instrument.

1. Similarity factor of standard solution and check standard solution should be between 0.95 and 1.05. 2. Meet requirement of system suitability under system precision.
3. The % RSD for content of known impurities, single maximum unknown impurity and total impurities should not be more than limit specified below as per result observed. ,
Result observed        % RSD
≤ 0.01%                      20%
0.01% to 0.10%          10%
≥ 0.10%                       5%
4. The overall % RSD for content of known impurities, single maximum unknown impurity, total impurities from method precision study and intermediate precision study together should not be more than limit specified below as per result observed.,
Result observed        % RSD
≤ 0.01%                         20%
0.01% to 0.10%             10%
≥ 0.10%                          5%

5.24.3 imit of Detection (LOD) and Limit of Quantitation (LOQ)

5.24.4 Linearity and Range

Experimental Design

Acceptance criteria

Perform the linearity of the standard from LOQ to 150% of limit level concentration (LOQ, 50%, 60%, 80%, 100%, 120%, 140%, 150% level). Six replicates injection at LOQ and 150% level. At other levels in duplicate.
Plot a graph of concentration against the average peak area.
Calculate correlation coefficient, y-intercept and % RSD of LOQ level and 150% level.

1. Similarity factor of standard solution and check standard solution should be between 0.95 and 1.05.
2. Meet requirement of system suitability under system precision
3. Correlation coefficient should be ≥0.990.
4. The y-intercept should be ± 3.
5. The % RSD of peak area should not be more than limit specified below.
Level                 % RSD
LOQ                   20%
150%                   5%

5.24.5 Accuracy

Experimental Design	Acceptance criteria
Prepare triplicate samples of drug substances spiked with impurities at LOQ, 50%, 100% and 150% of limit level concentration. Determine the %recovery for each impurity.	1. Similarity factor of standard solution and check standard solution should be between 0.95 and 1.05. 2. Meet requirement of system suitability under system precision 3. %Recovery of each impurity should be not more than limit specified below. Level % Recovery LOQ 80% to 120% 50%,100%,150% 85% to 115%

5.24.6 Robustness
5.24.6.1 Solution stability

Experimental Design

Acceptance criteria

Analyze sample solution and standard solution at interval between initial and predetermine time interval (e.g. 0, 12, 24, 36, 48, 60 and 72 hrs) against freshly prepared standard solution.
Note:
1. Time interval can be reduced for sensitive drug substances. 2. If method specify solution stability no need to do solution stability.

1. Similarity factor of standard solution and check standard solution should be between 0.95 and 1.05.
2. Meet requirement of system suitability under system precision
3. Overall RSD of Area of standard solution including initial replicate injections should not be more than 5.0%.
4. The overall RSD for content of known impurities, single maximum unknown impurity, total impurities at predetermine intervals should not be more than limit specified below as per result observed.
Result observed        % RSD
≤ 0.01%                      20%
0.01% to 0.10%          10%
≥ 0.10%                       5%

5.24.6.2 Variation in method parameter

Experimental Design

Acceptance criteria

Vary one parameter (as mention below) at a time and perform analysis of three samples spiked with all known impurities at limit level concentration. Also perform analysis of unspiked sample.
1. Determine the % RSD for content of known impurities, single maximum unknown impurity and total impurities.
2. Determine the overall % RSD for content of known impurities, single maximum unknown impurity and total impurities along with method precision under precision study.
Parameter:
a. pH of mobile phase/Buffer(±0.2)
b. Composition of Mobile phase (± 5%)
c. Column temperature(± 5°C)
d. Detection wavelength(±2 nm)
e. Flow rate (± 10%)
f. Different type and make of column

5.25 CHROMATOGRAPHIC PURITY / RELATED SUBSTANCES (BY HPLC) WHERE KNOWN IMPURITIES ARE NOT AVAILABLE – FOR DRUG PRODUCT.

5.25.1 Specificity
5.25.1.1 Specificity-I

Experimental Design	Acceptance criteria
Inject individual diluent, placebo, system suitability solution if applicable, standard solution, known impurities (at limit level concentration) and test solution. Carry out the test using PDA detector.	There should be no interference of any peaks due to diluent and placebo with known impurities, standard and test. The standard and test should be comparable with respect to retention time. Peak purity of analyte should pass, Peak purity index should be greater than single point threshold.

5.25.1.2 Specificity –II (By stress study / forced degradation)

5.25.2 Precision

5.25.2.1 System precision (System suitability)

Experimental Design	Acceptance criteria
1. Inject diluent, placebo and system suitability solution as applicable and determine system suitability. 2. Perform six replicate injection of standard solution at limit level concentration).	1. Parameters such as resolution, column efficiency, tailing factor should be determines. 2. The RSD for peak area should not be more than 5.0%.

5.25.2.2 Method precision (Repeatability)

Experimental Design

Acceptance criteria

Perform analysis of six samples.
Determine the % RSD for content of single maximum unknown impurity and total impurities.

5.25.2.3 Intermediate precision(Ruggedness)

Experimental Design

Acceptance criteria

Perform same exercise as that of Method precision (Repeatability) by a different analyst, different column, different day and on different instrument.

1. Similarity factor of standard solution and check standard solution should be between 0.95 and 1.05.
2. Meet requirement of system suitability under system precision.
3. The % RSD for content of single maximum unknown impurity and total impurities should not be more than limit specified below as per result observed.,
Result observed        % RSD
≤ 0.01%                      20%
0.01% to 0.10%          10%
≥ 0.10%                        5%
4. The overall % RSD for content of single maximum unknown impurity, total impurities from method precision study and intermediate precision study together should not be more than limit specified below as per result observed.
Result observed        % RSD
≤ 0.01%                      20%
0.01% to 0.10%          10%
≥ 0.10%                        5%

5.25.3 Limit of Detection (LOD) and Limit of Quantitation (LOQ)

5.25.4 Linearity and Range

Experimental Design

Acceptance criteria

Perform the linearity of the standard from LOQ to 150% of limit level concentration (LOQ, 50%, 60%, 80%, 100%, 120%, 140%, 150% level).
Six replicates injection at LOQ and 150% level. At other levels in duplicate. Plot a graph of concentration against the average peak area. Calculate correlation coefficient, y-intercept, and % RSD of LOQ level and 150% level.

1. Similarity factor of standard solution and check standard solution should be between 0.95 and 1.05.
2. Meet requirement of system suitability under system precision.
3. Correlation coefficient should be ≥0.990.
4. The y-intercept should be ± 3
5. The % RSD of peak area should not be more than limit specified below.
Level                 % RSD
LOQ                   20%
150%                   5%

5.25.5 Robustness
5.25.5.1 Solution stability

Experimental Design

Acceptance criteria

Analyze sample solution and standard solution at interval between initial and predetermine time interval (e.g. 0, 12, 24,36,48,60 and 72 hrs) against freshly prepared standard solution.
Note:
1. Time interval can be reduced for sensitive drug substances.
2. If method specify solution stability no need to do solution stability.

1. Similarity factor of standard solution and check standard solution should be between 0.95 and 1.05.
2. Meet requirement of system suitability under system precision.
3. Overall RSD of Area of standard solution including initial replicate injections should not be more than 5.0%.
4. The overall RSD single maximum unknown impurity, total impurities at predetermine intervals should not be more than limit specified below as per result observed.
Result observed        % RSD
≤ 0.01%                     20%
0.01% to 0.10%         10%
≥ 0.10%                       5%

5.25.5.2 Variation in method parameter

Experimental Design

Acceptance criteria

Vary one parameter (as mention below) at a time and perform analysis of three samples.
1. Determine the % RSD for content of single maximum unknown impurity and total impurities.
2. Determine the overall % RSD for content of single maximum unknown impurity and total impurities along with method precision under precision study.
Parameter:
a. pH of mobile phase/Buffer (± 0.2)
b. Composition of Mobile phase (± 5%)
c. Column temperature(± 5°C)
d. Detection wavelength(± 2nm)
e. Flow rate (±10%)
f. Different type and make of column

1. Similarity factor of standard solution and check standard solution should be between 0.95 and 1.05.
2. Meet requirement of system suitability under system precision.
3. The overall RSD for content of known impurities, single maximum unknown impurity, total impurities with method precision should not be more than limit specified below as per result observed.
Result observed        % RSD
≤ 0.01%                      20%
0.01% to 0.10%          10%
≥ 0.10%                       5%

5.26 CHROMOATOGRAPHIC PURITY/RELATED SUBSTANCES (BY TLC) FOR DRUG PRODUCT.

5.26.1 Specificity

Experimental Design	Acceptance criteria
Spot diluent, placebo, individual impurity and drug substance.	There should be no interference of diluent and placebo at the Rf value of known impurity and drug substance.All impurities should be well separated from drug substance.

5.26.2 Precision
5.26.2.1 System Precision (System suitability)

Experimental Design	Acceptance criteria
Spot diluent, placebo system suitability solution (If applicable) and determine system suitability.	Meet system suitability parameters.

5.26.2.2 Method precision (Repeatability)

Experimental Design	Acceptance criteria
Perform analysis of six samples spiked with all known impurities at limit level concentration. Also perform analysis of unspiked sample. Compare intensities of impurity spots visually.	1. The intensities of all impurities of same Rf value in six samples should be comparable. 2. The % RSD of Rf value of six samples solution should not be more than 10%.

5.26.2.3 Intermediate precision (Ruggedness)

Experimental Design	Acceptance criteria
Perform same exercise as that of Method precision (Repeatability) by a different analyst, different day.	1. The intensities of all impurities of same Rf value in six samples should be comparable. 2. The RSD of Rf value of six samples solution should not be more than 10% 3. The overall RSD of Rf value along with method precision study should not be more than 10%.

5.26.3 Limit of Detection (LOD)

Experimental Design	Acceptance criteria
Prepare solutions of different concentration of impurity and test solution. Check the lowest detection level where the spot clearly visible.	The concentration is acceptable as LOD, if the spot is clearly visible.

5.26.4 Robustness- Solution stability

Experimental Design	Acceptance criteria
Spot individual impurity solution and drug solution at predetermine time interval (e.g. 0, 4, 8, 12, 16, 20 and 24 hrs) Note:. 1. Time interval can be reduced for sensitive drug substances. 2. If solution stability is specified in method, no need to perform solution stability.	The intensities of all impurities of same Rf values should be comparable.

5.27 RESIDUAL SOLVENT (BY GC WITH HEAD SPACE)-FOR DRUG PROCUCT

5.27.1 Specificity

5.27.2 Precision
5.27.2.1 System precision (System suitability)

Experimental Design	Acceptance criteria
Perform six replicate injection of standard mixture solution at limit level concentration.	1. The resolution between two successive peaks should be not less than 1.0. 2. The RSD of peak area of each solvent should not be more than 15%. 3. The RSD of retention time of each solvent should not be more than 2%.

5.27.2.2 Method precision (Repeatability)

5.27.2.3 Intermediate precision(Ruggedness)

Experimental Design	Acceptance criteria
Perform same exercise as that of Method precision (Repeatability) by a different analyst, different column, different day and on different instrument.	1. The similarity factor between standard and check standard should be 0.85 to 1.15. 2. Meet requirement of system suitability under system precision. 3. The relative standard deviation for content of each solvent should not more than 15.0%. 4. The overall RSD for content of each solvent from method precision study and intermediate precision study together should be not more than 15%.

5.27.3 Limit of Detection (LOD) and Limit of Quantitation (LOQ)

5.27.4 Linearity and Range

Experimental Design	Acceptance criteria
Perform the linearity of the standard from LOQ to 150% of limit level concentration (LOQ, 50%, 60%, 80%, 100%, 120%, 140%, 150% level). Six replicates injection at LOQ and 150% level. At other levels in duplicate. Plot a graph of concentration against the average peak area. Calculate correlation coefficient, y-intercept and % RSD of LOQ level and 150% level.	1. The similarity factor between standard and check standard should be 0.85 to 1.15. 2. Meet requirement of system suitability under system precision. 3. Correlation coefficient should be ≥0.990. 4. The y-intercept should be ± 3. 5. The % RSD of peak area should not be more than limit specified below. Level % RSD LOQ 20% 150% 15%

5.27.5 Accuracy

Experimental Design	Acceptance criteria
Prepare triplicate samples of drug product spiked with all solvents at LOQ, 50%, 100% and 150% of limit level concentration. Determine the %recovery for each solvent.	1. The similarity factor between standard and check standard should be 0.85 to 1.15. 2. Meet requirement of system suitability under system precision. 3. % Recovery should be within below specified limit. Level % Recovery LOQ 80% to 120% 50%,100%,150% 85% to 115%

5.27.6 Robustness

Experimental Design

Acceptance criteria

Vary one parameter (as mention below) at a time and perform analysis of three samples spiked with all solvent at limit level concentration. Also perform analysis of unspiked sample. Determine the % RSD for content of each solvent.
Determine the overall % RSD for content of each solvent along with method precision under precision study.
Parameter:
1. Initial column temperature (±2°C)
2. Oven temperature of headspace sampler (±2°C)
3. Flow rate (± 10%)
4. Injector temperature (±2°C)
5. Transfer line temperature of headspace sampler (±2°C)
6. Needle temperature of headspace sampler (±2°C) Parameter for variation to be decided by department head /designee.

1. The RSD for content of each solvent should be not more than 15%.
2. The RSD of retention time of each solvent should not be more than 2%.
3. The overall RSD for content of each solvent from method precision study and intermediate precision study together should be not more than 15%.

5.28 CLEANING VALIDATION (FOR DRUG PRODUCTS)

5.28.1 Specificity

Experimental Design

Acceptance criteria

Check interference of analytical diluent, placebo, cleaning agent and swab.
1. Cleaning agent solution: Prepare 1ppm solution of cleaning agent in analytical diluent if cleaning agent is different than analytical reagent.
2. Swab solution: Wet the swab with diluent. Rub on the clean SS surface as per procedure and transfer into a volumetric flask. Add diluent and swirl.
3. Standard solution: Prepare as per method.

Preferably there should be no interference of diluent, placebo, cleaning agent and swab.
If observed it should be less than limit of detection of standard.

5.28.2 Precision
5.28.2.1 System suitability (System suitability)

Experimental Design	Acceptance criteria
For HPLC/GC: 1. Inject system suitability solution as applicable and determine system suitability. 2. Perform six replicate injection of standard solution at working level.	1. Parameters such as resolution, column efficiency, tailing factor should be determines. 2. The RSD for peak area should not be more than 5.0%.
For UV: Check six replicate absorbance of standard solution at working level.	The RSD for absorbance should not be more than 2.0%.

5.28.2.2 Method precision (Repeatability)

5.28.2.3 Intermediate precision (Ruggedness)

Experimental Design

Acceptance criteria

Perform same exercise as that of Method precision (Repeatability) by a different analyst, different column, different day and on different instrument.

5.28.3 Limit of Detection (LOD) and Limit of Quantitation (LOQ)

5.28.4 Linearity and Range

Experimental Design

Acceptance criteria

Perform the linearity of the standard from LOQ to 150% of working level concentration (LOQ, 50%, 60%, 80%, 100%, 120%, 140%, 150% level).
Six replicates injection/absorbance at LOQ and 150% level. At other levels in duplicate. Plot a graph of concentration against the average peak area/absorbance.
Calculate correlation coefficient, y-intercept, and slope, residual sum of squares and % RSD of LOQ level and 150% level.

1. The similarity factor between standard and check standard should be 0.95 to 1.05.
2. Meet requirement of system suitability under system precision
3. Correlation coefficient should be ≥0.990.
4. The y-intercept should be ± 3
5. The % RSD of peak area should not be more than limit specified below.
Level                 % RSD
LOQ                   20%
150%                   5%

5.28.5 Accuracy

Experimental Design

Acceptance criteria

Carry out recovery at LOQ, 50%, 150% of working level concentration in triplicate and 100% of working level concentration in six replicates. Apply known quantity of standard stock solution of drug substance in presence of placebo and apply onto a clean stainless steel cleaning validation pan which has an area of about 100 cm². The solution will be left to dry at room temperature. Wet a swab by dipping it into swabbing solvent (usually IPA or water) and squeeze out the excess solvent. Wipe the pan twice using the above wet swab (use one side of the swab to wipe the whole area in one direction, and then use the other side of the swab to wipe the whole area in a perpendicular direction). Transfer this swab into volumetric flask. Repeat the above procedure using three other swabs. Put all wiped swabs into the same volumetric flask. Add known amount of diluting solvent to volumetric flask, swirl and carry out determination.

1. The similarity factor between standard and check standard should be 0.95 to 1.05.
2. Meet requirement of system suitability under system precision% Recovery should be within below specified limit. Level % Recovery LOQ 80% to 120% 50%,100%, 85% to 115% & 150%

5.29 VERIFICATION OF STANDARD AND COMPENDIAL METHODS

Standard and compendial methods are not required to validate but merely verify their suitability under actual conditions of use and to ensure that the laboratory is capable of performing the analysis.
Verification of an analytical procedure is the demonstration that a laboratory is capable of replicating with an acceptable level of performance a standard method.
Method validation/ verification of the respective API is received from Vendor.
Method verification is performed for finished product as per IP/BP/USP with selected parameter of verification.
Verification under condition of use is demonstrated by meeting the system suitability specification; established for the method, as well as demonstration of Accuracy and precision or other method parameters for the type of method as mentioned below;

Categories	Parameter to be performed
Category I -Analytical method verification for quantitation of major components of bulk drug substances and finished drug products.	Precision, specificity, linearity, Accuracy
Category II -Analytical method verification for determination of impurities in bulk drug substances or in finished drug products.	Precision, specificity, linearity, LOD and LOQ, Accuracy
Category III -Analytical method verification for determination of performance characteristics (e.g. dissolution, drug release).	Precision, specificity, linearity, accuracy.
Category IV -Identification tests.	Specificity.

Acceptance criteria of method verification are same as per analytical method validation.

5.30 Numbering system for method validation and method verification is as follows;

AMVP/XX/YY/ZZ for protocol
AMVR/XX/YY/ZZ for report
Where,
- AMVP = for Analytical method validation/ method verification protocol
- AMVR = for Analytical method validation/ method verification report
- XX = for test which is validated
  - i.e. A = for Assay
  - RS = for Related substances/Chromatographic purity
  - D = for Dissolution
  - UOC =Uniformity of content
  - I = for Identification
  - OVI = Residual solvent
  - CV = Cleaning validation
- YY = for serial number
- ZZ = for year in which the method is validated

6.0 ABBREVIATIONS:

Abbreviation	Expanded form
SOP	Standard Operating Procedure
No.	Number
Q.C.	Quality Control
QA	Quality Assurance
ADL	Analytical development laboratory
Dept	Department
No.	Number
HPLC	High performance liquid chromatography
UV	Ultraviolet and visible spectrophotometer
GC	Gas chromatography
API	Active pharmaceutical ingredient
RSD	Relative standard deviation
ppm	Part per million
STEYX	The standard error of the predicted y-value for each x in the regression
LOD	Limit of detection
LOQ	Limit of quantitation