Sop for Analytical method validation

This Document describes (Standard Operating procedure) Sop for Analytical method validation

Sop for Analytical method validation.

I. Purpose & Scope:

• The Purpose of this SOP is to lay down the procedure for Analytical method validation.      
• This SOP is applicable for qualitative and quantitative analytical methods which are used to test finished products, in-process product and raw materials in support of registration documentation and cleaning methods.

II. Responsibilities:

• All Quality Control and Quality Assurance personnel shall be responsible to follow and implement this SOP.

III : Introduction and Procedural Part :

Materials And Equipment:

• Analytical instruments
• Compendial / non-compendial reference standards, working standards & impurity standards etc.

Definition’s:

Analytical method validation:

• Analytical method validation is the process of demonstrating that analytical procedures are suitable for their intended use.

Specificity:

• The specificity of analytical method is the ability to access unequivocally the analyte in the presence of components that may be expected to be present, such as impurities, degradation products and matrix components.

Linearity:

• 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.

Range:

• The range of the analytical method is the interval between upper and lower concentration of the analyte in the sample for which it has been demonstrated to be determined with a suitable level of precision, accuracy and linearity using the method as written.

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. OR 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.

Precision:

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

Precision – Repeatability:

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

Precision – Intermediate precision:

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

Limit of Detection:

• The detection limit of an individual analytical method 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 method is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.

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.

System Suitability Test:

• Set of parameters and criteria thereof to ensure the system is working properly.

Acceptance Criteria:

• Numerical limits, ranges, or other suitable measures for acceptance of analytical results.

Placebo:

• A dosage form that is identical to the drug product except the drug substance it may be replaced by inert ingredient.

Spiking:

• The addition of a small amount of a known compound to a standard, sample or placebo, typically for the purpose of confirming the performance of an analytical procedure.

Solution stability :

• Solution stability is to analyze the solution following the method and then and store it under the required conditions. It is re-analyzed after a specified period of time.

 Frequency of Analytical Method Validation:

• At the release of the new formulations/ projects developed by R&D.
• Any change in the Compendial methods, the suitability of the method will be verified.
• Any change in formulation, the method will be reviewed and assessed for the validation.

Numbering system for Analytical method validation/Verification:

• Number for Analytical method verification protocol shall be given as MVE/2019/01
• MVE stands for Method verification
• 2019 stands for year of Method verification
• 01 stands for serial number of the year
• Number for Analytical method validation shall be given as AMV/2019/01
• AMV stands for Analytical Method validation
• 2019 stands for year of Analytical Method validation
• 01 stands for serial number of the year.

Data elements required for validation:

• Compendial test requirements vary from highly exacting analytical determinations to subjective evaluations of attributes. Considering this broad variety, it is only logical that different test procedures requires different validation schemes. This chapter covers only the most common categories of tests for which validation data should be required. These categories as follows.
• Category I – Analytical procedures for quantitation of major components of bulk drug substances or active ingredients in finished pharmaceutical products.
• Category II – Analytical procedures for determinations of impurities in Bulk drug substances or degradation compounds in finished pharmaceutical products. These product includes quantitative assays and limit tests.
• Category III – Analytical procedures for determination of performance characteristics (e.g. Dissolution, drug release etc.)
• Category IV – Identification tests
• For each category, different analytical information is needed, Refer Annexure I. Data elements that are normally required for each of these categories.

Special conditions:

• Revalidation of the analytical method may be considered either of following conditions.
• Changes in the analytical procedures (Compendial or manufacturers reference).
• Change in the critical analytical instruments
• Change in sampling procedure.
• If the analytical method validation is submitted by the marketing authorization holder then only accuracy & precision repeatability and intermediate precision will be carried out.
• Compendial method verification shall be include
• Specificity
• Precision
• Accuracy
• Limit of detection (LOD) (Applicable for RS test)
• Limit of Quantitation (LOQ) (Applicable for RS test)
• Recovery of impurities (If applicable)
• Solution stability (If applicable)
• Above mentioned parameters of method verification will be vary depends upon applicability of the method
• Non Compendial (in house) method validation shall be include
• Specificity
• Precision
• Accuracy
• Linearity and Range
• Limit of detection (LOD)
• Limit of Quantitation (LOQ)
• Robustness
• Recovery of impurities
• Solution stability
• Forced degradation i.e. Acid Hydrolysis, Base Hydrolysis, Oxidation, light exposure and Heat degradation etc.
• Above mentioned parameters of analytical Method validation will be vary depends on applicability of the method
• Analytical Method Validation/ Verification protocol shall be prepared with respective of following parameters for the Identification tests, Quantitative tests for impurities, Limit tests for the control of impurities and Quantitative tests of the active drug substance of the selected component.
• Specificity
• Precision
• Repeatability/ System precision
• Intermediate Precision
• Reproducibility/ Method precision
• Accuracy
• Linearity and range
• Limit of Detection (LOD) shall be determine by any of the method as described below
• Visual Evaluation
• Signal to noise ratio
• Standard deviation of the response and the slope
• Standard deviation of the base
• Calibration curve
• Limit of Quantitation (LOQ) shall be determine by any of the method as described below
• Visual Evaluation
• Signal to noise ratio
• Standard deviation of the response and the slope
• Standard deviation of the base
• Calibration curve
• Robustness
• Recovery of impurities
• Solution stability
• System suitability testing
• Identification tests will ensure the identity of an analyte in a sample with respect to an authentic reference standard.
• For impurities either a quantitative test or a limit test will be implied for the impurity in a sample.
• Procedures will incorporate the measure major component(s) in the analyte given in the sample.

Procedure for Specificity:

• Prepare the placebo and treat it exactly as per sample. Analyze and record the observations.
• For HPLC method separately prepare sample solution and check the peak purity of analyte peak using PDA detector.
• If possible spike the sample with possible interfering substance like impurities and or degradation products and analyze the spiked and unspiked for percent agreement.
• Acceptance Criteria: There should not be any interference from placebo i.e. analyte peak should be pure when checked for peak purity test.

Procedure for Linearity:

• Prepare 3 different concentration of standard (equally spaced throughout the given interval and spanning the intended operating concentration of the assay method).
• Measure the response of each solution
• Plot a linearity curve and calculate the correlation coefficient.

Acceptance Criteria:

The correlation coefficient should be equal to or greater 0.99 for chemical method.

Procedure for Range:

• The range of the method will be validated by verifying the acceptable precision accuracy and linearity when applied to samples containing analyte at the extremes of the range as well as within the range.
• Determine the usable range for the method from accuracy, precision and linearity studies representing the formulation containing approximately 80% to 120% respectively of the nominal amount of the analyte. In some product 50% to 150 % shall be applied based on applicability of analytical method.

Procedure for Accuracy:

• Prepare 3 sets of samples having varying concentrations of analyte by spiking the placebo. 80%, 100%, 120% of label claim, Minimum three levels to the label claim to be performed. (In some product 50%, 100 % and 150 % shall be applied based on applicability of analytical method.
• Analyze all the above samples using proposed method.
• Calculate the percentage recovery for individual concentration and mean percentage recovery. Also calculate SD and % RSD.

Accepted criteria for Assay method :

• Acceptance criteria may differ from method to method and test to test but normally accepted criteria for assay method are as follows:
• Percentage recovery: should be Between 98.0% to 102.0%
• Percentage RSD: Individual Level and all Level Should be not more than 2.0%

Acceptance Criteria for Dissolution Method :

Accepted criteria for Dissolution method are as follows:

• % Recovery (individual) and % mean recovery (For less than 70% Release) should be between 90.0% and 110.0%
• % Recovery (individual) and % mean recovery (For more than 70%  Release) should be between 95.0% and 105.0%
• % RSD (individual level) and %RSD all level should be Not More than 5.0%

Acceptance Criteria for Related Substances:

Accepted criteria for Related Substances method are as follows:

% level	% Recovery	% RSD
LOQ	70.0 %-130.0%	≤15.0%
≥0.10 % to 0.50 %	80.0 %-120.0%	≤10.0%
>0.50 % to 1.0 %	85.0 %-115.0%	≤7.5%
More than 1.0%	90.0 %-110.0%	≤5.0%

Procedure for Precision:

• Determine the system precision for the method by analyzing 6 replicate of the same aliquot of the standard solution and calculate the mean, Standard Deviation (SD) and Relative Standard Deviation (RSD) of the response obtained.
• For method precision collect the homogenous sample.
• Analyze separately the six sample preparations and calculate the results
• Calculate the Mean, SD and % RSD.

Procedure for Limit of Detection:

• Prepare 3 different concentrations of standard which are in the lowest quarter of the linearity curve.
• Inject each standard solution six times and record the response and calculate the SD.
• Plot the linearity curve and calculate slope and correlation coefficient. Calculate average SD of three solutions. Calculate the system noise as follows :
• Noise = Average SD / Slope
• Alternatively calculate signal to noise ratio by injecting blank for HPLC methods.
• Prepare a standard solution having concentration of 3 x noise factor Analyze in duplicate and check for detection.

Acceptance Criteria:

The detection limit is signal-to-noise ratio 2:1 or 3:1

Procedure for Limit of Quantification:

• Prepare a standard solution having concentration of 10 x noise factor.
• Inject each standard solution 6 times and record the response and calculate Relative Standard Deviation.

Acceptance Criteria:

% RSD should not be more than 10.0%.

Procedure for Robustness:

• Repeat the precision check procedure using same sample but under different conditions.
• For Assay by HPLC test robustness parameter shall be modified as Change in flow rate, change in Buffer pH, Change in wavelength, column temperature and change in mobile phase composition. Any three parameter shall be performed. For UV method any one parameter shall be followed.
• For Related substances by HPLC test robustness parameter shall be modified as Change in flow rate, change in Buffer pH, Change in wavelength, column temperature and change in mobile phase composition. Any three parameter shall be performed
• For Dissolution by HPLC test robustness parameter shall be modified as Change in flow rate, change in Buffer pH, Change in wavelength, column temperature, Change in RPM, Change in Dissolution medium Temperature and change in mobile phase composition. Any three parameters shall be performed. For UV method any three parameter shall be followed.
• Prepare the solutions of analyte and check the response at different time intervals. Calculate the % RSD.

Acceptance Criteria:

% RSD should be less than 2.0% and difference between the average of original test and repeat test shall be insignificant.

Procedure for Force Degradation:

• Force Degradation is also known as Stress testing and drug is degraded forcefully by applying artificial methods. It is useful tool to predict the stability of any Active Pharmaceutical Ingredient (API) or Formulation product. It helps to know about the impurities developed during the storage of drug products in various Conditions.
• Force Degradation test shall be treated for Test Sample and Placebo.

Conditions for Forced Degradation Studies:

 Hydrolytic Degradation in Acidic condition:

• Hydrolysis is Common   Degradation Process by the reaction of Chemicals with drug Reacted at Acidic Condition.
• 0.1 M to 1.0 M Hydrochloric Acid or Sulphuric acid shall be used to create the Acidic Condition. The concentration of acid shall be varied based on degradation pattern or if not meets the predefined acceptance criteria.
• The Analysis shall be done as soon as Possible after termination of degradation process.

Hydrolytic Degradation in Basic condition:

• Hydrolysis is Common Degradation Process by the reaction of Chemicals with drug Reacted at Basic Condition.
• 0.1 M to 1.0 M Sodium Hydroxide or Potassium Hydroxide shall be used to create the Basic Condition. The concentration of base shall be varied based on degradation pattern or if not meets the predefined acceptance criteria.
• The Analysis shall be done as soon as Possible after termination of degradation process.

Oxidation Degradation:

• H₂O₂ (Hydrogen Peroxide) shall be widely used   oxidizing agent. 0.1% to 3.0%   Solution of hydrogen peroxide applied for the treated of Test Sample and Placebo. The concentration of oxidize substance shall be varied based on degradation pattern or if not meets the predefined acceptance criteria.
• The Analysis shall be done as soon as Possible after termination of degradation process.

 Thermal Degradation:

The Temperature effect of the drug shall be done on 50°C to 105°C for 24 hours kept on oven in open dish. The temperature value shall be selected based on the melting point or range of active material. This value shall be referred from literature available.

 Photolytic Degradation:

• Photolytic degradation of any drug shall be done to determine the effect of light on the product during its storage. The light exposure time for LUX is should not be less than 1.2 million LUX hours. The light exposure time for UV light intensity should not be less than 200 watt-hours per sq meter.
•  The forced degradation parameters shall be same for Related substances   test and Assay test.

 Acceptance Criteria

• Peak purity of principal peak as determined by PDA software in
• Degraded samples should pass. Mass Balance Should be Achieve.
• The mass balance shall be between 90.0% to 110.0%

Procedure for System suitability testing:

• The system suitability testing mainly depends upon the type of the test.
• For the chromatographic methods following factors shall be considered, it can be variable depends of analytical method
• Tailing factor
• Relative retention
• Resolution
• Relative Standard Deviation
• Capacity factor
• Number of Theoretical plates
• K prime
• It will be checked before start of run and to be verified after new name.
• The procedure to be verified as per described in respective Pharmacopoeia.

• Note: For analytical method validation of compendial method it is not necessary to study robustness as this parameter is well considered during the standardization of a procedure before it is submitted to Pharmacopoeia.

IV: Annexure:

Annexure I: Data Elements Required for Analytical Method Validation

 

Annexure –I

Data Elements Required for Analytical Method Validation

 

Analytical performance / Characteristics	Category I	Category II		Category III	Category IV
		Quantitative	Limit tests
Accuracy	Yes	Yes	*	*	No
Precision-repeatability	Yes	Yes	No	Yes	No
Precision – Intermediate precision	Yes	Yes	No	Yes	No
Specificity	Yes	Yes	Yes	*	Yes
Detection Limit	No	No	Yes	*	No
Quantitation Limit	No	Yes	No	*	No
Linearity	Yes	Yes	No	*	No
Range	Yes	Yes	*	*	No

 

*= May be required depending on the nature of the specific test.