Analytical Method Validation
1.0 What
This document details the procedure for the Analytical Method Validation.
2.0 WHY
It is the policy of your Company that the written procedure shall be followed for the validation of analytical method used for the analysis for the consistency of the method and must meet standards of accuracy and reliability of test protocols with predefined norms.
3.0 WHEN
This analytical method validation is applicable at the time of
i) Introduction of New Product and validation data not provided by tie up company. (Other than Pharmacopoeial products).
ii) Addition of new Raw material (other than Pharmacopoeias product).
iii) Ruggedness of pharmacopoeias drugs and drug product (if required).
4.0 Responsibility
Following persons are responsible for preparation, operation and implementation of this procedure.
S. No | Designation | Responsibility |
01 | Executive – Corporate Quality Assurance | Prepare the SOP for analytical method validation and follow the SOP accordingly. |
02 | Manager – Quality Control | Conducting the validation activity and maintain the relevant record. |
03 | Manager – Quality Assurance | To ensure the implementation of SOP |
04 | Manager – Corporate Quality Assurance | To ensure the implementation of SOP |
5.0 PROCEDURE
5.1 Analytical Parameter (Definition and Procedure)
5.1.1 Linearity and Range
Definition of Linearity: Linearity of an analytical method is its ability to elicit test result that are directly or by a well defined mathematical transformation, proportional to the concentration of analyte in samples within a given range.
Definition of Range : The range of an analytical method is the interval between the upper and lower level of analyte that have been demonstrated to be determined with a suitable level of precision, accuracy and linearity using the method.
Procedure : The linearity should established the relationship between the response of an analyte and its concentration. For assay method linearity and range shall be done in the range of 80 to 120% (for content uniformity 80 to 120%) of standard / sample preparation.
For related substances linearity and range should be done in the range of limit of Quantification and 120% of specification limits. A minimum of 5 data points is required in the range as specified above.
5.1.2 Precision
System Precision: The system precision is the agreement of five or more replicates that are obtained under identical conditions using some test methods.
Method Precision: The precision of an analytical method is the degree of agreement among individual test result when the method is applied repeatedly to multiple sampling of a homogeneous sample.
Intermediate Precision: The Intermediate Precision of the method is established by estimating the assay of six different sample preparations of the same batch by different Chemist on a different system, different column and on a different day.
Procedure: The precision of an analytical method is by assaying a sufficient number of aliquots of a homogeneous sample to be able to calculate statistical estimates of standard deviation or relative standard deviation (coefficient of variation) of a series of measurement. Precision may be measured by the following three levels.
i)Repeatability or method precision : It refers to use of the analytical procedure within a laboratory over a short period time using the same analyst with of concern analytical method. The sample should be assessed using minimum of nine determinations covering the specified range for the procedure (i.e. three concentrations and three replicate of each concentration using minimum six determinations at 100 % of the test concentration).
ii) Intermediate precision or ruggedness: The precision method on different days by different analyst and using different instruments. The sample shall prepared and analysed as per the method, six times by two different analysts on different days and on different instruments.
iii) Reproducibility or system precision : Reproducibility refers to the use of the analytical procedure in different laboratories.
5.1.3 Accuracy
Definition: Accuracy of an analytical method is the closeness of test results obtained by that method to true values.
Procedure: For Active Pharmaceutical Ingredient (API), the accuracy is inferred from specificity, linearity, and precision data.
For drug product the accuracy shall be derived by performing, recovery experiments by spiking API in the placebo in triplicate at minimum five different concentrations of assay sample preparation. For Related Substances (API and Drug Product) the accuracy shall be shown by performing recovery experiment by spiking known impurities in the same in triplicate at minimum five different concentrations.
5.1.4 Specificity
Definition: The specificity is define as the ability to assess unequivocally the analyte in the presence of components that may be expected to be present such as impurities, degradation product and matrix components.
Procedure : Assay Method : In case of assay demonstration of specification requires that it can be shown that the procedure is unaffected by presence of impurities/related substances or excipient with the analyte peak. In practice this can be done by spiking the drugs substances with appropriate level of impurity (1%) or excipient in triplicate and demonstrating that the assay result is unaffected by the presence of these extraneous materials.
Related Substances method: This is done by spiking all known impurities at 1% level and showing the separation of all impurities from the main peak and from each other and demonstrating peak purity.
5.1.5 Limit of detection
Definition: It is the lowest amount of analyte in a sample that can be detected but not necessary to be quantified. Several approaches for determining the detection limit are possible, depending on whether the procedure is a non- instrumental. Approaches other than those listed below may be acceptable.
5.1.5.1 Based on Standard Deviation of the Response and the slope
For non-instrumental method the detection limit is generally determined by the analysis of sample with known concentration of analyte and by establishing the minimum level at which the analyte can be reliably detected.
In case of instrumental analytical procedures measure the magnitude of analytical back ground response by analysis number of analyte samples of different concentrations at detection level (1:3 signal of noise ratio) and calculating limit of detection by following method.
LOD = 3.3. x S.E.
Slope
Where S.E. (Standard Error) and slop are calculated by number of concentration at LOD level.
5.1.5.2 Based on Visual Evaluation
Visual evaluation may be used for non – instrumental methods but may also be used with instrumental methods.
The detection limit is determined by the analysis of the samples with unknown concentrations of analyte and by establishing the minimum level at which the analyte can be reliably detected.
5.1.5.3 Based on Signal to Noise
This approach can only be applied to analytical procedures which exhibit baseline noise.
Determination of the signal – to –noise ratio is performed by comparing measured signals from samples with known low concentrations of analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably detected. A signal – to – noise ratio between 3:1 is generally considered acceptable for estimating the detection limit.
5.1.5.4 Based on the Standard Deviation of the Blank
Measurement of the magnitude of analytical background response is performed by analyzing an appropriate number of blank samples and calculating the standard deviation of these responses.
5.1.5.5 Based on the Calibration Curve
A specific calibration curve should be studied using samples containing an nalyte in the range of detection limit. The residual standard deviation of a regression line or the standard deviation of y- intercepts of regression lines may be used as the standard deviation.
5.1.6 Limit of Quantification
Definition: It is the lowest amount of analyte in a sample at which that can be determined with acceptable precision and accuracy under the stated experimental conditions. 5 to 10% precision is acceptable.
5.1.6.1 Based on Standard Deviation of the Response and the slope
For non-instrumental method the detection limit is generally determined by the analysis of sample with known concentration of analyte and by establishing the minimum level at which the analyte can be reliably detected.
In case of instrumental analytical procedures measure the magnitude of analytical back ground response by analysis number of analyte samples of different concentrations at detection level (10:1 signal of noise ratio) and calculating limit of detection by following method.
LOD = 10.0 x S.E.
Slope
Where S.E. (Standard Error) and slop are calculated by number of concentration at LOD level.
5.1.6.2 Based on Visual Evaluation
Visual evaluation may be used for non – instrumental methods but may also be used with instrumental methods.
The detection limit is determined by the analysis of the samples with unknown concentrations of analyte and by establishing the minimum level at which the analyte can be reliably detected.
5.1.6.3 Based on Signal to Noise
This approach can only be applied to analytical procedures, which exhibit baseline noise.
Determination of the signal – to –noise ratio is performed by comparing measured signals from samples with known low concentrations of analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably detected. A signal – to – noise ratio between 10 :1 is generally considered acceptable for estimating the detection limit.
5.1.6.4 Based on the Standard Deviation of the Blank
Measurement of the magnitude of analytical background response is performed by analyzing an appropriate number of blank samples and calculating the standard devition of these responses.
5.1.6.5 Based on the Calibration Curve
A specific calibration curve should be studied using samples containing an nalyte in the range of detection limit. The residual standard deviation of a regression line or the standard deviation of y- intercepts of regression lines may be used as the standard deviation.
5.1.7 Robustness
Definition: It to show the ability of method to remain unaffected by small but deliberate variations in the method parameters.
Procedure: The robustness of method shall be done by following deliberately change in the method.
The stability of analyte in solution should be demonstrated to show the robustness of the method.
i) Change in flow rate ± 0.2 ml/min.
ii) Change in pH buffer/mobile phase by ± 0.2 unit.
iii) Change in organic content of mobile phase by ± 5%.
iv) Change in wavelength by ± 2 nm.
5.2 VALIDATION PLAN
5.2.1 Following analytical parameter shall be validated before implementation of analytical method.
i) Assay
ii) Determination of related substances.
iii) Dissolution Rate tests.
5.2.2 If the assay procedure for content uniformity is different from the composite assay the test method shall be validated.
5.2.3 For pharmacopoeial drugs and drugs products only robustness parameter shall be validated.
5.2.4 For non-pharmacopoeial drugs and drug product, the method shall be validated for the following parameters.
5.2.5 Raw Material
Assay
i) System Suitability
ii) Specificity
iii) Linearity & Range
iv) Precision - System precision
Method precision
Intermediate precision
v) Accuracy
vi) Robustness
Related substances
i) System Suitability
ii) Specificity
iii) Linearity & Range
iv) Limit of Detection
v) Limit of Quantification
vi) Precision - System precision
Method precision
Intermediate precision
vii) Accuracy
5.2.6 Finished Product
Assay of active material(s) and preservative (s) (if any)
iv) System Suitability
v) Specificity
vi) Linearity & Range
iv) Precision - System precision
Method precision
Intermediate precision
v) Accuracy
vi) Robustness
5.2.7 Dissolution Rate
i) System suitability ii) Specificity iii) linearity & range
iv) Precision - System precision
Method precision
Intermediate precision
v) Accuracy
vi) Robustness
5.3 Revalidation
A partial or complete revalidation of the analytical method shall be done according to following defined change and revalidation criteria.
CHANGE PARAMETER TO BE VALIDATED
i) Change in composition of drugs - Specifications
- Linearity
- Precision
ii) Revision in specification - Linearity
- Accuracy
iii) Change in Master Formula (Excipients) - Specifications
- Linearity - Precision
iv) Change in analytical Procedure - Complete validation
5.4 Acceptance Criteria
5.4.1 Acceptance criteria for HPLC analysis
The acceptance criteria for the analytical method validation will be followed:
Characteristics | No. of replicates to be Injected | Acceptance Criteria |
System Suitability | ||
Peak Symmetry | Standard X 6 replicates | NMT 2.0 |
Theoretical Plates | NLT 1500 | |
% RSD ( for assay and dissolution rate tests) | NMT 2.0 | |
% RSD (for Impurity Profile) | NMT 10.0 | |
Linearity | 5 Concentrations X 3 replicates | R2 > 0.99, similar response ratio |
Precision – System | Standard X 6 replicates | RSD NMT 2% |
Precision – Method | Standard X 6 replicates Sample X 6 replicates | RSD NMT 2% (For Assay) RSD NMT 10% (For RS) |
Precision – Intermediate | Standard X 6 replicates Sample X 6 replicates | RSD < 2% (For Assay) RSD < 15% (For Related Substances) |
Accuracy | Placebo X 1 injection 5 Standard X 3 replicates 5 spiked samples at different concentration X 3 replicates | 97 to 103 % (for Assay) 80 to 120 % (for RS) |
Characteristics | No. of replicates to be Injected | Acceptance Criteria |
Specificity | Blank X 3 replicates Placebo X 3 replicates Standard X 3 replicates Placebo spiked with analyte X 3 replicates | No interference |
Quantification | Standard X 6 replicates | |
Detection | Standard X 6 replicates | Signal to noise ratio 3:1 |
Range | 5 Concentrations X 3 replicates | Concentration where data can be reliably determine (97 to 103 % recovery) |
Robustness | For each parameter: Standard X 3 replicates Sample X 3 replicates | Overall RSD < 2% (for Assay) Overall RSD < 15% (for RS) |
5.4.2 Acceptance criteria for ICP-MS analysis
The acceptance criteria for the analytical method validation will be followed:
Characteristics | No. of replicates to be Injected | Acceptance Criteria |
System Suitability | ||
% RSD | Standard X 6 replicates | NMT 2.0 |
Limit of Detection (Intensity / Blank Response) | Standard X 6 replicates | |
Limit of Quantification (Intensity / Blank Response) | Standard X 6 replicates | |
Specificity | Blank X 3 replicates Standard X 3 replicates Sample X 3 replicates Sample + Satndard X 3 replicates | No Interference |
Precision | ||
System Precision | Standard X 6 replicates | RSD NMT 10.0% |
Method Precision | Standard calibration curve Sample X 6 replicates | RSD NMT 10.0% |
Intermediate Precision | Standard calibration curve Sample X 6 replicates | RSD NMT 15.0% |
Linearity | 5 Concentrations X 3 replicates | R2 <0.99, similar response ratio |
Range | 5 Concentrations X 3 replicates | |
Accuracy | Standard calibration curve 5 spiked samples at different concentration X 3 replicates | 80 to 120% |
Characteristics | No. of replicates to be Injected | Acceptance Criteria |
Dilution Integrity (%RSD & Accuracy) | Dilution 2 Times x 3 Replicates Dilution 4 Times x 3 Replicates | NMT 15.0 % % Accuracy – 85 – 115% |
Stability (%RSD & Accuracy) | Stability (for 0, 4, 6… hours) Standard x 3 replicates Sample x 3 replicates | NMT 10.0 % % Accuracy – 90 – 110% |
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