To provide a procedure for the validation of analytical methods under use
I. PURPOSE :
To provide a procedure for the validation of analytical methods under use
II. SCOPE :
The procedure is to guide the analytical method validation studies of all the analytical methods being developed and used for the analysis of different products. The procedure also provides the steps involved in the qualification of the compendial methods.
III . RESPONSIBILITY :
A. Quality Control shall :
i. Prepare, Review and approve the method validation protocol with an assigned protocol number.
ii. Carry out analysis & provide supporting data with a conclusive report on method validation carried out.
B. Quality assurance shall :
i. Review and approve the method validation protocol.
ii. Should authorized any documents being used during the validation study
ii. Review and approve the method validation report
IV.DEFINITIONS :
Method : A set of all the written procedures and instructions involved in the collection, processing, storage and analysis of chemical matrix for an analyte.
System Suitability : Specific tests to ascertain the suitability and effectiveness of the operating system when employing chromatographic methods such as pressurized liquid chromatography and gas chromatography.
Validation: The obtaining and documenting of evidence to demonstrate that a method can be relied upon to produce the intended result under stated conditions within defined limits. It is the process of establishing that the performance characteristics of a method (expressed in terms of analytical parameters) to meet the requirements for the intended application of the method. The normal analytical parameters used in method validation are defined as follows .
Protocol: A pre approved document ,which should define the scope, objective, responsibilities, method details , experimental design and acceptance criteria with consideration to the intended application of the study is being performed.
Report: An approved document ,which should explain the details found during the study and also should be concluded the status of the study against to the predetermined acceptance criteria
Specificity: The ability of a method to measure accurately and specifically the analyte (the constituent being tested or analysed) in the presence of components that may be expected to be present in the sample. It is a measure of the degree of interference in the analysis of complex sample mixtures such as blank, bulk drug substances containing
degradation products, related substances etc.
Precision: The degree of agreement among individual test results when a method is applied to multiple samplings of a homogeneous sample. It is a measure of either the degree of reproducibility (agreement under different conditions) or of repeatability (agreement under same conditions) of the method.
Limit of detection (LOD) : The lowest concentration of analyte in a sample that can be detected, but not necessarily quantitated under the stated experimental conditions. It is a parameter of limit tests.
Limit of Quantitation (LOQ) :: The lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions. It is a parameter of quantitative determinations of low levels of compounds in sample mixtures, such as impurities in bulk drugs .
Linearity: The ability of a method to produce results that are directly or indirectly proportional to the concentration of the analyte in samples within a given range.
Range : The interval between the upper and lower levels of analyte (including these levels) that have been shown to be determined with precision, accuracy, and linearity using the method as written..
Accuracy: The closeness of test results obtained by a method to the true value. It is a measure of the exactness of the method.
Ruggedness ( Intermediate precision ) : The Intermediate precision of an analytical method is the degree of reproducibility of test results obtained by the analysis of the same samples under a variety of normal test conditions ( with the combination of different analyst, different instrument, different lots of reagents, different day at normal laboratory conditions etc.) Intermediate precision is normally expressed as the lack of influence on test results of operational and environmental variables of the analytical method.
Robustness : The robustness of an analytical procedure is a measure of its capacity to retain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.
V. PROCEDURE :
01. Before an analytical method is validated, the method must be completely developed and a detailed procedure shall be documented.
02. A method must be validated for intended use, employing an approved protocol.
03. Based on the analyte, the technique, a specific procedure shall be identified to assure the optimum operation of the system employed (as system suitability).
04. Validation studies shall be unique for each method, and shall be consistent with the methods purpose, concentration range to be tested, the sample and the technique used for analyte determination.
05. Validation experimental plans (protocols) shall be supported by appropriate statistical methodologies to assist in the interpretation of the results obtained.
06. Any modification of an analytical method shall be subject to revalidation.
07. Documentation of the successful completion of laboratory studies is a basic requirement for determining whether a method is suitable for its intended applications.
08. The procedure used to evaluate each validation parameter as described in the approved protocol will be focused into the following steps :
i. Identification of appropriate validation parameters.
ii. Design of experimental plan for parameters evaluation (validation protocol).
iii. Execution of the validation.
iv. Documentation to support of the conclusion that the method has been validated.
v. The documentation shall contain the following :
a. Approved protocol
b. Raw data – Analysis work books/test data sheet, Chromatograms,Charts, Thermogram, Histograms (if any) etc.,
c. Validation report with conclusion of the study.
09. The experimental design and acceptance criteria of validation parameters can be altered to other than the conditions specified in this SOP
VI. EXPERIMENTAL DESIGN:
A) For Inhouse methods :
1.1 Determine the category (see Table I ) of the test/s which are to be validated. Prepare a Method Validation Protocol in the specimen format given in the Annexure for each item. Indicate in the protocol the experimental design comprising the tests or methods to be validated, the relevant analytical performance parameters to be checked, on the basis of the category given in the Table, the experimental details for carrying out these checks and the acceptance criteria for each parameter. Guidelines for experimental designs for some commonly done tests and assays are given below
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Category of tests and selection of validation parameters for In-house methods
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Category of test |
Specifi- city |
Precision |
Linearity & range |
Accuracy |
Intermediate precision |
Robust-ness |
LOQ |
LOD |
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Assay by HPLC |
Yes |
Yes |
Yes |
No |
Yes |
Yes** |
No |
No |
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Chromatographic purity by HPLC
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Yes |
Yes |
Yes |
Yes |
Yes |
Yes** |
Yes |
Yes |
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OVI &Residual solvent test |
Yes |
Yes |
Yes |
Yes |
Yes |
Yes |
Yes |
Yes |
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Chromatographic purity (By TLC)
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Yes |
Yes |
No |
No |
Yes |
No |
No |
Yes |
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Assay Potentiometry
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Yes |
Yes |
Yes |
No |
Yes |
Yes |
No |
No |
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Particle size by laser diffraction |
No |
Yes |
No |
No |
Yes |
Yes |
No |
No |
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Rinse analysis in cleaning validation
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Yes |
Yes |
Yes |
Yes |
Yes |
No |
Yes |
Yes |
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Swab analysis in cleaning validation
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Yes |
Yes |
Yes |
Yes |
Yes |
No |
Yes |
Yes |
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** Carry out stability study for both mobile phase and test solution.
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B) For Pharmacopoeial methods :
1.2 Determine the category (see Table I ) of the test/s which are to be validated. Prepare a Method Validation Protocol in the specimen format given in the Annexure for each item. Indicate in the protocol the experimental design comprising the tests or methods to be validated, the relevant analytical performance parameters to be checked, on the basis of the category given in the Table, the experimental details for carrying out these checks and the acceptance criteria for each parameter.
1.3 Guidelines for experimental designs for some commonly done tests and assays are given below
Category of tests and selection of analytical parameters for Pharmacopoeial methods
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Category of test |
Specifi- city |
Precision |
Linearity & range |
Accuracy |
Intermediate precision |
Robust-ness |
LOQ |
LOD |
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Assay by HPLC |
Yes |
Yes |
No |
No |
No |
No** |
No |
No |
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Chromatographic purity by HPLC
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Yes |
Yes |
No |
Yes |
No |
No** |
Yes |
Yes |
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Chromatographic purity (By TLC)
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Yes |
Yes |
No |
No |
No |
No |
No |
Yes |
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Assay by Potentiometry
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Yes |
Yes |
Yes |
No |
Yes |
No |
No |
No |
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** Carry out stability study for both mobile phase and test solution.
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Experimental design for the method validation of Assay by HPLC
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Analytical parameters |
Experimental design |
Acceptance criteria |
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System suitability
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. 1. Establish the system suitability through out the validation followed by 2 procedures.
2.Procedure1: Establish the system suitability Whenever the chromatographic system changed or disturbed.
3. Procedure2: When there is no disturbance in chromatographic system , establish the online system suitability between each parameter by injecting the standard solution(single time) and calculate the %RSD for the values obtained from initial values and each online standard.
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As applicable (As per method details) 1 e.g. : For chromatographic methods suitable parameters such as theoretical plates, tailing factor must be determined.
2. As applicable (As per method details)
3. The cumulative RSD for the values obtained from initial (system suitability)values and the individual online standard solution should be less than 2.0%
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Experimental design for an assay by HPLC method validation
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Analytical parameters |
Experimental design |
Acceptance criteria |
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Specificity |
2. Separate injection of individual impurities for identification and assay in case of chromatographic method.
3. Compare the Relative retention times of each component obtained from individual solutions and blend solution. |
1. There should be no interference of the diluent or impurities & the RT of drug substance.
2.The impurities of impurities should not interfere at the retention time of main compound
3. The relative retention times (with respect to drug substance) of each impurity obtained from the blend solution should be comparable(within + 0.06) to the relative retention times obtained from the individual solutions of each impurity. |
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Precision
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3. Method precision of atleast 6 replicate assay dermination of a sample. Determine the %RSD of assays. |
1 System suitability as applicable
2 The % RSD for peak area response of replicate standards should not be more than 2.0 %
3 % RSD of assay should not be more than 2.0 %
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Experimental design for the method validation of Assay by HPLC
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Analytical parameters |
Experimental design |
Acceptance criteria |
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Linearity & range |
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100 x y intercept = ------------------------------------- Response of assay level
2. The % RSD for peak area response of three replicates of each level should not be more than 2.0 % .
3. The % RSD for peak area responses of five replicates of first & last level should not be more than 2.0 %
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Experimental design for the method validation of Assay by HPLC
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Analytical parameters |
Experimental design |
Acceptance criteria |
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Ruggedness (Intermediate precision) |
Standard solution at assay level concentration. Determine the % RSD of peak area response & retention time.
3. Intermediate precision of Six replicate determination of assay of a sample (Which is assayed under precision study) with the combination of different analyst / day / instrument / different column of same make. Determine the %RSD of assays.
4. Determine the cumulative% RSD of assays of precision study and Intermediate precision study together. |
1.System suitability as applicable
2 . The % RSD for peak area response of replicate standards should not be more than 2.0 %
3 % RSD of assay should not be more than 2.0 %
4 Cumulative % RSD of assay of precision study &intermediate precision should not be more than 2.0 %
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Experimental design for the method validation of Assay by HPLC
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Analytical parameters |
Experimental design |
Acceptance criteria |
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Robustness |
a Mobile phase composition (higher concentration by 2 % or lower concentration by 5-10 %)
b pH of mobile phase (± 0.2 )
c Column oven temperature (± 2.0 °C)
d Mobile phase flow rate
3. Method precision of three replicate determination of assay of a sample (Which is analysed under precision study) under each individual condition of the robustness study . Determine the %RSD of assays.
4.Determine the cumulative % RSD of Assay of Precision study & Robustness study together. |
1. Select as per the applicability of the method
2. System suitability as applicable. ( The % RSD for peak area response of replicate standards should not be more than 2.0 %
3 % RSD of assay should not be more than 2.0 %
4. The cumulative % RSD of assay of precision study along with the results of Robustness study should not be more than 2.0 %
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Experimental design for the method validation of Assay by HPLC
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Analytical parameters |
Experimental design |
Acceptance criteria |
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Solution stability |
calculate the assay of each preparation and note down the result as initial assay and store one sample is on bench top and another one is in refrigerator simultaneously for about 24 hours. After the specified time interval evaluate the assay of each sample using the same chromatographic conditions(same mobile phase, column &system) with freshly prepared standard solution. Also monitor the pattern of chromatogram of sample solution
The time intervals can be reduced and altered for sensitive drug substances .
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1 System suitability as applicable.
2. a. The % variation between the initial results and the results of solution stability(individually) should be less than 1.0%
b .If any extraneous peak of impurities or degradants are observed in chromatogram of sample than that of initial pattern consider the solution is not stable.
3. Report the solution stability in hours (solution stability is the last time interval of the test solution is found stable)
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Experimental design for the method validation of Assay by HPLC
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Analytical parameters |
Experimental design |
Acceptance criteria |
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Mobilephase stability |
of Standard solution at assay level concentration.
4. Method precision of three replicate determination of assay of a sample (Which is assayed under precision study) using each storage condition of mobile phase. Determine the %RSD of assays.
5. Determine the cumulative% RSD of assays of each condition of mobile phase stability study and precision together. |
1. Establish the mobile phase stability for 2 to 7 days at each condition
2. System suitability as applicable
3 .The % RSD for peak area response of replicate standards should not be more than 2.0 %
4. % RSD of assay should not be more than 2.0 %
5. Cumulative % RSD of assay of precision study & each condition of the mobile phase stability study should not be more than 2.0 %
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Experimental design for the method validation of Assay Titremetry
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Analytical parameters |
Experimental design |
Acceptance criteria |
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Specificity |
Check the blank interference of diluent.
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The Burette reading value should be negligible ( Around 0.1 ml ). * Blank reading should be taken for the calculation of assay . |
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Precision |
Method precision of at least 6 replicate assay determination of a sample. Determine the % RSD of assays. |
% RSD of assay should not be more than 2.0 %
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Linearity & range |
Determination of minimum 6 levels of standard in the range of at least 50 to 150% of the assay level concentration including assay level concentration. Carry out linearity for first & last level in six replicates & other levels in triplicate and take mean burette reading for calculation. Determine linearity co-efficient and % y intercept.
Determine the precision of assay content of lower (1st level) and higher level (last level) in six replicates. |
Linearity co-efficient of mean burette reading of replicate determination plotted against respective concentration should not be less than 0.999.The %y-intercept as obtained from the linearity data (without extrapolation through origin 0,0) should be within ± 2.0 % determined using the formula : 100 x y intercept = ------------------------------------- B.R. of assay level The % RSD for % assay of replicate (1st level) and higher level (last level) should not be more than 2.0 %.
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Ruggedness (Intermediate precision) |
Reproducibility of Six replicate determination of assay of a sample (Which is assayed under precision study) by a different analyst / day at normal laboratory condition / instrument (if available)Determine the cumulative % RSD of assays of precision study and Intermediate precision study together. |
1 System suitability as per method
2 % RSD of assay should not be more than 2.0 %
3 Cumulative % RSD of assay of precision study & intermediate precision should not be more than 2.0 % |
Experimental design for the method validation of chromatographic purity/
Related substances by HPLC
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Analytical parameters |
Experimental design |
Acceptance criteria |
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System suitability
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. 1. Establish the system suitability through out the validation followed by 2 procedures.
2.Procedure1:Establish the system suitability when ever the chromatographic system changed or disturbed.
3. Procedure2: When there is no disturbance in chromatographic system , establish the online system suitability between each parameter and observe the compliance of system suitability.
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1. As applicable (As per method details) e.g. : For chromatographic methods suitable parameters such as resolution, RRT , Peak to valley ratio must be determined. If not specified keep following criteria .
2. As applicable (As per method details)
3. Should complies the system suitability |
Experimental design for the method validation of chromatographic purity/
Related substances by HPLC
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Analytical parameters |
Experimental design |
Acceptance criteria |
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Specificity
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Analytical method validation, validation
