ANALYTICAL METHOD VALIDATION

  1.       Principle

1.1   This appendix presents some information on the characteristics that should be considered during validation of analytical methods. Approaches other than those specified in this appendix may be followed and may be acceptable. Manufacturers should choose the validation protocol and
procedures most suitable for testing of their product.

1.2   The manufacturer should demonstrate(Through validation) that the analytical procedure is suitable for its intended purpose.

1.3   Analytical methods, whether or not they indicate stability, should be validated.

1.4   The analytical method should be validated by research and development before being transferred to the quality control unit when appropriate.

       2.       General

2.1   There should be specifications for both, materials and products. The tests to be performed should be described in the documentation on standard test methods.

2.2   Specifications and standard test methods in pharmacopoeias (“pharmacopoeial methods”), or suitably developed specifications or test methods (“non-pharmacopoeial methods”) as approved by the national drug regulatory authority may be used.

2.3   Well-characterized reference materials, with documented purity, should be used in the validation study.

2.4   The most common analytical procedures include identification tests, assay of drug substances and pharmaceutical products, quantitative tests for content of impurities and limit tests for impurities. Other analytical procedures include dissolution testing and determination of particle size.

2.5   The results of analytical procedures should be reliable, accurate and reproducible. The characteristics that should be considered during validation of analytical method are discussed in paragraph 6.

2.6   Verification or revalidation should be performed when relevant, for example, when there are changes in the process for synthesis of the drug substance; changes in the composition of the finished product; changes in the analytical procedure; when analytical methods are transferred from one laboratory to another; or when major pieces of equipment instrument change.

2.7   The verification or degree of revalidation depend on the nature of the change(s).

2.8   There should be evidence that the analysts, who are responsible for certain tests, are appropriately qualified to perform those analyses (“analyst proficiency”).

      3.       Pharmacopoeial methods

3.1   When pharmcopoeial mentods are used, evidence should be available to prove that such methods are suitable for routine use in the laboratory (verification).

3.2   Pharmacopoeial method used for determination of content or impurities in pharmaceutical products should also have been demonstrated to be specific with respect to the substance under consideration (no placebo interference).

      4.       Non-pharmacopoeial methods

4.1   Non-pharmacopoeial methods should be appropriately validated.

      5.       Method validation

5.1   validation should be performed in accordance with the validation protocol. The protocol should include procedures and acceptance criteria for all characteristics. The results should be documented in the validation report.

5.2   Justification should be provided when non-pharmacopoeial methods are used if pharamcopoeial methods are available. Justification should include data such as comparisons with the pharamcopoeial or other methods are available.

5.3   Standard test methods should be described in detail and should provide sufficient information to allow properly trained analysts to perform the analysis in a reliable manner. As a minimum, the description should include the chromatographic conditions (in the case of chromatographic tests), reagents needed, reference standards, the formulae for the calculation of results and system suitability tests.

      6.       Characteristics of analytical procedures

6.1   Characteristics that should be considered during validation of analytical methods include:

-          Specificity

-          Linearity

-          Range

-          Accuracy

-          Precision

-          Detection limit

-          Quantitation limit

-          Robustness

6.1.1          Accuracy is the degree of agreement of test results with the true value, or the closeness  of the results obtained by the procedure to true value. It is normal established on  samples of the material to be examined that have been prepared to quantitative  accuracy. Accuracy should be established across the specified range of the analytical  procedure.

6.1.2          Precision is the degree of agreement among individual results. The complete procedure  should be applied repeatedly to separate, identical samples drawn from the same  homogeneous batch of material. It should be measured by the scatter of individual  results from the mean (good grouping) and expressed as the relative standard deviation (RSD).

6.1.2.1    Repeatability should be assessed using a minimum of nine determinations covering the specified range for the procedure e.g. three concentrations/ three replicates each, or a minimum of six determinations at 100% of the test concentration.

6.1.2.2    Intermediate precision expresses within-laboratory variations (usually on different days, different analysts and different equipment).

6.1.2.3    Reproducibility is assessed, a measure of intermediate precision is not required.

6.1.3          Robustness (or ruggedness) is the ability of the procedure to provide analytical results of  acceptable accuracy and precision under a variety of conditions. The results from  separate sample are influenced by changes in the operational or environmental  conditions. The results from separate samples are influenced by changes in the  operational or environmental conditions. Robustness should be considered during the  development phase, and should show the reliability of an analysis when deliberate  variations are made in method parameters.

6.1.3.1    Factors that can have an effect on robustness when performing chromatographic  analysis include;

-          Stability of test and standard sample and solutions;

-          Reagents (e.g. different suppliers);

-          Different columns (e.g. different lots and/or suppliers);

-          Extraction time;

-          Variations of pH of a mobile phase;

-          Variations in mobile phase composition;

-          Temperature; and

-          Flow rate.

6.1.4          Linearity indicates the ability to produce results that are directly proportional to the  concentration of the analyte in samples. A series of samples should be prepared in  which the analyte concentration span the claimed range of the procedure. If there is a  linear relationship, test results should be evaluated by appropriate statistical methods. A  minimum of five concentrations should be used.

6.1.5          Range is an expression of the lowest and highest levels of analyte that have been  demonstrated to be determinable for the product. The specified range is normally  derived from linearity studies.

6.1.6          Specificity (selectivity) is the ability to measure unequivocally the desired analyte in the  presence of components such as excipients and impurities that may also be expected to  be present. An investigation of specificity should be conducted during the validation of  identification tests, the determination of impurities and assay.

6.1.7          Detection limit (limit of detection) is the smallest quantity of an analyte that can be  detected, and not necessarily determined, in a quantitative fashion. Approaches may  include instrumental or non-instrumental procedures and could include those based on:

-          Visual evaluation;

-          Signal to noise ratio;

-          Standard deviation of the response and the slope;

-          Standard deviation of the blank; and

-          Calibration curve.

6.1.8          Quantitation limit (Limit of Quantitation) is the lowest concentration of an analyte in sample that may be determined with acceptable accuracy and precision. Approaches may include instrumental or non-instrumental procedures and could include those based on:

-          Visual evaluation;

-          Signal to noise ratio;

-          Standard deviation of the response and the slope;

-          Standard deviation of the blank; and

-          Calibration curve.

6.2   Characteristics (including tests) that should be considered when using different types of analytical procedures are summarized in Table 1.

Table 1 Characteristics to consider during analytical validation
Type of Analytical procedure	Identification	Testing for impurities	Testing for impurities	Assay - dissolution (measuremen only) - Content/potency
Characteristics		Quantitative test	Limit tests
Accuracy	-	+	-	+
Precision Repeatability Intermediate precision	- -	+ +	- -	+ +
Specificity	+	+	+	+
Detection limit	-	-b	+	-
Quantitation limit	-	+	-	-
Linearity	-	+	-	+
Range	-	+	-	+
- Characteristic is normally not evaluated; + Characteristic should normally be evaluated. -a In cases where a reproducibility study has been performed, intermediate precision is not needed. -b May be needed in some cases.

6.3   System suitability testing:

System suitability testing is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analysed constitute an integral system that can be evaluated as such. System suitability test parameters that need to be established for a particular procedure depend on the type of procedure being evaluated, for instance, a resolution test for an HPLC procedure.