WHY VALIDATE ANALYTICAL PROCEDURES ?
There are many reasons for the need to validate analytical procedures. Among them
are regulatory requirements, good science, and quality control requirements. The
Code of Federal Regulations (CFR) 311.165c explicitly states that “ the accuracy,
sensitivity, specifi city, and reproducibility of test methods employed by the fi rm shall
be established and documented. ” Of course, as scientists, we would want to apply
good science to demonstrate that the analytical method used had demonstrated
accuracy, sensitivity, specifi city, and reproducibility. Finally management of the
quality control unit would defi nitely want to ensure that the analytical methods that
the department uses to release its products are properly validated for its intended
use so the product will be safe for human use.
CYCLE OF ANALYTICAL METHODS
The analytical method validation activity is not a one - time study. This is illustrated
and summarized in the life cycle of an analytical procedure in Figure 1 . An analytical
method will be developed and validated for use to analyze samples during the early
development of an active pharmaceutical ingredient (API) or drug product. As drug
development progresses from phase 1 to commercialization, the analytical method
will follow a similar progression. The fi nal method will be validated for its intended
use for the market image drug product and transferred to the quality control laboratory
for the launch of the drug product. However, if there are any changes in the
manufacturing process that have the potential to change the analytical profi le of the
drug substance and drug product, this validated method may need to be revalidated
to ensure that it is still suitable to analyze the API or drug product for its intended
Ø Validation is the act of demonstrating and documenting a procedure that operates effectively.
Ø The discussion of the validation of analytical procedures is directed to the four most common types of analytical procedure:
R Identification tests
R Quantitative tests for impurities content
R Limit tests for the control of impurities
R Quantitative tests of the active moiety in samples of drug substance or drug product or other selected components in the drug product.
Ø Typical validation characteristics which should be considered are:
R Quantitation Limit
R Linearity and Range
· Method Validation Parameter for the assay of Mebendazole:
Ø Linearity: Mebendazole to be analyzed as per proposed method. The results obtain is used to statistically evaluate for coefficient of determination (r2), standard error of estimate and y intercept.
Ø Precision: Precision of the chemical method is ascertained by carrying out the analysis as per the procedure and as per normal weight taken for analysis. Repeat the analysis five times. Calculate the % assay, mean assay, % Deviation and % relative standard deviation and %RSD.
Ø Accuracy: Accuracy of the method is ascertained by standard addition method at 3 levels. Standard quantity equivalent to 80%, 100% and 120% is to be added in sample.
· Method Validation Parameter for residual solvent by GC for Mebendazole:
Ø Specificity: Resolution of the analyte peak from the nearest peak: Solution of each of the analyte was injected separately and their retention time is noted. The standard working solution containing a mixture of the component being analyze is also injected and each of analyte peaks is check for its resolution from the nearest.
R Repeatability: Six replicate injections of standard solution for system precision should analyze as per the proposed method and from the chromatograms obtained the percentage % RSD is calculated.
R Intermediate precision: The purpose of this test is to demonstrate the intermediate precision of the method when method is executed by a different analyst and on different day. Results obtained will be compared.
Ø Linearity and Range: Solution of analyte solvent, having different concentration should make separate from L.O.Q. concentration, which is 50% to 150%. The result obtained is statistically evaluated for coefficient of determination (r2), standard error of estimate and y intercept.
Ø LOD & LOQ:
R The limit of Detection (L.O.D.) was calculated as per below equation:
LOD = 3.3 X SD
R The limit of Quantification (L.O.Q.) was calculated as per below equation:
LOQ = 10 X SD
Ø Accuracy / % Recovery (By Standard Addition Method): Accuracy of the method was ascertained by standard addition method at 3 levels.
R Standard solution quantity equivalent to 50%, 100% and 150% are added in sample.
R The solutions amount is analyzed by the proposed method and chromatogram obtained.
R The amount recover by the method is compared to the amount added. Percent deviation is calculated at each levels and a grand average across all the levels are also calculated.
Methanol standard concentration –– 3000 ppm
Acetic acid standard concentration –– 5000 ppm
DMF standard concentration –– 880 ppm
R The evaluation of robustness should be considered during the development phase and depends on the type of procedure under study. It should show the reliability of an analysis with respect to deliberate variations in method parameters.
R If measurements are susceptible to variation in analytical conditions, the analytical condition should be suitably controlled or a precautionary statement should be included in the procedure.
R One consequence of the robustness should be that a series of system suitability parameters (e.g. resolution test) is established to ensure that the validity of the analytical procedure is maintained whenever used.
PROCESS OF ANALYTICAL METHOD VALIDATION
The typical process that is followed in an analytical method validation is chronologically listed below:
1. Planning and deciding on the method validation experiments
2. Writing and approval of method validation protocol
3. Execution of the method validation protocol
4. Analysis of the method validation data
5. Reporting the analytical method validation
6. Finalizing the analytical method procedure
The method validation experiments should be well planned and laid out to ensure
effi cient use of time and resources during execution of the method validation. The best way to ensure a well - planned validation study is to write a method validation protocol that will be reviewed and signed by the appropriate person (e.g., laboratory management and quality assurance).
The validation parameters that will be evaluated will depend on the type of
method to be validated. Analytical methods that are commonly validated can be
classifi ed into three main categories: identifi cation, testing for impurities, and assay. Table 3 lists the ICH recommendations for each of these methods.
Execution of the method validation protocol should be carefully planned
to optimize the resources and time required to complete the full validation
study. For example, in the validation of an assay method, linearity and accuracy may be validated at the same time as both experiments can use the same standard solutions.
A normal validation protocol should contain the following contents at a
(a) Objective of the protocol
(b) Validation parameters that will be evaluated
(c) Acceptance criteria for all the validation parameters evaluated
(d) Details of the experiments to be performed
(e) Draft analytical procedure
The data from the method validation data should be analyzed as the data are
obtained and processed to ensure a smooth fl ow of information. If an experimental error is detected, it should be resolved as soon as possible to reduce any impact it may have on later experiments. Analysis of the data includes visual examination of the numerical values of the data and chromatograms followed by statistical treatment of the data if required.
Upon completion of all the experiments, all the data will be compiled into a
detailed validation report that will conclude the success or failure of the validation
exercise. Depending on the company ’ s strategy a summary of the validation data may also be generated. Successful execution of the validation will lead to a final analytical procedure that can be used by the laboratory to support future analytical work for the drug substance or drug product.
There are various circumstances under which a method needs to be revalidated.
Some of the common situations are described below:
1. During the optimization of the drug substance synthetic process, signifi cant
changes were introduced into the process. To ensure that the analytical method
will still be able to analyze the potentially different profi le of the API, revalidation
may be necessary.
2. If a new impurity is found that makes the method defi cient in its specifi city,
this method will need to be modifi ed or redeveloped and revalidated to ensure
that it will be able to perform its intended function.
3. A change in the excipient composition may change the product impurity
profi le. This change may make the method defi cient in its specifi city for the
assay or impurity tests and may require redevelopment and revalidation.
4. Changes in equipment or suppliers of critical supplies of the API or fi nal drug
product will have the potential to change their degradation profi le and may
require the method to be redeveloped and revalidated.