Analytical method validation is a process used to prove through scientific study that the method is suitable for its intended use. Compendial methods have to be verified for suitability under actual conditions of use and for a particular formulation.
The validation of analytical procedures is directed to the four most common types of analytical procedures:
- Identification tests.
- Quantitative tests for impurities’ content.
- Limit tests for the control of impurities.
- Quantitative tests of the active moiety in samples of drug substance or drug product or other selected component(s) in the drug product.
Although there are many other analytical procedures, such as dissolution testing for drug products or particle size determination for drug substance, these have not been addressed herein.
Typical validation characteristics which should be considered are listed below:
- limit of quantitation,
- limit of detection,
- ruggedness, and
Accuracy of the method is the exactness of the analytical method to the true value. Accuracy is determined by spiking known amounts of the analyte into a placebo matrix (without the active) and analyzing the samples according to the method. The analyte should be spiked into the placebo matrix at concentration values ranging from 50, 75, 100, 125, and 150% of the expected range.
Precision is the degree of repeatability of the analytical method when it is performed on multiple samples obtained from a homogeneous mixture and is expressed as the relative standard deviation. Method precision is determined by analyzing at least six individual samples at 100% level, which are carried through all the steps from sample preparation to the final results according to the analytical procedure.
Linearity of the analytical method is determined by using linear regression analysis to deduce the relationship between instrument response and known concentrations of the analyte (usually 50–150% of the expected concentration).
Specificity is the ability of the analytical method to measure the analyte in the presence of other components that are expected to be present. For the determination of specificity, samples are prepared by spiking possible interfering agents (impurities, degradation products, excipients, etc.) or by using forced degraded samples.
LOD and LOQ
Limit of detection (LOD) is the lowest concentration of the analyte in a sample that can be detected, but not quantitated. Limit of quantitation (LOQ) is the lowest concentration of the analyte in a sample that can be determined with acceptable precision. LOD and LOQ are determined by analyzing at least three low concentrations (12.5, 25, and 50% of the expected range for impurities/degradation) samples.
The ruggedness of the analytical method is evaluated by having a second analyst independently repeat the accuracy, precision, and linearity measurements. The experiment must be conducted using different HPLC instruments and columns. The ability of the second analyst to reproduce the validation results of the primary analyst is taken as a proof of the ruggedness of the assay method.
Robustness is the ability of the method to withstand small, deliberate changes in the method parameters. It is evaluated by making small and deliberate changes in the method parameters and studying the effect on the system suitability requirements. These can include variations in flow rate, pH of the buffer, auto sampler/column temperatures, and organic composition of the mobile phase as well as use of selective columns from different vendors.
System suitability test parameters to be established for a particular procedure depend on the type of procedure being validated.
Furthermore revalidation may be necessary in the following circumstances:
- changes in the synthesis of the drug substance;
- changes in the composition of the finished product;
- changes in the analytical procedure;
The degree of revalidation required depends on the nature of the changes. Certain other changes may require validation as well.