Analytical Test Method Validation for API Raw Material, In Process Control and Early Intermediate Material Tests
Linearity, Range and Specificity
Introduction
This procedure provides guidance for the validation of analytical test methods. These analytical test methods include those tests which evaluate API Raw Materials, In Process samples (e.g. reaction monitoring) and early intermediate materials (prior to the introduction of the first critical intermediate).
Per ICH Q7A, the degree of analytical validation performed should reflect the purpose of the analysis and the stage of the API production process.
During method development, selection of the assay range is linked to its linearity. At the completion of linearity studies, the appropriate concentration range for the standards and instrumental parameters (e.g. injection volume for chromatographic methods) can be set for all subsequent studies.
It is recommended that a linear relationship be evaluated across the range of the analytical procedure.
Linearity can be evaluated by visual inspection of a plot of signals as a function of analyte concentration or content. If the relationship appears linear on visual inspection, the test results should be evaluated by appropriate statistical methods, for example, by calculation of a regression line by the method of least squares as described in ICH Q2B.
Recommended Linearity Data
It is suggested that standard solutions of multiple (at least 5) concentrations encompassing the concentration range of interest be tested. Test the standard solution using the method conditions.
Rationale to establish linearity using less than 5 different concentrations may be provided by considering the specification range (i.e. if the specification range is sufficiently narrow).
The correlation coefficient, y-intercept, slope of the regression line and the residual sum of squares should be submitted. A plot of the data should also be included. In addition, an analysis of the deviation of the actual data points from the regression line is also helpful for evaluating linearity. The determination of linearity should be done using the method of quantification specified in the procedure.
Evaluation of linearity for TLC may be performed by visual examination of the plate, if this is consistent with the method of quantification specified in the procedure. A linear relationship for the main standard spot may be confirmed visually by an increase in intensity with concentration. The sample concentration may then be “estimated” by comparison with the standards.
Recommended Linearity Acceptance Criteria
The assay need not give results that are directly proportional to the concentration (amount) of analyte in the sample for the test method to be valid. However, the desire to have a linear relationship reflects a practical consideration, since a linear relationship should be accurately described with fewer standards.
A validated method may be sufficiently linear to meet accuracy requirements in the concentration range in which it is intended to be used. When inferring accuracy from a linearity study, linearity could be considered acceptable if results, as compared to a standard, meet the accuracy criteria. A plot of the data should visually appear to be linear. Suggested acceptance criteria (for API Raw Material, In Process Control, and early intermediate material tests) for an acceptable linear relationship may be a test method having a minimum correlation coefficient (r) of > 0.95.
Range:
The range is the interval between the upper and lower levels of analyte concentration for which acceptable linearity, accuracy (recovery), and precision are obtained. It is recommended that the range be established to include all specification limits for a method and the expected results. The range should include at least five points to establish linearity. Values outside of the validated range can be reported as estimates. Range should be established by summarizing the accuracy (where appropriate), the linearity, and the precision data.
The following minimum specified ranges are taken from ICH and may be considered as minimum start points for test methods within the scope of this document.
– For the assay, the ICH range is normally from 80% to 120% of the test concentration. If assay and purity are performed together as one test and only a 100% standard is used, linearity should cover the range from the reporting level of the impurities to 120% of the assay specification.
– For determination of an impurity; the range of concentrations used to evaluate the linearity should consist of the quantitation limit and at least 120% greater than the concentration that would be the impurity specification limit.
– For example, if the concentration at the specification limit was 0.2% w/w, and the limit of quantitation was 0.08% w/w then the range should span 0.08% (w/w) to 0.24% w/w. For example, the concentrations for the linearity experiment might be 0.08%, 0.12%, 0.16% 0.20% and 0.24%. More solutions may be evaluated if the linearity range must be extended.
– In cases where specified impurities/degradation products are not available a surrogate material such as a compound with similar structure or API may be used to demonstrate linearity. In these cases, a rationale for the use of a surrogate should be given.
– For impurities known to be unusually potent or to produce toxic or unexpected pharmacological effects, the detection/quantitation limit should be commensurate with the level at which the impurities must be controlled.
– For validation of impurity test procedures carried out during development, it may be necessary to consider the range around a suggested (probable) limit.
Recommended Range Data and Acceptance Criteria:
Accuracy, precision, and linearity experiments may include the ranges as described above and should meet their respective criteria.
Specificity
Specificity is the ability of the method to assess unequivocally the analyte in the presence of components, which may be expected to be present. Typically these might include impurities, degradants, matrix, etc. It is suggested to assay the analyte in the presence of known precursors and synthetic impurities or degradation products and demonstrate that there is no interference from those materials. The discriminatory ability of a test method may be confirmed by obtaining positive results (perhaps by comparison with a known reference material) from samples containing the analyte, coupled with negative results from samples which do not contain the analyte.
For example, use of the processing solvents minus the component under analysis may be used as a “blank sample” to demonstrate lack of interference for reaction monitoring samples.
Suggested Specificity Data:
When the analyte and the closest eluting peak are of similar height, a suitable specificity may be shown when the resolution between the analyte and the closest eluting peak is at least 1.3. Non-specific methods may be used if they are used in conjunction with methods that permit the determination of a specific impurity(s). In such instances resolution may be less than 1.3 between the analyte and the closest eluting impurity(s). The components of interest should be sufficiently resolved such that they can be quantified without significant interference. Peaks should be baseline resolved or peaks can be consistently integrated separately even at LOQ levels.
For non-chromatographic methods, compare the responses in the presence and absence of precursors/impurities/degradation products/other materials that might be present. For example, for TLC method specificity may include examination of the TLC plate to confirm that spots have well defined borders with no overlap from other spots.