STABILITY-INDICATING METHOD VALIDATION PARAMETERS
The FDA requires that all CGMP facilities perform potency testing utilizing methods that are stability-indicating, specific, accurate, and precise.
Validation of an analytical procedure is the process by which it is established, by laboratory studies, that the performance characteristics of the procedure meet the requirements for the intended analytical applications. The method must also be appropriately validated to ensure that quantitation of the analyte is reproducible, and no interference occurs from excipients, degradants, or impurities. Forced degradation studies are used during method development to accelerate the formation of degradants to demonstrate the specificity of the method (i.e., adequate separation of the analyte from the degradants that form under accelerated conditions).
†ICH Q2(R1) provides general guidance for analytical method validation. The various components of method validation are described in the following sections:
System suitability assesses the ability of the test equipment and method to separate analytes and produce accurate results. System suitability tests are based on the concept that the equipment, electronics, analytical operations, and samples to be analyzed constitute an integral system that can be evaluated. Testing is conducted by preparing a solution of API reference standard in a mixture of excipients or placebo preparation at the same active ingredient to excipient ratio as included in the compounded preparation and at the same dilution as would be used for the analysis of the compounded preparation.
The ICH documents define specificity as the ability to assess unequivocally the analyte in the presence of
components that may be expected to be present, such as impurities, degradation products, and matrix components. In the case of the assay, demonstration of specificity requires that it can be shown that the procedure is unaffected by the presence of impurities or excipients. In practice, this can be done by spiking the drug substance or product with appropriate levels of impurities or excipients and demonstrating that the assay result is unaffected by the presence of these extraneous materials.
If impurity or degradation product standards are unavailable, specificity may be demonstrated by comparing the test results of samples containing impurities or degradation products to a second well-characterized procedure (e.g., a pharmacopeial or other validated procedure). These comparisons should include samples stored under relevant stress conditions (e.g., light, heat, humidity, acid/base hydrolysis, and oxidation). In the case of the assay, the results should be compared; in the case of chromatographic impurity tests, the impurity profiles should be compared.
The ICH documents state that when chromatographic procedures are used, representative chromatograms should be presented to demonstrate the degree of selectivity, and peaks should be appropriately labeled.
Accuracy expresses the closeness of the test result obtained to the true value. The accuracy of a method is determined by preparing spiked solutions of API in the placebo metrics at intervals of the assay sample concentration (e.g., 20% intervals from 80% to 120%). Accuracy is calculated as the percentage of recovery by the assay of the known added amount of analyte in the sample, or as the difference between the mean and the accepted true value, together with confidence intervals. The ICH documents recommend that accuracy should be assessed using a minimum of nine determinations over a minimum of three concentration levels, covering the specified range (i.e., three concentrations and three replicates of each concentration).
The precision of an analytical procedure is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings of a homogeneous sample. The precision of an analytical procedure is usually expressed as the standard deviation or relative standard deviation (coefficient of variation) of a series of measurements.
Precision may be a measure of either the degree of reproducibility or of repeatability of the analytical procedure under normal operating conditions. In this context, reproducibility refers to the use of the analytical procedure in different laboratories, as in a collaborative study.
Intermediate precision (also known as ruggedness) expresses within-laboratory variation, as on different days, or with different analysts or equipment within the same laboratory. Repeatability refers to the use of the analytical procedure within a laboratory over a short period of time using the same analyst with the same equipment.
The precision of an analytical procedure is determined by assaying a sufficient number of aliquots of a homogeneous sample to be able to calculate statistically valid estimates of standard deviation or relative standard deviation (coefficient of variation). Assays in this context are independent analyses of samples that have been carried through the complete analytical procedure from sample preparation to the final test result.
The ICH documents recommend that repeatability should be assessed using a minimum of nine determinations covering the specified range for the procedure (i.e., three concentrations and three replicates of each concentration) or using a minimum of six determinations at 100% of the test concentration.
Linearity and Range
The linearity of an analytical procedure is its ability to elicit test results that are directly, or by a well-defined mathematical transformation, proportional to the concentration of analyte in samples within a given range. The range of an analytical procedure is the interval between the upper and lower levels of analyte (including these levels) that have been demonstrated to be determined with a suitable level of precision, accuracy, and linearity using the procedure as written. The range of the procedure is validated by verifying that the analytical procedure provides acceptable precision, accuracy, and linearity when applied to samples containing analyte at the extremes of the range as well as within the range. ICH recommends that, for the establishment of linearity, a minimum of five concentrations normally be used.
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in procedural parameters listed in the procedure documentation and provides an indication of its suitability during normal usage. Robustness may be determined through deliberate variations of selected operating parameters such as mobile phase composition, buffer pH, flow rate, column temperature, or alternate columns.
The detection limit is a characteristic of limit tests. It is the lowest amount of analyte in a sample that can be detected but not necessarily quantitated, under the stated experimental conditions. Thus, limit tests merely substantiate that the amount of analyte is above or below a certain level. For non-instrumental procedures, the detection limit is generally determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be reliably detected. In the case of instrumental analytical procedures that exhibit background noise, the ICH documents describe a common approach, which is to compare measured signals from samples with known low concentrations of analyte with those of blank samples. The minimum concentration at which the analyte can reliably be detected is established.
The quantitation limit is a characteristic of quantitative assays for low levels of compounds in sample matrices, such as impurities in bulk drug substances and degradation products in finished pharmaceuticals. It is the lowest amount of analyte in a sample that can be determined with acceptable Precision and Accuracy under the stated experimental conditions. For non-instrumental procedures, the quantitation limit is generally determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be determined with acceptable Accuracy and Precision. In the case of instrumental analytical procedures that exhibit background noise, the ICH documents describe a common approach, which is to compare measured signals from samples with known low concentrations of analyte with those of blank samples. The minimum concentration at which the analyte can reliably be quantified is established.
† Refer to ICH.org for the latest general guidance for analytical method validation.
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