Analytical Methods: A Statistical Perspective on the ICH Q2A and Q2B Guidelines for Validation of Analytical Methods - - BioPharm International

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Analytical Methods: A Statistical Perspective on the ICH Q2A and Q2B Guidelines for Validation of Analytical Methods


BioPharm International
Volume 19, Issue 12

LINEARITY AND RANGE

The linearity of an analytical procedure is its ability, within a given range, to obtain test results that are directly proportional to the concentration of analyte in the sample. De facto, the range is the smallest and largest concentration that maintains a linear relationship between the concentration and the response of the method. The ICH guidelines do not require any proof of precision, though it is clear that without sufficient precision, the linear relationship cannot be guaranteed. The most common method used for demonstrating linearity is least squares regression. Sometimes it is necessary to transform the data to get a linear fit. The guidelines recommend a minimum of five dose levels throughout the range, with each tested for a minimum of three independent readings. It is possible to use these samples to test the accuracy of the method. Accuracy is the lack of bias at each level, and as long as the bias is consistent along the range of the assay, the method is considered linear. Residual analysis, or the observed value minus the predicted (from the linear equation) can help to assess if there is sufficient linearity in the data. Daniel and Wood give an excellent explanation of residual analysis.7

ACCURACY

Accuracy is the difference between the measured value and the true value. This is different from trueness, which is the difference between the mean of a set of measured values and the true mean value. Accuracy is usually presented as a percent of nominal, although absolute bias is also acceptable. Accuracy in the absence of precision has little meaning. Accuracy claims should be made with acceptable precision. ICH guidelines suggest testing three replicates at a minimum of three concentrations. If the same data from the linearity experiment are used, then there would be five levels. Precision of the data should be compared to observed precision from previous studies or development runs, to confirm the observed precision and validity of the accuracy runs.

ICH guidelines recommend using confidence intervals for reporting accuracy results. Confidence intervals are used for probability statements about the population mean—for example, that the average percentage recovery should be 95–105%.

Tolerance intervals can be used to set appropriate accuracy specifications. These say, for example, that no individual percentage recovery should be less than 80% or greater than 120%.

Tolerance intervals make a statement about the proportion of the population values with a fixed confidence. Therefore, one would say that x% of the population will be contained in the tolerance limits with y% confidence. Tolerance intervals are computed from the sample mean and sample standard deviation. A constant k is used such that the interval will cover p percent of the population with a certain confidence level. The general formula for a tolerance interval is: x-mean kS

Values of the k factor as a function of p and percent confidence are tabulated in Dixon and Massey.8

PRECISION

The most important part of any analytical method validation is precision analysis. The ICH guidelines break precision into two parts: repeatability and intermediate precision. Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision. Intermediate precision expresses within-laboratory variations: different days, different analysts, different equipment, etc. Additionally, the ICH Q2A guideline defines reproducibility as the precision among laboratories (collaborative studies, usually applied to standardization of methodology).4 This lab-to-lab precision could be combined into the estimate of intermediate precision, because it is possible that a particular test method could be run in more than one laboratory. The suggested testing consists of a minimum of two analysts on two different days with three replicates at a minimum of three concentrations. If lab-to-lab variability is to be estimated, the experimental design should be performed in each lab. The analyst and day variability combine to give the intermediate precision (lab-to-lab, if estimated, is added here), whereas the variation after accounting for the analyst and day is the repeatability.

Variance components, or decomposition of variance, is a statistical method to partition the different sources of variation into their respective components. Statistical programs such as Minitab are commonly used to calculate variance components. In Minitab, the option to calculate variance components is contained in the analysis of variance (ANOVA) menu option. Box, Hunter, and Hunter provide an excellent source for additional information on how to calculate variance components.9 It is important to remember that variances can be added or averaged, but not the standard deviations.


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