Bioanalytical method validation design for the simultaneous quantitation of analytes that may undergo interconversion...

In the analysis of post-dose biological samples for quantitative determination of two analytes that can potentially undergo interconversion, it is essential to minimize the interconversion during the multiple steps of the bioanalytical method. However, even after optimizing the conditions of each step, some interconversion may be unavoidable. Even then, a method can be developed for the accurate simultaneous determination of the two analytes in post-dose biological samples if the composition, in terms of the ratio of the concentrations of the two analytes, of the calibration standards and quality control (QC) samples are selected judiciously, in relation to the composition of the unknown samples to be analyzed. As an example of such interconverting analytes, a delta-hydroxy acid compound (analyte 1) and its delta-lactone (analyte 2) were selected as model compounds that can potentially undergo interconversion. The effects of changing the relative concentrations of the two analytes in QC samples vis-à-vis the calibration standards on the performance of the method under conditions were investigated where: (a) the interconversion between the two analytes was minimized; (b) the conversion of analyte 2 to analyte 1 was enhanced; (c) the interconversion between the two analytes was enhanced. The results showed that the method performance, as measured by the accuracy and precision of the QC samples, was not acceptable when the ratio of concentration of analyte 1 to that of analyte 2 in the QC samples was different from that in the calibration standards and the conditions used facilitated the conversion of one analyte to the other. However, when the relative concentration of the two analytes in the QC samples was identical to that of the calibration standards, the method performance was acceptable under all three conditions of interconversion. This was because the same degree of interconversion took place in the QC samples and calibration standards. The purpose of QC samples in bioanalytical methods is to gauge how the method will perform for the analysis of post-dose test samples and hence, ideally, the relative concentrations of the analytes in QC samples, should be selected to mimic the anticipated concentrations in the test samples. However, the relative concentrations of the analytes in test samples may not be known a-priori, or may change from sample to sample; therefore, it is not always possible to construct QC samples that exactly mimic the relative concentrations of analytes in the test samples. Thus, in order to cover the variety of test samples, the method should include, in addition to QC samples that contain the analytes at the same relative concentration as in the calibration standards, QC samples with relative concentrations that are different from those in the calibration standards, including those that contain only analyte 1 and only analyte 2. In addition, the conditions adopted for the method should favor the minimization of the conversion of the analyte that is expected to be the major component in the post-dose test samples.