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

In the analysis of post-dose biological samples for quantitative determinat ion of two analytes that can potentially undergo interconversion, it is ess ential to minimize the interconversion during the multiple steps of the bio analytical method. However, even after optimizing the conditions of each st ep, some interconversion may be unavoidable. Even then, a method can be dev eloped For the accurate simultaneous determination of the two analytes in p ost-dose biological samples if the composition, in terms of the ratio of th e concentrations of the two analytes, of the calibration standards and qual ity control (QC) samples are selected judiciously, in relation to the compo sition of the unknown samples to be analyzed. As an example of such interco nverting analytes, a delta-hydroxy acid compound (analyte 1) and its delta- lactone (analyte 2) were selected as model compounds that can potentially u ndergo interconversion. The effects of changing the relative concentrations of the two analytes in QC samples vis-a-vis the calibration standards on t he performance of the method under conditions were investigated where: (a) the interconversion between the two analytes was minimized; (b) the convers ion of analyte 2 to analyte 1 was enhanced; (c) the interconversion between the two analytes was enhanced. The results showed that the method performa nce, as measured by the accuracy and precision of the QC samples, was not a cceptable 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 sa mples was identical to that of the calibration standards, the method perfor mance was acceptable under all three conditions of interconversion. This wa s because the same degree of interconversion took place in the QC samples a nd calibration standards. The purpose of QC samples in bioanalytical method s is to gauge how the method will perform for the analysis of post-dose tes t samples and hence, ideally, the relative concentrations of the analytes i n QC samples should be selected to mimic the anticipated concentrations in the test samples. However, the relative concentrations of the analytes in t est 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 conce ntration as in the calibration standards. QC samples with relative concentr ations that are different from those in the calibration standards, includin g those that contain only analyte 1 and only analyte 2. In addition, the co nditions adopted for the method should favor the minimization of the conver sion of the analyte that is expected to be the major component in the post- dose test samples. (C) 2000 Elsevier Science B.V. All rights reserved.