2.5. Calibration, quantification, and limit of detection

The developed analytical method was validated using the Food and Drug Administration (FDA) bioanalytical method validation guidelines [39,40]. To determine the limit of detection (LOD), multiple concentrations of A-967079 were prepared in plasma and analyzed by HPLC-MS/MS. The LOD was defined as the lowest concentration of A-967079 that reproducibly produced a signal-to-noise (S/N) of 3. The noise was determined as peak-to-peak noise in the blank samples over the elution time of A-967079.

For the calibrators, a working standard of A-967079 (1 mM) was prepared in rabbit plasma. From the working solution, calibration standards of A-967079 with concentration range of 0.025–500 μM (0.025, 0.05, 0.1, 0.2, 0.3, 0.5, 1, 3, 5, 10, 30, 50, 100, 200 and 500 μM) were prepared in rabbit plasma. All calibration standards were analyzed in triplicate. The average peak areas were plotted as a function of A-967079 concentration. Both non-weighted and weighted (1/x and 1/x²) calibration curves were constructed using linear least squares. The linearity, accuracy, and precision of the calibration standards were used to determine the best model for quantification.

The lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) were defined as satisfying the inclusion criteria of <15% relative standard deviation (RSD, as a measure of precision), and a percent error (as a measure of accuracy) of 100 ± 20% back-calculated from the nominal concentration for all calibration standards within the linear range. The goodness-of-fit of the calibration curves was determined using percent residual accuracy (PRA) (i.e., PRA values ≥ 90% are indicative of a good fit) [41,42].

The accuracy and precision of the method were evaluated by analyzing three different quality control (QC) standards, i.e., 2 μM (low QC), 20 μM (medium QC), and 60 μM (high QC), not included in the calibration curve. QCs were analyzed in quintuplicate over three days (within 7 calendar days). Intraassay precision and accuracy were calculated for each individual day, and interassay precision and accuracy were calculated from comparison of the data gathered over three separate days.