Calibration curves were fit to the equation y = ax + b using a linear regression in which y and x were the HPLC analyte peak area relative to that of the internal standard (IS) and compound concentration, respectively (Fig. ). To determine the limits of detection (LOD) and limits of quantitation (LOQ), standard stock solutions were serially diluted in methanol and injected into the HPLC system for analysis. LOD was defined as the concentration at which the least intense detectable peak in the chromatogram had a signal-to-noise (S/N) ratio of three. LOQ was defined as the lowest quantitative level with an S/N ratio of 10. Intra-day precision and accuracy were examined by analysing the standard solutions five times within a single day. Inter-day precision and accuracy were determined by measuring samples on five different days. Recovery tests were performed to evaluate the accuracy of the method by spiking known quantities of mixed standards into samples with a known quantity of each standard. The spiked samples were then extracted and analysed immediately. The added standards were prepared at three different concentrations and each concentration was analysed five times. Stability of the standard methanol solutions was evaluated at 15, 20, 25, 30, 35, and 40°C after aging for 30 and 10 days, respectively.