3.5. Method Validation

The developed UPLC-QQQ-MS/MS method was validated by calibration curves, LODs and LOQs, precision, stability, and recovery [30]. A stock solution containing thirteen standard compounds was diluted to a series of appropriate concentrations with 50% methanol/water to establish calibration curves. Then, the calibration curve was constructed by the ratio of the peak area of the analyte to the IS to the concentration of the corresponding analyte solution. Each concentration was determined in triplicate. The limits of detection (LODs) and limits of quantification (LOQs) were determined by a signal-to-noise (S/N) ratio of 3 and 10, respectively. The analysis of the intra- and inter-day precisions of the method was evaluated with 6 replicate injections within one day (n = 6) and 3 consecutive days (n = 3), respectively. The intra- and inter-day precisions, expressed as relative standard deviations (RSDs), were less than 2.0%. The stability of the method was performed with 6repetitive injections at 0, 2, 4, 8, 12, 24, and 48 h under the same conditions, confirming the repeatability. Variations were expressed by RSD. In order to assess the accuracy of the method, three different concentration levels (50%, 100%, and 150%) of the thirteen standard solutions were added into 0.05 g of sample powder for recovery tests. According to the above method, the spiked samples were extracted and measured. Eventually, the average recovery was calculated by the formula: Recovery (%) = (amount found − un-spiked amount)/amount spiked × 100%, and RSD (%) = (SD/mean) × 100%.