*2.7. Method Validation*

The method was validated for linearity, limit of detection (LOD), limit of quantification (LOQ), precision (inter-day and intra-day precision), stability, and accuracy following the International Conference on Harmonization (ICH) guideline and the previous reports [26,27].

Linearity was examined through the triplicate analysis of mixed standard solutions at six different concentrations, and the calibration curves were constructed by linear regression analysis of the integrated peak areas (*y*) versus concentrations (*x*). LOD and LOQ for each analyte under the HPLC conditions were determined at the signal-to-noise ratio (S/N) of 3 and 10, respectively.

Intra-day and inter-day variations were chosen to evaluate the precision of the developed HPLC method. Intra-day precision was assessed by six injections of a mixed standard solution within a single day, and inter-day precision was validated with the mixed standard solution used above once a day for 3 consecutive days. The precision of this method was expressed as relative standard deviation (RSD). Stability was evaluated by analyzing the same sample at 0, 2, 4, 6, 8, and 12 h at room temperature. The accuracy test was performed by the standard addition method. The authentic standards at three different concentration levels (low, medium, and large) were added to the sample, then extracted and quantified based on the established procedures. The recovery was counted according to the following formula:

$$\text{Recovery} \left( \% \right) = \frac{\text{total detected amount} - \text{original amount}}{\text{added amount}} \times 100 \tag{3}$$
