## 2 Materials and Methods
### Quantitative HPLC-PDA Assay
#### 2.4.3 Method Validation  
The analytical method was validated by determination of the selectivity, linearity, precision, accuracy, and stability of each analyte, according to the International Council for Harmonisation (ICH) Guidance for Industry [18] and some previous literature reports [19].  
The standard stock solution of compounds **1** and **2** was prepared in triplicate in deionised water and serially diluted (in two replicates) with the same solvent to six concentration levels (2%, 10%, 25%, 50%, 75%, and 100% of the stock concentration) within the ranges of 2.98-149.30 \(\upmu\)g/mL and 3.14-157.20 \(\upmu\)g/mL, respectively. The replicate solutions were injected three times into the HPLC system. Calibration tests were run at the beginning, midpoint, and at the end of the analytical tests. A calibration graph was constructed by plotting the mean peak area versus concentration. The linearity was tested by the regression method of least squares and the statistical \(F\)- and \(t\)-tests at the 99% confidence level. The residuals from linear regression models were tested ex post to check the validity of the assumptions of normality, independence and homoscedasticity of the response variables. The possible matrix effects were evaluated by constructing the sensitivity plots for the standards dissolved in the real sample of the rhizome of \(G\). _rivale_. Concentration ranges of the added standards were the same as those used for calibration. Statistical differences between the slopes of the matrix-matched linear regression equations and the calibration curves were tested by the Tukey's honest significant difference (HSD) test at the 95% confidence level.  
The limit of detection (LOD) and limit of quantitation (LOQ) values were estimated from the standard solution diluted with water to provide serial solutions with their concentrations decreasing to the smallest detectable peaks. The LODs were accepted with the 3-signal-to-noise (S/N) ratio, while the LOQs were accepted if the relative standard deviation (RSD) values for peak area were less than 15% for both intraday and inter-day variability with S/N ratio greater than 10, according to [20].  
Stability of the standards was tested using standard stock solution. The solution was kept at 4 \({}^{\circ}\)C for 30 days and the content of compounds **1** and **2** was determined every fifth day and compared with the initial concentration.  
The precision tests were performed in triplicate using standard solution at two concentration levels (10% and 100% of the stock concentration) and the real sample of the rhizome of _G. vivax_. The replicate solutions were injected three times into the HPLC system and the RSD values were calculated for both retention time and peak area, and considered as a measure of precision. The repeatability (intra-day variability) was determined by analysing each sample within 24 h, while the reproducibility (inter-day variability) was measured on three non-consecutive days.  
The accuracy of the method was tested in the extract of _G. vivax_ rhizome by means of the standard addition/recovery procedure at three different levels (0.25, 1.25, or 2.5 mg) of each standard, corresponding to the investigated analytical range. The samples were prepared in triplicate according to the procedure described above (Section 2.4.2) with the standards added to the plant material prior to the extraction. The replicate samples were injected in triplicate, and the accuracy was evaluated by calculating the mean recovery of the analytes from the spiked extracts versus the non-spiked samples.