## 3 Results and Discussion
### Validation of the Method  
Selectivity of the method and peak purity were analysed by the comparison of retention times and UV-VIS spectra with reference compounds using an automated match system. The comparison of the spectra upslope, apex, and downslope, and between the peak spectral data at different wavelengths confirmed that all analyte peaks of the real samples eluted as pure bands. Representative chromatograms (Figure 5), including that of the most complex extract of _V. myrtillus_, show good resolution of the matrix peaks and high selectivity of the developed method for the quantification of OA and UA. Calibration standards were prepared in methanol. Linearity of the calibration curves was tested using two linear regression models (\(y=ax+b\); \(y=ax\)), and the \(F\)- and \(t\)-tests were applied to check the statistical significance of the regression equations, slopes, and intercepts at the 99% confidence level. The correlation coefficient \(r\) of the accepted equations was 0.99998 for both analytes, and the chromatographic responses were linear in wide concentration ranges (Table 4). Since no significant differences (\(p<0.05\)) were found between the slopes of the calibration curves and the matrix-matched sensitivity plots, the calibration standards prepared in methanol were assumed to be appropriate for the quantification of the target triterpenes in plant materials. The high sensitivity of the method was demonstrated with the low LODs (47-51 \(\mu\)g/mL; 0.14-0.15 ng) and LOQs (374-405 \(\mu\)g/mL; 1.12-1.22 ng). The LODs and LOQs of both compounds were similar, as were the slopes of their regression curves, which is consistent with their close structural similarity and, thus, similar UV-VIS absorption. In comparison with the previously developed HPLC methods with UV- (Table 5) and ELS-detection [25], as well as with the only one available to date UHPLC method [28] (Table 5), the sensitivity was highly improved. Lower LODs and LOQs could have been obtained for native \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Response 1** & **Predicted Values** & **Experimental Values** & **Error 2 (\%)** \\ \hline \(ST\) (min) & 6.090 & 6.200 & 1.81 \\ \(R_{\mathrm{s}}\) & 1.490 & 1.500 & 0.67 \\ \(D\) & 0.495 & 0.490 & 1.01 \\ \hline \hline \end{tabular}  
* \(ST\): separation time (retention time of the last peak; \(R_{\mathrm{s}}\): resolution OA/UA acc. to Equation (5); \(D\): total response acc. to Equation (2). 2 Percent error between predicted and experimental values.  
\end{table} 
Table 3: Experimental and predicted responses under optimal conditions. Figure 5: Representative chromatograms in the optimised conditions at \(\lambda\) = 215 nm of: (**a**) standards at concentrations of 149.40 \(\mu\)g/mL (OA), 324.30 \(\mu\)g/mL (UA), (**b**) _A. uwa usri_ leaves (12 mg/mL), and (**c**) _V. myrtillus_ leaves (30 mg/mL). OA and UA (43-95 ng/mL; 0.017-0.038 ng) with MS-detection [26], and for fluorophore derivatives (0.8-6.3 ng/mL; 0.004-0.063 ng) with FL-detection [17; 22; 23; 24]. Stability of the standards (expressed as recovery of the initial concentration) was tested at 4 \({}^{\circ}\)C during 30 days in five-day intervals. All analytes were found stable in neutral solutions, with the 30 th-day recovery >96%. Precision of the method was tested for the standard solution and real sample of _A. uva__ursi_. The RSD values for intraday and inter-day variabilities were <1% for the retention times and in the range of 0.4%-3.2% for the concentrations (Table 6). Accuracy was tested by determination of recovery by the standard addition technique. The average total recovery of the method was 95.3%-100.9% for the both analytes, with RSD < 5% (Table 6).