## 2 Results and Discussions
### Comparing UV and ELSD in Quantitative Determination of Saponins in PV  
Under the above-mentioned HPLC-ELSD/UV conditions, the performance of both detectors in the detection of saponins in PV were compared. With both ELSD and UV detection, the results showed good linearity (R\({}^{2}\)\(\geq\) 0.98 in ELSD and R\({}^{2}\)\(\geq\) 0.9997 in UV detection, Table 1). The UV detector showed a better sensitivity for PPT and PPD saponins with the LOQ in the range of 0.001-0.002 mg/mL and LOD in the range of 0.0002-0.0004 mg/mL while ELSD has lower sensitivity with LOD and LOQ 10-15 times higher than that of the UV detector. However, ELSD showed advances in detection of M-R2 with LOD and LOQ of 0.004 mg/mL and 0.019 mg/mL, respectively, which was two times lower than those of the UV detector. Moreover, ELSD could quantitatively determine other minor OCT-type saponins, including M-R1, V-R2, V-R11, and p-RT4, whereas UV failed to do so. Figure 3: Peak area of main saponins of PV extracted at different extraction temperatures (**A**), duration (**B**), and extraction at 1 st, 2 nd, and 3 rd times (**C**). The results of **A** and **B** expressed at the mean and standard deviation of a triplicated experiment. Table 2 exhibits the reproducibility of the optimized quantitative method using the ELSD and UV detectors presented by % R.S.D. of each compound. With the UV detection, intra- and inter-day variations were less than 2 and 6%, respectively, while those of ELSD were both less than 4%. The developed method had good accuracy with the overall recovery of 89.4-107% in ELSD and of 96-106.3% for UV detection (Table 3). However, some minor saponins such as N-R1, M-R1, and G-Re were detected under the LOQ of ELSD. Therefore, the detection of these saponins is just for qualitative purposes. Regarding the validation data, ELSD showed superior performance and, therefore, was chosen for quantitative determination of saponins in PV for its precision and sensitivity. \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline  & \multicolumn{6}{c}{**ELSD**} \\ \cline{2-7}
**Saponin** & \multicolumn{2}{c}{**Equation**} & \multicolumn{2}{c}{**R2**} & \multicolumn{2}{c}{**Range**} & \multicolumn{2}{c}{**LOQ**} & \multicolumn{2}{c}{**LOD**} \\  & & & & **(mg/mL)** & **(mg/mL)** & **(mg/mL)** \\ \hline N-R1 & y = 16,095,158 x1.6081 & 0.9985 & 0.013–1.040 & 0.0220 & 0.0060 \\ M-R1 & y = 18,280,672 x1.5972 & 0.9804 & 0.013–1.010 & 0.0220 & 0.0060 \\ G-Rg1 & y = 14,505,168 x1.5687 & 0.9983 & 0.007–0.955 & 0.0220 & 0.0070 \\ G-Re & y = 15,540,853 x1.6196 & 0.9977 & 0.013–1.075 & 0.0220 & 0.0070 \\ M-R2 & y = 20,958,157 x1.6685 & 0.9984 & 0.019–0.61 & 0.0190 & 0.0040 \\ P-RT4 & y = 20,123,608 x1.6351 & 0.9990 & 0.009–0.56 & 0.0160 & 0.0065 \\ V-R11 & y = 4,707,057 x1.6007 & 0.9994 & 0.055–1.69 & 0.0550 & 0.0260 \\ V-R2 & y = 22,774,081 x1.6510 & 0.9994 & 0.007–0.56 & 0.0130 & 0.0035 \\ G-Rb1 & y = 30,016,270 x1.6916 & 0.9991 & 0.007–0.495 & 0.0130 & 0.0038 \\ G-Rd & y = 28,974,603 x1.7116 & 0.9993 & 0.007–0.495 & 0.0120 & 0.0039 \\ \hline  & \multicolumn{6}{c}{**UV**} \\ \hline N-R1 & y = 8,225,894x \(-\) 12,872 & 1 & 0.0004–1.0400 & 0.0020 & 0.0004 \\ M-R1 & ND & ND & ND & ND & ND \\ G-Rg1 & y = 9,069,242x \(+\) 12,434 & 0.9997 & 0.0019–1.9100 & 0.0018 & 0.0004 \\ G-Re & y = 9,069,242x \(+\) 12,434 & 0.9997 & 0.0019–1.9100 & 0.0018 & 0.0004 \\ M-R2 & y = 188,174.90x \(-\) 166.66 & 1 & 0.009–2.4400 & 0.04 & 0.010 \\ P-RT4 & ND & ND & ND & ND & ND \\ V-R11 & ND & ND & ND & ND & ND \\ V-R2 & ND & ND & ND & ND & ND \\ G-Rb1 & y = 6,675,167x \(-\) 221 & 0.9999 & 0.00097–0.9900 & 0.0010 & 0.0002 \\ G-Rd & y = 7,278,735x \(+\) 1457 & 1 & 0.00098–1.0000 & 0.0010 & 0.0002 \\ \hline \hline \end{tabular} ND—not detected.  
\end{table} 
Table 2: Inter- and intra-day precision of the HPLC-ELSD-UV method.