## 3 Results and Discussion
### Method Validation
#### 3.2.4 Recovery  
Recovery testing was performed in triplicate using the method developed by spiking HST samples with reference standards. The average recoveries of berberine, palmitine, geniposide, and paeoniflorin ranged from 92.33% to 116.72% and RSD values from 0.731% to 4.705% (Table 3). Therefore, the developed assay method was found to be suitable for analyzing levels of the four compounds in HST samples.  
\begin{table}
\begin{tabular}{l c c c c c c c c} \hline \hline \multirow{2}{*}{**Compound**} & \multirow{2}{*}{\begin{tabular}{c} **\(\mathbf{\lambda^{a}}\)** \\ **(nm)** \\ \end{tabular} } & \multirow{2}{*}{\begin{tabular}{c} **\(\mathbf{\mathbf{f_{R}}^{b}}\)** \\ **(\%)** \\ \end{tabular} } & \multirow{2}{*}{\begin{tabular}{c} **RSD\({}^{c}\)** \\ **(\%)** \\ \end{tabular} } & \multirow{2}{*}{\begin{tabular}{c} **Linear Range** \\ **(ug/mL)** \\ \end{tabular} } & \multirow{2}{*}{\begin{tabular}{c} **Regression** \\ **Equation\({}^{d}\)** \\ \end{tabular} } & \multirow{2}{*}{\begin{tabular}{c} **\(\mathbf{\mathbf{\mu^{2}e}}\)** \\ **(ug/mL)** \\ \end{tabular} } & \multirow{2}{*}{\begin{tabular}{c} **LOD** \\ **(ug/mL)** \\ \end{tabular} } & \multirow{2}{*}{\begin{tabular}{c} **LOQ** \\ **(ug/mL)** \\ \end{tabular} } \\ \hline berberine & 230 & 20.20 & 0.26 & 2.50–25.00 & \begin{tabular}{c} Y = 59,041.58 x + \\ 7174.76 \\ \end{tabular} & 0.9998 & 0.131 & 0.398 \\ palmatine & 230 & 21.07 & 0.25 & 2.50–25.00 & \begin{tabular}{c} Y = 44,562.64 x + \\ 7204.29 \\ \end{tabular} & 0.9999 & 0.179 & 0.543 \\ geniposide & 240 & 22.26 & 0.39 & 5.00–50.00 & \
\begin{tabular}{c} Y = 23,807.44 x + \\ 6159.4 \\ \end{tabular} & 0.9998 & 0.217 & 0.659 \\ paeoniflorin & 230 & 24.11 & 0.38 & 5.00–50.00 &
\begin{tabular}{c} Y = 19,555.98 x - \\ 2874.87 \\ \end{tabular} & 0.9994 & 0.296 & 0.898 \\ \hline \hline \end{tabular}  
* Detection wavelength; b Retention times; c Relative standard deviation; d Y: peak area, x: concentration (ug/mL).; e Coefficient of determination.  
\end{table}
Table 1: Retention times, linear range, calibration curves, limits of detection (LODs), and limits of quantitation (LOQs) of the four compounds (\(n=5\)).  
\begin{table}
\begin{tabular}{l c c c c c c c} \hline \hline \multirow{2}{*}{**Compound**} & \multirow{2}{*}{\begin{tabular}{c} **Con.** \\ **(ug/mL)** \\ \end{tabular} } & \multicolumn{4}{c}{**Intra-Day (\(n=3\))**} & \multicolumn{4}{c}{**Inter-Day (\(n=3\))**} \\ \cline{3-8}  & & \begin{tabular}{c} **Measured** \\ **(ug/mL)** \\ \end{tabular} & \begin{tabular}{c} **RSD** \\ **(\%)** \\ \end{tabular} & \begin{tabular}{c} **Accuracy** \\ **(\%)** \\ \end{tabular} & \begin{tabular}{c} **Measured** \\ **(\%)** \\ \end{tabular} & \
\begin{tabular}{c} **RSD** \\ **(\%)** \\ \end{tabular} &
\begin{tabular}{c} **Accuracy** \\ **(\%)** \\ \end{tabular} \\ \hline berberine & 3.91 & 3.92 & 2.348 & 102.53 & 3.85 & 2.281 & 100.45 \\  & 5.21 & 5.02 & 0.697 & 98.28 & 5.04 & 1.068 & 98.70 \\  & 7.81 & 7.68 & 0.111 & 100.36 & 7.68 & 0.946 & 100.38 \\ \hline palmatine & 3.91 & 3.90 & 2.127 & 101.79 & 3.86 & 2.840 & 100.83 \\  & 5.21 & 4.99 & 0.826 & 97.71 & 5.01 & 1.774 & 98.22 \\  & 7.81 & 7.69 & 1.230 & 100.48 & 7.69 & 1.109 & 100.49 \\ \hline geniposide & 7.81 & 7.78 & 1.135 & 101.63 & 7.68 & 1.467 & 100.34 \\  & 10.42 & 9.88 & 0.280 & 96.74 & 10.00 & 1.061 & 97.95 \\  & 15.63 & 15.50 & 1.526 & 101.22 & 15.41 & 1.118 & 100.64 \\ \hline paeoniflorin & 7.81 & 7.81 & 1.693 & 101.96 & 7.64 & 3.593 & 99.76 \\  & 10.42 & 10.03 & 1.731 & 98.23 & 10.09 & 1.274 & 98.85 \\  & 15.63 & 15.30 & 2.857 & 99.91 & 15.38 & 2.444 & 100.42 \\ \hline \hline \end{tabular}  
* \end{table}
Table 2: Precision and accuracy of the HPLC-PDA method for the four compounds.