# Simultaneous Determination of 12 Marker Components in Yeonkyopaedok-san Using HPLC-PDA and LC-MS/MS
## 3 Results and Discussion
### Method Validation for Quantitative Analysis by HPLC  
In the established HPLC analytical method, the values of the system suitability parameters for the 12 marker components, namely, \(k^{\prime}\), \(\alpha\), \(N\), \(Rs\), and \(T\!f\) were 4.02-14.44, 1.02-1.34, 2.7624792 \(\times\)\(10^{5}\)-1.36925323 \(\times\)\(10^{6}\), 4.16-50.58, and 1.09-1.29, respectively (Table S4). The calibration curve of each analyte was determined by plotting the peak area (y) versus the corresponding concentration (x) using the standard solution, in the different concentration levels (Table 1). The linearity of these components was \(\geq\)0.99. The results showed good linearity. The LOD and LOQ ranges were calculated to be 0.05-0.20 \(\upmu\)g/mL and 0.16-0.60 \(\upmu\)g/mL, respectively (Table 1). The accuracy (recovery) test of the marker components was conducted using the standard addition method, and the recoveries averaged 92.30-101.30% with the RSD (%) values of \(<\)3.00 (Table 2). Furthermore, the RSD (%) values of repeatability and intra- and inter-day precisions showed good precision within 4.79% (Table 3). These results demonstrated that the developed HPLC assay is suitable for the simultaneous analysis of the 12 marker components in YPS.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Compound**} & \multicolumn{2}{c}{**Linear Range**} & \multirow{2}{*}{**Regression Equation \({}^{\text{a}}\)**\(y\)=ax+b**} & \multirow{2}{*}{\(r^{\text{2}}\)} & \multicolumn{2}{c}{LOD} & \multicolumn{1}{c}{**LOQ**} \\  & \multicolumn{2}{c}{**(\(\upmu\)g/mL)**} & & & **(\(\upmu\)g/mL)** & **(\(\upmu\)g/mL)** \\ \hline
3-CQA & 1.56–100.00 & \(y=3.168268\times 10^{4}x-2.279203\times 10^{4}\) & 0.9986 & 0.19 & 0.57 \\ CFG & 0.78–50.00 & \(y=1.589998\times 10^{4}x-3.69893\times 10^{4}\) & 0.9993 & 0.13 & 0.41 \\ LlQA & 1.56–100.00 & \(y=1.435196\times 10^{4}x-6.7471\times 10^{5}\) & 0.9991 & 0.16 & 0.49 \\ FA & 0.78–50.00 & \(y=7.557159\times 10^{4}x-1766964\times 10^{4}\) & 0.9991 & 0.09 & 0.26 \\ NAR & 0.78–50.00 & \(y=1.698577\times 10^{4}x-4.07642\times 10^{3}\) & 0.9990 & 0.05 & 0.16 \\
5-MVS & 0.78–50.00 & \(y=1.850794\times 10^{4}x-4.67958\times 10^{3}\) & 0.9990 & 0.11 & 0.32 \\ NAG & 2.34–150.00 & \(y=1.779584\times 10^{4}x-1.182341\times 10^{4}\) & 0.9991 & 0.09 & 0.27 \\ NHES & 1.56–100.00 & \(y=2.215239\times 10^{4}x-1.007198\times 10^{4}\) & 0.9991 & 0.10 & 0.31 \\ OPDH & 0.78–50.00 & \(y=2.949189\times 10^{4}x-6.27553\times 10^{3}\) & 0.9994 & 0.15 & 0.47 \\ ARC & 0.78–50.00 & \(y=1.0918938\times 10^{4}x-2.73577\times 10^{3}\) & 0.9989 & 0.16 & 0.49 \\ GA & 1.56–100.00 & \(y=8.69885\times 10^{3}x-3.5352\times 10^{2}\) & 0.9993 & 0.20 & 0.60 \\
6-GIN & 0.78–50.00 & \(y=8.57485\times 10^{3}x-2.32865\times 10^{3}\) & 0.9990 & 0.06 & 0.18 \\ \hline \hline \end{tabular}  
* \(y\): peak area (mAU) of compounds; \(x\): concentration (\(\upmu\)g/mL) of compounds.  
\end{table}
Table 1: Linear range, regression equation, \(r^{\text{2}}\), LODs, and LOQs for marker compounds using HPLC–PDA (\(n=3\)).  
Figure 1: HPLC chromatograms of the standard solutions (**A**) and YPS sample (**B**). 3-CQA (**1**), CFG (**2**), LlQA (**3**), FA (**4**), NAR (**5**), 5-MVS (**6**), NAG (**7**), NHES (**8**), OPDH (**9**), ARC (**10**), GA (**11**), and 6-GIN (**12**).