# Simultaneous Determination of 12 Marker Components in Yeonkyopaedok-san Using HPLC-PDA and LC-MS/MS
## 3 Results and Discussion
### UPLC-MS/MS Confirmation  
Table 3 shows that five compounds (3-CQA, LIQA, NAR, NAG, and GA) were detected in the negative ion mode ([M-H]\({}^{-}\)) at _m/z_ 353.2, 549.3, 579.3, 279.2, and 821.6, respectively, whereas seven compounds (CFG, FA, 5-MVS, NHES, OPDH, ARC, and 6-GIN) were detected in the positive ion mode ([M+H]\({}^{+}\)) at _m/z_ 469.3, 195.0, 453.0, 611.1, 304.9, 373.2, and 295.3, respectively. The precursor ion (Q1) and product ion (Q3) for the LC-MS/MS MRM method are shown in Table S3. Briefly, the Q3 peak of 3-CQA was detected at _m/z_ 191.0 [M-H-caffeoyl]\({}^{-}\) by elimination of the caffeoyl group from the Q1 peak [27]. CFG and 5-MVW were set at the Q3 peaks at _m/z_ 307.2 and 291.3 in the form of [M+H-Glc]\({}^{+}\), and these components released one glucose group at the Q1 peak [28,29]. FA and GA were detected at _m/z_ 177.0 [M+H-H\({}_{2}\)O]\({}^{+}\) and 351.2 [M-H-2 Glc]\({}^{-}\) by the loss of one H\({}_{2}\)O and two glucose groups from the Q1 peak, respectively [30,31]. LIQA was detected at _m/z_ 549.3 (Q1) as [M-H]\({}^{-}\), formed by the loss of the apiosy-glucosyl function group from Q1 [32]. NAR, NAG, and NHES were apt to lose the rutinose to give alycone ions of _m/z_ 271.2 [M-H-rutinose]\({}^{-}\), 271.0 [M-H-rutinose]\({}^{-}\), and 303.2 [M+H-rutinose]\({}^{+}\), respectively [33, 34, 35]. The characteristic MS fragmentations of OPDH, ARC, and 6-GIN were observed at _m/z_ [M+H-C\({}_{5}\)H\({}_{9}\)O-OH]\({}^{+}\), 137.0 [dimethoybenzyl group-H]\({}^{+}\), and \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Compound** & \begin{tabular}{c} **Original Amount** \\ **(**mg/mL)** \\ \end{tabular} & \begin{tabular}{c} **Spiked Amount** \\ **(**mg/mL)** \\ \end{tabular} & \
\begin{tabular}{c} **Found Amount** \\ **(**mg/mL)** \\ \end{tabular} &
\begin{tabular}{c} **Recovery** \\ **(**\%)** \\ \end{tabular} & \multirow{2}{*}{**SD**} & \multirow{2}{*}{**RSD (\%)**} \\ \hline
3-CQA & 44.98 & 8.00 & 52.65 & 95.87 & 1.44 & 1.51 \\
44.98 & 20.00 & 64.02 & 95.16 & 2.85 & 2.99 \\  & & 40.00 & 82.68 & 94.24 & 0.56 & 0.60 \\ \hline  & & 1.00 & 6.50 & 95.28 & 2.35 & 2.47 \\ CFG & 5.55 & 2.00 & 7.52 & 98.62 & 2.00 & 2.02 \\  & & 4.00 & 9.49 & 98.61 & 1.37 & 1.39 \\ \hline  & & 6.00 & 36.05 & 96.14 & 1.59 & 1.65 \\ LIQA & 30.28 & 15.00 & 44.73 & 96.33 & 1.61 & 1.67 \\  & & 30.00 & 59.70 & 98.05 & 0.91 & 0.93 \\ \hline  & & 1.00 & 4.36 & 98.22 & 1.77 & 1.80 \\ FA & 3.38 & 2.00 & 5.28 & 94.85 & 0.68 & 0.72 \\  & & 4.00 & 7.29 & 97.85 & 1.43 & 1.47 \\ \hline  & & 2.00 & 13.83 & 101.22 & 1.35 & 1.34 \\ NAR & 11.81 & 5.00 & 16.81 & 100.14 & 1.87 & 1.87 \\  & & 10.00 & 21.85 & 100.41 & 0.60 & 0.60 \\ \hline  & & 1.00 & 6.98 & 97.34 & 2.38 & 2.45 \\
5-MVS & 6.01 & 2.00 & 7.96 & 97.66 & 1.63 & 1.67 \\  & & 4.00 & 9.83 & 95.58 & 2.53 & 2.65 \\ \hline  & & 8.00 & 52.09 & 100.32 & 2.90 & 2.89 \\ NAG & 44.07 & 20.00 & 63.74 & 98.39 & 0.95 & 0.97 \\  & & 40.00 & 84.25 & 100.46 & 1.26 & 1.25 \\ \hline  & & 6.00 & 37.06 & 93.48 & 1.33 & 1.42 \\ NHES & 31.45 & 15.00 & 45.51 & 93.71 & 0.33 & 0.35 \\  & & 30.00 & 59.14 & 92.30 & 0.97 & 1.05 \\ \hline  & & 1.00 & 7.31 & 96.39 & 1.42 & 1.47 \\ OPDH & 6.35 & 2.00 & 8.26 & 95.73 & 1.29 & 1.34 \\  & & 4.00 & 10.26 & 97.69 & 0.36 & 0.37 \\ \hline  & & 2.00 & 11.16 & 99.56 & 2.80 & 2.81 \\ ARC & 9.17 & 5.00 & 14.08 & 98.38 & 1.44 & 1.46 \\  & & 10.00 & 18.80 & 96.38 & 1.04 & 1.08 \\ \hline  & & 6.00 & 34.91 & 93.76 & 1.11 & 1.19 \\ GA & 29.28 & 15.00 & 43.30 & 93.41 & 1.05 & 1.13 \\  & & 30.00 & 56.99 & 92.36 & 0.67 & 0.72 \\ \hline  & & 1.00 & 2.15 & 101.30 & 1.31 & 1.29 \\
6-GIN & 1.14 & 2.00 & 3.12 & 99.26 & 2.44 & 2.45 \\  & & 4.00 & 5.11 & 99.29 & 2.49 & 2.51 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Recovery test for the assay of 12 components in YPS using HPLC–PDA. 177.0 [M+H-C\({}_{6}\)H\({}_{12}\)O\({}_{2}\)]\({}^{+}\); this was assigned to the loss of a C\({}_{5}\)H\({}_{9}\)O molecule and OH group, and C\({}_{13}\)H\({}_{15}\)O\({}_{4}\) and C\({}_{6}\)H\({}_{12}\)O\({}_{2}\) molecules from the Q1 peak, respectively [36; 37; 38].