# Simple HPLC-PDA Analysis to Determine Illegal Synthetic Dyes in Herbal Medicines
## 3 Results and Discussion
### Confirmation by LC-MS/MS Analysis  
The LC-MS/MS analysis was conducted to identify analytes from positive samples of synthetic dyes with suspected adulteration after HPLC analysis. This study established the precursor and production of each synthetic pigment by referring to the LC-MS/MS analysis of detecting synthetic dyes in foodstuffs or herbal medicines (Table 4). Most dyes were ionized under negative ESI modes, while only Auramine O was analyzed under positive ESI modes. These findings confirmed that ten synthetic pigments in the mixed standard solution can be identified through the LC-MS/MS analysis using the MRM conditions  
\begin{table}
\begin{tabular}{c c c c c c c c c c c} \hline \hline \multirow{2}{*}{**Colorants**} & \multirow{2}{*}{ \begin{tabular}{c} **Spiked Conc.** \\ **(ug/mL)** \\ \end{tabular}  } & \multicolumn{4}{c}{**Hawthorn Fruit (\%)**} & \multicolumn{4}{c}{**Cornus Fruit(\%)**} & \multicolumn{4}{c}{**Schisandra Fruit (\%)**} \\ \cline{3-10}  & & **Recovery** & **RSD** & **RSDr** & **Recovery** & **RSD** & **RSDr** & **Recovery** & **RSD** & **RSDr** \\ \hline \multirow{3}{*}{Tartrazine} & 5 & 132.1 & 0.9 & 0.7 & 109.6 & 0.4 & 0.3 & 113.3 & 0.5 & 0.5 \\  & 10 & 128.8 & 1.4 & 1.1 & 109.7 & 1.0 & 0.9 & 111.8 & 1.8 & 1.6 \\  & 25 & 122.6 & 6.9 & 5.7 & 111.2 & 0.7 & 0.6 & 115.0 & 1.5 & 1.3 \\ \hline \multirow{3}{*}{Auramine O} & 5 & 98.6 & 0.3 & 0.3 & 80.9 & 2.7 & 3.4 & 99.0 & 0.3 & 0.3 \\  & 10 & 97.3 & 0.8 & 0.8 & 78.3 & 2.5 & 3.2 & 99.1 & 1.6 & 1.6 \\  & 25 & 98.8 & 2.3 & 2.3 & 85.0 & 1.3 & 1.5 & 102.4 & 0.2 & 0.2 \\ \hline \multirow{3}{*}{Metani yellow} & 5 & 102.8 & 0.5 & 0.5 & 103.4 & 0.3 & 0.3 & 101.3 & 0.3 & 0.3 \\  & 10 & 101.8 & 0.7 & 0.7 & 102.2 & 0.2 & 0.2 & 100.3 & 1.1 & 1.1 \\  & 25 & 102.4 & 1.5 & 1.4 & 102.8 & 0.5 & 0.5 & 103.1 & 0.3 & 0.3 \\ \hline \multirow{3}{*}{Amaranth} & 5 & 111.5 & 0.6 & 0.5 & 93.6 & 0.9 & 0.9 & 90.6 & 1.0 & 1.1 \\  & 10 & 109.7 & 0.9 & 0.8 & 93.8 & 0.3 & 0.3 & 90.0 & 2.3 & 2.5 \\  & 25 & 110.3 & 1.2 & 1.1 & 95.8 & 0.8 & 0.8 & 92.7 & 1.6 & 1.8 \\ \hline \multirow{3}{*}{New occine} & 5 & 99.3 & 0.4 & 0.4 & 92.5 & 1.2 & 1.3 & 87.8 & 0.7 & 0.8 \\  & 10 & 98.2 & 0.8 & 3.9 & 92.3 & 0.1 & 0.1 & 85.7 & 1.5 & 1.8 \\  & 25 & 99.6 & 1.2 & 2.9 & 94.0 & 0.8 & 0.9 & 88.3 & 1.4 & 1.6 \\ \hline \multirow{3}{*}{Sunset yellow} & 5 & 104.3 & 0.3 & 3.6 & 102.3 & 0.8 & 0.7 & 97.3 & 0.8 & 0.8 \\  & 10 & 103.6 & 0.8 & 3.7 & 100.7 & 0.2 & 0.2 & 95.7 & 1.1 & 1.1 \\  & 25 & 103.3 & 2.0 & 1.2 & 101.9 & 0.7 & 0.7 & 98.4 & 0.4 & 0.4 \\ \hline \multirow{3}{*}{Azorubine} & 5 & 99.2 & 0.3 & 0.3 & 98.1 & 1.1 & 1.2 & 94.0 & 0.5 & 0.5 \\  & 10 & 98.5 & 0.7 & 0.2 & 97.6 & 0.5 & 0.5 & 93.5 & 1.2 & 1.3 \\  & 25 & 100.4 & 1.1 & 0.7 & 99.0 & 0.7 & 0.7 & 96.7 & 0.7 & 0.7 \\ \hline \multirow{3}{*}{Erythrosine B} & 5 & 96.0 & 0.2 & 0.2 & 95.2 & 1.4 & 1.5 & 75.9 & 0.9 & 1.2 \\  & 10 & 94.3 & 0.8 & 0.8 & 94.2 & 1.4 & 1.5 & 74.6 & 2.2 & 3.0 \\  & 25 & 97.6 & 2.1 & 2.1 & 97.0 & 0.9 & 1.0 & 79.8 & 1.3 & 1.6 \\ \hline \multirow{3}{*}{Acid red 73} & 5 & 100.5 & 0.2 & 0.2 & 97.0 & 0.9 & 0.9 & 91.4 & 0.4 & 0.5 \\  & 10 & 99.9 & 0.9 & 96.1 & 0.4 & 0.4 & 90.8 & 1.1 & 1.2 \\  & 25 & 101.0 & 1.2 & 1.2 & 98.1 & 0.7 & 0.7 & 94.0 & 0.7 & 0.7 \\ \hline \multirow{3}{*}{Orange II} & 5 & 104.1 & 0.4 & 0.4 & 100.5 & 0.5 & 0.5 & 100.1 & 0.7 & 0.7 \\  & 10 & 103.3 & 1.0 & 1.0 & 99.9 & 0.2 & 0.2 & 99.9 & 1.1 & 1.1 \\ \cline{1-1}  & 25 & 103.9 & 1.4 & 1.4 & 101.5 & 0.6 & 0.6 & 102.7 & 0.5 & 0.4 \\ \hlineshown in Figure 4. The sample solution obtained after pretreating samples spiked with the mixed color solution was assumed to be a positive sample solution in the present study. In order to evaluate the reliability of the developed LC-MS/MS analysis, the positive sample solution was analyzed using the MRM conditions suggested in Table 4. The results revealed that ten synthetic dyes in the sample solution were successfully identified through LC-MS/MS analysis by indicating target ions and reference ions identical to those of the mixed standard solution in each synthetic dye.