# Decomposition of Flavonols in the Presence of Saliva
## 2 Materials and Methods
### Apparatus  
The analytical method used in the presented work was developed in our laboratory and discussed in detail in a previous paper on the study of the phenolic compounds [32]. Chromatographic analyses were carried out using LC-MS/MS system consisted of binary pumps LC20-AD, degasser DGU-20A5, column oven CTO-20 AC, and autosampler SIL-20 AC, connected to 3200 QTRAP Mass spectrometer (Applied Biosystem/MDS SCIEX). Compounds were separated on Chromolith Performance C18 column (100 \(\times\) 2 mm, Merck) at 30 \({}^{\circ}\)C. Formic acid (8 mM, pH 2.8) as eluent A and methanol as eluent B were used. Samples incubated at 37 \({}^{\circ}\)C were kept in the same temperature before analysis in an autosampler. The flow rate of mobile phase was 0.2 mL/min and the gradient mode was as follow: 0-3 min, 10% B; 20-25 min, 50% B; 26-40 min, 10% B. LC system was connected to the 3200 QTRAP Mass spectrometer (Applied Biosystem/MDS SCIEX) with electrospray ionization (ESI) working in negative mode. ESI conditions were as follows: capillary temperature of 450 \({}^{\circ}\)C, curtain gas at 0.3 MPa, auxiliary gas at 0.3 MPa, and negative ionization mode source voltage of 4.5 kV. Nitrogen was used as curtain and auxiliary gas. Analyst 1.4.2 software was used for data acquisition. LC-MS/MS analysis were carried out by comparing retention time and _m_/\(\underline{\text{z}}\) values obtained by MS and MS\({}^{2}\) with the mass spectra of standards obtained under the same conditions. Because some degradation products such as depsides are not commercially available, the presence of these compounds was confirmed by comparison of retention times, masses, and fragmentation spectra of potential oxidation products with literature.  
Quantification of compounds was done using the selected reaction monitoring mode (SRM). For each compound, the optimum conditions for SRM mode were determined in infusion mode and two SRM pairs were chosen as representatives (SRM1 and SRM2) (Table 1). Due to the higher intensity of peak obtained using the SRM1 pairs, they were chosen for qualitative analyses. Calibration curves were drawn from the analysis of 5 \(\mu\)L volumes at concentration ranging from 0.5 to 50 mg/L (n = 7) measured in triplicated. Coefficients of linearity (R\({}^{2}\)) for the calibration curves were \(\geq\) 0.996. LODs were estimated by decreasing the concentration of the analytes to the smallest detectable peaks, and then its concentration was multiplied by three. LODs ranged 0.1-0.5 mg/L.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Compound** & **Retention Time, Min** & **Q1 Mass** & **Q3 Mass** & **DP, V** & **CE, V** \\ \hline Gallic acid & 3.0 & 169 & 125 & \(-\)45 & \(-\)20 \\  & & & 97 & \(-\)45 & \(-\)26 \\ Protocatechic acid & 4.4 & 153 & 109 & \(-\)15 & \(-\)18 \\  & & & 66 & \(-\)15 & \(-\)26 \\
4-hydroxybenzoic acid & 7.1 & 137 & 93 & \(-\)25 & \(-\)18 \\  & & & 119 & \(-\)25 & \(-\)8 \\ Vanillic acid & 9.8 & 167 & 152 & \(-\)30 & \(-\)16 \\  & & & 107 & \(-\)30 & \(-\)24 \\ Hyperoside & 16.6 & 463 & 300 & \(-\)80 & \(-\)32 \\  & & & 271 & \(-\)80 & \(-\)56 \\ Spiraeoside & 18.4 & 463 & 300 & \(-\)80 & \(-\)32 \\  & & & 151 & \(-\)80 & \(-\)48 \\  & 18.9 & 609 & 300 & \(-\)65 & \(-\)32 \\  & & & 270 & \(-\)65 & \(-\)74 \\ Myricetin & 20.1 & 317 & 151 & \(-\)20 & \(-\)26 \\  & & & 179 & \(-\)20 & \(-\)22 \\ Quercitrin & 20.4 & 447 & 300 & \(-\)60 & \(-\)26 \\  & 270 & \(-\)60 & \(-\)56 \\ Quercetin & 22.8 & 301 & 151 & \(-\)40 & \(-\)30 \\  & & & 179 & \(-\)40 & \(-\)24 \\ Kaempfer & 25.0 & 285 & 151 & \(-\)45 & \(-\)25 \\  & & & 117 & \(-\)45 & \(-\)53 \\ Isorhamnetin & 25.6 & 315 & 300 & \(-\)35 & \(-\)24 \\  & & & 199 & \(-\)35 & \(-\)32 \\ \hline \hline \end{tabular}
\end{table}
Table 1: LC-MS/MS characteristics of phenolic compounds in the negative mode.