# Study of the Phenolic Compound Profile of _Arbutus unedo_ L.
## 3 Results and Discussion
### Analysis of Phenolic Compounds by HPLC-TQ-MS/MS
#### 3.3.1 Development of the HPLC-TQ-MS/MS Method
\begin{table}
\begin{tabular}{c c c c c c c} \hline \multirow{2}{*}{**Class**} & \multirow{2}{*}{**Analyte**} & \multirow{2}{*}{**Rt (min)**} & \multicolumn{2}{c}{**Molecular Ion;**} & \multicolumn{2}{c}{**Linear**} & \multirow{2}{*}{**Linearity; R2**} & \multirow{2}{*}{**LOD (mg**} & \multirow{2}{*}{**LOQ (mg**} \\  & & & **Product Ions** & **Range** & & & L\({}^{-1}\)) & L\({}^{-1}\)) \\  & & & **(Coll. Energy, eV) (m/z)** & **(mg L\({}^{-1}\))** & & & \\ \hline Hydroxybenzoic acids & 4- & & & & & & \\ hydroxybenzoic acids & hydroxybenzoic acid & 10.6 & 136.8; 92.9*(16V) & 0.025–10.0 & y = 2.39E8x + 1.71E6; & 0.099 & 0.008 & 0.025 \\  & Gallic acid & 8.8 & 168.9; & & & & \\  & & 1247.7*(14V) /166.7*(10V) & 0.05–10.0 & y = 1.90E8 x - 1.16E7; & 0.015 & 0.050 \\  & Protocatechuric acid & 152.7; & & & & & \\  & & & 106.72*(14V)/ & 0.025–10.0 & y = 2.27E8x - 7.78E6; & 0.008 & 0.025 \\  & Syringic acid & 9.5 & 197.0*(16V)/ & 0.1–10.0 & y = 6.26E6x - 3.21E5; & 0.030 & 0.100 \\  & & & 158.2(14V)/ 121.7*(16V) & & & & \\  & Vanillic acid & 9.5 & 165.7*(5V)/151.7*(14V)/ & 0.25–10.0 & y = 8.61E6x - 8.81E5; & 0.075 & 0.250 \\  & & & 122.8(12V) & & & & \\ Hydroxycinamic acids & p-coumaric acid & 9.9 & 160.810(10V)/ & 118.8*(16V) & 0.01–10.0 & y = 3.62E8x + 3.08E7; & 0.003 & 0.010 \\  & Chlorogenic acid & 9.9 & 24.43(2V) & & & & \\  & & 353.1; & & & & & \\  & acid & 190.7*(16V)/ & 189.5(20V) & 0.025–10.0 & y = 1.43E8x - 7.52E6; & 0.008 & 0.025 \\ \hline \end{tabular}  
* \end{table} 
Table 3: Mass spectrum and analytical parameters for the HPLC-TQ-MS/MS method developed for the determination of the target polyphenols. Secondly, different elution gradients using ACN as organic solvent and Milli-Q water (both containing 2 mM ammonium acetate and 0.1% formic acid) were evaluated to achieve the chromatographic separation of the 21 target PC. For this purpose, the mobile phase elution gradient started with a high aqueous content (90% of water) and the organic solvent gradually increased up to 100% of ACN in 25 min. With this gradient elution, most of the analytes presented good peak symmetry, but eluted between 12-14 min (except gallic acid that eluted at 2.7 min but with a very wide and low-symmetrical peak). Therefore, the gradient elution was optimized to improve the separation of the compounds. In this sense, the initial organic composition of the mobile phase started with 5% of ACN, then increased linearly to 100% in the first 18 min, and finally returned to initial conditions in 2 min. With this elution gradient, gallic acid eluted at 8.8 min with good peak symmetry, and good separation between the compounds was achieved, as it is shown in Table 3 and Figure S1. The linear concentration ranges and calibration curves obtained for each analyte are shown in Table 3. Good linear regression was achieved for all PC, with R\({}^{2}\) > 0.99. LODs and LOQs ranged from 0.003-0.075 mg/L and 0.01-0.25 mg/L, respectively, except for procyanidin B2 (1.5 mg/L and f 5 mg/L, respectively). Precision was assessed in terms of repeatability and reproducibility at an intermediate concentration level, and satisfactory results were achieved with RSD values lower than 9% and 14%, respectively. From the above results, it can be concluded that the proposed method can be successfully used for the quantification of PC.