## 3 Results and Discussion
### UHPLC-MS/MS Analytical Method Validation for Melatonin Quantification  
Different parameters were evaluated for the analytical method validation, such as assessing recovery, linearity, the limit of detection (LOD), the limit of quantification (LOQ) and repeatability. Recovery of the purification step was evaluated by comparing the area of the standard before and after the purification process (Figure 1). Moreover, the recovery has been evaluated in the real matrix by spiking it with melatonin standard. In detail, the areas obtained after the purification of the fortified extract (spike), the purification of the normal extract of _H. perforatum_ II (blank) and the standard solution at the same spiking concentration were compared. In particular, the percentage of analyte recovered (%R) was calculated by relating the difference between the area of the spike and the area of the blank(St. John's Wort extract) to the area of the standard solution multiplied by 100, as shown in Formula (1). The purification gave excellent results, with a recovery of 91.7% and an RSD of 2.8%. \[\%R=\frac{Area\ spike-Area\ blank}{Area\ standard}\times 100 \tag{1}\]  
The linearity was calculated by injecting different concentrations of the external standard of melatonin (0.0001-1 ug/mL). The calibration curves were constructed by plotting the analyte peak areas against the analyte concentrations and the respective determination coefficients (R\({}^{2}\)) were measured. The observed linearity of the target compounds was evidenced by R\({}^{2}\) values equal to or more than 0.9921. The LOQ and LOD were calculated by injecting low concentrations of the standard and calculated the signal-to-noise ratio \begin{table}
\begin{tabular}{c c c c c} \hline
**No.** & **Compounds** & **_H. perforatum_** & **_H. perforatum_** & **_H. perforatum_** \\  & & **I** & **II** & **III** \\ \hline
1 & Gallic acid & 93.28 & 140.36 & 50.80 \\
2 & Neochlorogenic acid & 3892.00 & 4095.90 & 1680.42 \\
3 & Delphindin-3-galactoside & n.d. & n.d. & n.d. \\
4 & (+)-Catechin & 349.86 & 519.22 & 129.04 \\
5 & Procyanidin B2 & 1319.85 & 1184.41 & 260.01 \\
6 & Chlorogenic acid & 282.52 & 612.38 & 203.74 \\
7 & p-Hydroxybenzoic acid & 78.57 & 184.91 & 138.17 \\
8 & (-)-Epicatechin & 519.49 & 551.41 & 117.56 \\
9 & Cyanidin-3-glucose & n.d. & n.d. & n.d. \\
10 & Petunidin-3-glucose & n.d. & n.d. & n.d. \\
11 & 3-Hydroxybenzoic acid & n.d. & n.d. & n.d. \\
12 & Caffe acid & 29.04 & 77.52 & 23.17 \\
13 & Vanillic acid & 84.12 & 204.61 & 128.20 \\
14 & Resveratrol & n.d. & n.d. & n.d. \\
15 & Pelargonidin-3-glucose & n.d. & n.d. & n.d. \\
16 & Pelagonidin-3-rutinoside & n.d. & n.d. & n.d. \\
17 & Malvidin-3-galactoside & n.d. & n.d. & n.d. \\
18 & Syringic acid & 10.47 & 24.33 & 18.87 \\
19 & Procyanidin A2 & 84.68 & 83.99 & 64.29 \\
20 & p-Coumaric acid & 38.56 & 108.93 & 44.55 \\
21 & Ferulic acid & 6.16 & 13.46 & 5.57 \\
22 & 3.5-Dicaffeoylquinic acid & n.d. & 0.95 & 0.82 \\
23 & Rutin & 5843.49 & 9573.17 & 2405.53 \\
24 & Hyperoside & 10,743.95 & 18,726.59 & 6334.01 \\
25 & Isoquercitrin & 6518.96 & 11,895.02 & 4028.77 \\
26 & Delphindin-3,5-diglucoside & 5805.38 & 10,619.51 & 3616.57 \\
27 & Phloridzin & 6.32 & 11.12 & 3.47 \\
28 & Quercitrin & 851.13 & 2069.36 & 976.78 \\
29 & Myricetin & 15.12 & 15.32 & 4.43 \\
30 & Naringin & n.d. & n.d. & n.d. \\
31 & Kaempfer-3-glucose & 112.56 & 241.29 & 112.70 \\
32 & Hesperidin & 32.58 & n.d. & 42.87 \\
33 & Ellagic acid & n.d. & n.d. & n.d. \\
34 & trans-cinamic acid & 13.79 & 15.11 & 10.46 \\
35 & Quercetin & 2007.43 & 4195.62 & 2433.79 \\
36 & Phloretin & 0.49 & 0.43 & 0.15 \\
37 & Kaempfer & 59.80 & 111.60 & 73.24 \\
38 & Isofhamnetin & n.d. & n.d. & n.d. \\ \hline
**Total** & & 38,799.59 & 65,276.51 & 22,857.19 \\ \hline \end{tabular}
\end{table} 
Table 6: Results of 38 polyphenols UHPLC-MS/MS triple quadrupole. Data were expressed in ug/g of the dried extract. (SNR) using MassHunter Qualitative Analysis B.04.00 software from Agilent Technology (Santa Clara, CA, USA).