## 2 Materials and Methods
### Efficiency of Anthocyanins Extraction from Peels
#### 2.5.2 Characterization of the Anthocyanin Extract  
Based on the response of the optimizer and seeking to maximize the concentration of total monomeric anthocyanins, an ultrasound-assisted extraction was carried out with 60% ethanol as a solvent with pH adjusted to 6 and a ratio of 80 mL of solvent per gram of dried peels, at 20 \({}^{\circ}\)C for 15 min. These conditions were determined to be better according to the analysis of the response surface methodology. Once the extraction was complete, the phases were separated in a refrigerated centrifuge Heal Force (Shanghai, China) at 5000 rpm at 5 \({}^{\circ}\)C for 10 min. The residue was extracted two more times under identical conditions.  
The pH and total soluble solids were measured according to the AOAC Methods. A pHmeter Accurate pH 900, Horiba, (Kyoto, Japan) and a digital refracometer ATAGO Pocket (Tokyo, Japan) were employed. The antioxidant activity was measured by the TEAC assay previously described [26]. The total phenolic content was determined by the Folin-Ciocalteau method described by [38], and the total monomeric concentration was determined by the differential pH method described by [39] using the UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). The total monomeric anthocyanins were expressed as mg of Cyanidin 3 gliosido (C3G) per 100 g dry weight (DW) of the dry epicarp or lyophilized peel. All measurements were carried out by triplicates.  
UPLC-ESI-MS/MS Profiling of the Peel Fruits Extract from S. odorifera Naudim Vell. \"kurugua\": UPLC-ESI-DAD-MS/MS Peel Extract Analyses  
The analyses were carried out through a Waters (Milford, CT, USA) Acquity UPLC system coupled with an Acquity PDA eA detector and a Xevo TQD Qq-MS mass spectrometer with an electrospray ionization (ESI) source. Chromatographic separations were performed with a Phenomenex KINETEX core-shell EVO-C18 (2.1 \(\times\) 100 mm, 1.7 \(\mu\)m) column at a flow rate of 0.3 mL/min. The column temperature was maintained at 40 \({}^{\circ}\)C, and the injection volume was 10 \(\mu\)L. The mobile phase was MeOH (phase A) and water (phase B), both containing 0.1% formic acid and 10 mM ammonium formate, with a gradient elution as follows: 0-0.7 min, 80-80% A; 0.7-3.2 min, 80-60% A; 3.2-7.6 min, 60-20% A; 7.6-8.3 min, 20-0% A; 8.3-10.4 min, 0-80% A, 10.4-12 min, 80-80% A. The extracts at 5 mg/mL were filtered through 0.22 \(\mu\)m nylon syringe filters for injection. The compounds were monitored at 280, 320, and 500 nm. Spectra from 200 to 500 nm were recorded for peak characterization. The MS spectra were acquired using MS scan mode (_m/z_ 80-800) in negative and positive polarities. The mass of the major compounds was submitted to MS/MS analysis through daughter ion mode (scan time 0.1 s) for fragmentation data collection. The collision gas was argon. Nitrogen was employed as a nebulizer and drying gas. The MS conditions were as follows: electrospray capillary voltage was 2.5 kV, source temperature was 150 \({}^{\circ}\)C, desolvation temperature was 350 \({}^{\circ}\)C, cone gas flow was 80 L/h, and desolvation gas flow was 900 L/h. The cone voltage was set at 30 V, while the collision energy (CE) was 25 V for flavonoid derivatives and 30 V for aglycones. The system was controlled using Waters Masslynx V4.1 software.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Independent Variable**} & \multicolumn{3}{c}{**Level**} & \multirow{2}{*}{**References**} \\ \cline{2-2} \cline{4-4}  & \(-\)**1** & & **0** & **1** \\ \hline Solvent concentration (\%) (\(\gamma\)/\(\delta\)) & 20 & 45 & 70 & [10,27,30] \\ pH & 1 & 4 & 7 & [31] \\ Temperature (\({}^{\circ}\)C) & 20 & 40 & 60 & [32,33] \\ Time (min) & 5 & 10 & 15 & [34,35] \\ Liquid–solid ratio (mL/g) & 5 & 27.5 & 50 & [10,36,37] \\ \hline \hline \end{tabular}
\end{table}
Table 1: Levels of the independent variables considered in the anthocyanin extraction process.