## 2 Materials and Methods
### Polyphenols in Almond Skin
#### 2.1.6 Ultra-High-Performance Liquid Chromatography--Mass Spectrometry  
Polyphenol extracts were analyzed by Ultra-Performance Liquid Chromatography (UPLC) using an ACQUITY UPLC(r) system (Waters Corp., Milford, MA, USA) linked simultaneously to both a diode array detector 190-800 nm (UPLC DAD, Waters, Milford, MA, USA) and a Bruker Daltonics HCT (High Capacity Ion Traps) Ultra Ion Trap Mass equipped with an electrospray ion source (UPLC DAD/ESI-MSn). Analysis was carried out using an ACQUITY UPLC(r) BEH C18 column (100 mm \(\times\) 2.1 mm, i.d, 1.7 \(\mu\)m; Waters Corp., Milford, MA, USA). The column temperature was set at 35 \({}^{\circ}\)C. The injection volume was fixed at 5 \(\mu\)L. The mobile phase was water containing 0.1% formic acid (solvent A) and Acetonitrile (solvent B) at the flow rate of 0.4 mL/min. Gradient conditions were as following: A, 99-75%, 0-14 min; A, 75-20%, 14-18 min; A, 20-0%, 18-19 min; A, 099%, 19-19.5 min. The ion trap was operated in the Ultra Scan mode from _m/z_ 100 to 1500. The ICC target was set to 100,000 with a maximum accumulation time of 50 ms. Nitrogen (99.99% purity) was used as the desolvation gas. The ionization was achieved using an ESI source in negative. The ionization source parameters were set as followed: dry temperature 365 \({}^{\circ}\)C, nebulizer pressure 50 psi, dry gas flow 9 L\(\backslash\)min \({}^{-1}\), capillary voltage \(-\)2 kV. The chromatographic peaks were tentatively identified by their MS and MS\({}^{2}\) mass spectra and when this was possible, by injection of standard. Quantification was performed with UV signal on the basis of the calibration curve of catechin, epicatechin, and vanillic acid at 280 nm, chlorogenic acid at 330 nm, isorhamnetn, kaempfer, and naringenin at 360 nm. Other compounds for which standards were not available (procyanidins, heterosides) are expressed as equivalent of the injected standards. Polyphenols content: 15 \(\pm\) 1 mg of ultrasonicated almond UA, non-sonicated almonds NUA and natural skin NS freeze-dried extracts were mixed with 0.6 mL of MeOH-0.2% of formic acid. The mixtures were put in small tubes, vortexed and sonicated for 5 min each. After centrifugation, supernatants were injected for analysis. Samples from blanched almond extracts BA were mixed with 0.8 mL of MeOH-0.2% of formic acid and were diluted twice before injection. Procyanidins content: procyanidins in the four extracts were quantified as described by [PERSON] et al. 2011 [18] with some modifications. The following protocole was used: first, A solution of 0.2 N HCl in MeOH, containing 50 g/L phloroglucinol and 10 g/L ascorbic acid was prepared. Second, 15 \(\pm\) 1 mg of lyophilized extracts (blanched almonds BA, ultrasonicated almonds UA and non-ultrasonicated almonds NUA) and natural almond skin (NS) were mixed with 400 \(\upmu\)L of the previous solution. The procyanidin of interest was reacted in this solution at 50 \({}^{\circ}\)C for 20 min. The reaction was then stopped with 400 \(\upmu\)L of ammonium formate solution (12.6 g/L). After stirring and centrifuging, the supernatant was diluted twice and injected for analysis. Procyanidin concentration (_PC_) in almond skin was calculated as shown in the following equations [19]:  
\[PC=\left(\textit{[upper and extension units]}_{M}+\textit{[end units]}_{M}\right) \tag{3}\]  
\[PC=\left(\textit{[cat p]}-\textit{[cat c]}\right)+\left(\textit{[cpicat p]}-\textit{[cpicat c]}\right)+\textit{[Phloroglucinol-cat/picat]} \tag{4}\]  
where _[cat p]_ and _[picat p]_ are the concentrations of catechin and epicatechin after phloroglucynolysis, _[cat C]_ and _[epicat C]_ are the concentrations of crude catechin and epicatechin before treatment, and _[Phloroglucinol-cat/picat]_ is the concentration of phloroglucinol-catechin + phloroglucinol-epicatechin. In addition to the concentration of procyanidins, this reaction allows to determine the mean degree of polymerization (_mDP_), the average number of units in the polymer.