Cto 30a

The phenolic compounds were analyzed using a ultra-high performance liquid chromatography (UPLC) system (CBM-20A, Shimadzu) with two gradient pump systems (LC-30AD, Shimadzu), a UV-detector (SPD-M30A, Shimadzu), an auto sample injector (SIL-30AC, Shimadzu), and a column oven (CTO-30A, Shimadzu). Separation was achieved on an XR-ODS column (3.0 × 100 mm, 1.8 μm, Shimadzu) using a linear gradient elution program with a mobile phase containing solvent A (0.1%, v/v, trifluoroacetic acid in distilled deionized water) and solvent B (0.1%, v/v, trifluoroacetic acid in acetonitrile). The phenolic compounds were separated using the following gradient: 0–5 min, 10–15% B; 5–10 min, 15–20% B; 10–15 min, 20–30% B; 15–25 min, 30–50% B; 25–30 min, 50–75% B; 30–35 min, 75–100% B; 35–40 min, 100–5% B; and 40–45 min, 5–0% B. The phenolic compounds and anthocyanins were detected at 280 nm and 520 nm, respectively. The chlorogenic acid (CGA), caffeic acid (CA), delphinidin-3-sambubioside (Dp3-Sam), delphinidin-3-glucoside (Dp3-Glu), cyanidin-3-sambubioside (Cy3-Sam), all obtained from Sigma-Aldrich Co. (St. Louis, MO, USA) were identified based on the retention times of commercial standards (UV spectrum). Gallocatechin (GC) and gallic acid (GAL) were identified as described in a previous study [16 (link)] and by their UV-visible spectral characteristics.

The UPLC-MS-MS system consisted of Nexera UPLC (LC-30A), degasser (DGU-20A5), autosampler (SIL-30A) and column oven (CTO-30A) coupled with a Shimadzu triple Quadrupole Mass Spectrometer (model 8030 Shimadzu, Kyoto, Japan). Electro Spray Ionisation (ESI) and Atmospheric Pressure Chemical Ionisation (APCI) interfaces were included in the system.

Phenolic compounds were extracted following the International Oleic Council method [18 ] and hydrolyzed according to the method proposed by Rovellini et al. [19 ]. The hydrolyzed sample was then analyzed by UHPLC using an Agilent Poroshell 120 EC-C18 reversed-phase column (2.7 µm particle size, 4.6 × 150 mm) on a Shimadzu Nexera UHPLC System (Shimadzu Nexera, Kyoto, Japan) equipped with dual pump LC-30AD, on-line degasser DGU-20AS, column oven CTO-30A, autosampler SIL-30AC and diode array detector (SPD-M20A). Gradient separation was created from solvent A (water with 2% of acetic acid) and solvent B (acetonitrile) as follows: starting from 95% A; 0.01–12 min linear gradient from 5% to 70% B; 12–13 min linear gradient from 70% to 90% B; isocratic condition kept up to 17 min; 17 min back to initial condition at 5% B; isocratic step kept up to 22 min for column re-conditioning. The mobile phase flow rate was 450 μL min⁻¹. The column temperature was 30 °C. Injected volumes for each sample was 5 μL. The detector was set at 280 nm. Polyphenols quantification was obtained using calibration curves obtained by injection on the column of different amounts of both tyrosol and hydroxytyrosol (10–600 ng) with R² values higher than 0.999, in all cases.

An ultra-high-performance liquid chromatography (LC-30A, Shimadzu, Japan) equipped with a binary high-pressure pump (LC-30AD), column temperature chamber (CTO30A), and an autosampler (SIL30AC) was used. The composition of mobile phase was ultrapure water containing 0.1% formic acid and 5 mM of ammonium acetate (A), and acetonitrile with 0.1% formic acid (B). The mixture of drugs was separated by gradient elution with the following elution procedure: 0–2 min, 60% B; 2–3 min, 60–90% B; 3–4 min, 90% B; 4.0–4.1 min, 90–60% B; 4.1–5.1 min, 60% B. The column temperature chamber was set at 40 °C, and the injection volume was 6 µL. Chromatographic separation was performed using a column XSelect HSS T3 (2.1 mm × 100 mm, 2.5 μm, Waters) at a flow rate of 0.25 mL/min.