4.6. Determination the Content of Monomer Anthocyanins

The chromatographic analyses of anthocyanins were performed using LC-20AT HPLC system (Shimadzu, Japan), consisting of Pump LC-20AT, Central controller CBM-20A, Auto sampler SIL-20A, Column oven CTO-20A, Detector (PDA) SPD-M20A, equipped with a reversed phase column (Synergi Hydro-RP C18, 250 × 4.6 mm, 4 μm). The mobile phase was ultrapure water:acetonitrile:methanoic acid (800:100:25) as solvent A, and ultrapure water:acetonitrile:methanoic acid (400:500:25) as solvent B. The elution profile had the following proportions (v/v) of solvent B: 0.00–15 min, 0%–10%; 15–30 min, 10%–20%; 30–45 min, 20–35%; 45–46.00 min, 35%–100%; 46.00–50.00 min, 100%. The column was held at 35 °C and at a flow rate of 1 mL/min. The injection volume was 20 µL and analyses were detected at 520 nm. Before injection, the extracts were filtered through 0.22 m filters (cellulose acetate and nitrocellulose, CAN).

All phenolic compounds were identified by comparison of their order of elution and retention time with those of standards, the weight of molecular ion and the fragment ion with standards and references. Quantitative determinations were made by the external standard method with the commercial standards. The calibration curves were obtained by injecting of standard solutions under the same conditions as for the samples analyzed, observing the range of concentrations. Anthocyanins, flavan-3-ols, were respectively expressed as micrograms of malvidin-3-O-glucoside (ME), catechin equivalence (CE)/L of grape skins.