3.6. Analysis of Anthocyanins in Grape Berries by HPLC-PDA Analysis

The identification and quantification of anthocyanins were performed according to Lago-Vanzela et al. [2]. The analysis was carried out on a Luna 5 μ C18 column (250 × 4.6 mm) with a guard column. A binary gradient of a mixture of water/acetonitrile/formic acid was used. The linear gradient was A 83/7/10; B: 40/50/10 (v/v/v); A%: 0 min. 94%, 20 min. 80%, 35 min. 60%, 35 min. 40%, 40 min. 90%, and 45 min and 55 min 94%. The flow rate was set at 0.5 mL/min. Chromatographic separation and detection were performed using an Agilent Series 1100 chromatographic system equipped with a quaternary pump, a degassing device, a 20-μL injection loop (Rheodyne, Cotati, CA, USA), and a PDA detector (200–650 nm).

Stock solution of malvidin-3-glucoside (5 mg/L) was prepared with (5% v/v) perchloric acid solution to construct external standard curve (y = 269.37 × –19.353, and correlation coefficient R² = 0.9997). This solution of malvidin-glucoside was diluted to obtain standard solutions (100–500 µg/mL for the preparation of calibration curves. These solutions were freshly prepared every day. The final anthocyanin concentration is expressed as milligram per kilogram fresh weight (fw) of extracts. All analyses were done in triplicate.