HPLC Analysis of Anthocyanins in Wine

LC-20AT HPLC system (Shimadzu, Japan) was used for the analysis of anthocyanins according to our published paper [6 (link),31 (link)]. An Agilent SB-C18 column (250 × 4.6 mm², 5 µm, Santa Clara, CA, USA) was used for the separation of anthocyanins with a flow rate of 1 mL/min. The injection volume was 20 µL and the column temperature was maintained at 35°C. The mobile phase consisted of (A) 2% formic acid in water and (B) acetonitrile. The gradient program was set as follows: 0–30 min, 0–20% B; 30–45 min, 20–35% B; 45–46 min, 35–100% B; 46 51 min, isocratic 100% B; 51–52 min, 100% to 0% B; and 52–57 min, isocratic 0% B. Malvidin-3-O-glucoside was used as the external standard for quantitation of individual anthocyanins. An Accela 600 HPLC system coupled with a Thermo Fisher LTQ XL ion trap mass spectrometer (Thermo Fisher Scientific Inc, San Jose, CA, USA) was used for the identification of anthocyanins in the wine samples based on the published methods [14 ,23 (link)].