Agilent 1260 infinity chromatograph

Anthocyanins were determined by HPLC-DAD using an Agilent 1260 Infinity chromatograph (Agilent, Palo Alto, CA, USA) equipped with a Licrospher® 100 RP-18 reversed-phase column (250 × 4.0 mm; 5 μm packing; Agilent) with a Licrospher® 100 RP-18 pre-column (4 × 4 mm; 5 μm packing; Agilent), both thermostated at 40 °C. The chromatographic procedure for anthocyanin analysis was the same as that described by Portu, López-Alfaro, Gómez-Alonso, López & Garde-Cerdán (2015) . 10 μL of wine samples which had previously been filtered (0.22 µm, Easyprep. Quebec, Canada) were injected into the system with a flow rate of 0.630 mL min -1 . The eluents used were: acetonitrile-water-formic acid (3:88.5:8.5, v/v/v) (eluent A) and acetonitrile-waterformic acid (50:41.5:8.5, v/v/v) (eluent B) and the following linear solvent gradient was established: 0 min, 6% B; 15 min, 30% B; 30 min: 50% B; 35 min, 60% B; 38 min, 60% B; 46 min, 6% B. Anthocyanins were identified according to the retention times of pure compounds and the UV-Vis characteristics obtained from authentic standards. Quantification was made using the DAD chromatograms recorded at 520 nm and calibration graph of the malvidin-3-O-glucoside.