Flavan-3-ols in Vitis seeds: Their extraction and analysis by HPLC-ESI-MS/MS

An orthogonal L₁₆4³ × 2² test design was applied to select the optimum conditions for extracting flavan-3-ols in grape seeds. Highest yield of flavan-3-ols was achieved with 80% methanol, a ratio [1:30 (g/mL)] of sample-to-solvent, sonication for 20 min, and extraction at 25 °C for 12 h in darkness. The optimized analytical method for HPLC separation was a multistep gradient elution using 1% formic acid (A) and acetonitrile containing 1% formic acid (B), at a flow rate of 0.6 mL/min in 36 min. Moreover, fourteen flavan-3-ols were separated and identified using HPLC-ESI-MS/MS, including four monomers ((+)-catechin, (-)-epicatechin, epigallocatechin gallate and epicatechin gallate) and ten oligomers (three dimers, four trimers, two tetramers and one pentamer). The optimized method was used to determine flavan-3-ols content and compositions among ten representative cultivars. The new wine grape - Beihong, had higher flavan-3-ols content and polymerization than classic wine grapes - Cabernet Sauvignon, Merlot, Semillon and Riesling.