Quantitative Analysis of Polymeric Procyanidins (Tannins) from Grape (Vitis vinifera) Seeds by Reverse Phase High-Performance Liquid Chromatography

Abstract

A reverse phase C₁₈ HPLC method with potential for high automated throughput has been developed for the quantitative analysis of polymeric procyanidins (tannins) in grape seed extracts. Chromatography gave rise to 13 distinct UV-absorbing peaks with good baseline separation. The UV-absorbing peak eluting last is distinct and therefore easily quantified. Biochemical analyses including ultrafiltration, protein precipitation, and Sephadex LH20 chromatography combined with electrospray mass spectrometric analyses establish that this peak predominantly contains polymeric procyanidins. The polymers, which appear to be galloylated to various degrees and seem to fragment in a characteristic manner during electrospray mass spectrometry, are well separated from catechins and procyanidin oligomers of up to 4 units. The recovery of polymeric grape seed tannins with this HPLC method was 86%, which is similar to the 89% recovery achieved with commercial quebracho tannins. The concentration of tannins in seeds from ripe Vitis vinifera cv. Shiraz grapes ranged from 1360 to 2830 mg/kg of berries. Keywords: Proanthocyanidin; liquid chromatography electrospray mass spectrometry; Sephadex LH20 chromatography; ultrafiltration; protein precipitation; PVPP; Shiraz; Pinot Noir