Mass spectrometric evidence for the formation of pigmented polymers in red wine

The polymeric materials (wine tannin) were isolated from a three-year-old Pinot Noir using Toyopearl TSK HW 40-F size exclusion chromatography. The wine tannin was analysed by reversed-phase and gel permeation high performance liquid chromatography, UV-Vis spectrophotometry and mass spectrometry. Analysis by gel permeation chromatography indicated that the isolate was in large part polymeric, and also indicated the presence of pigmented material monitored at a wavelength of 520 nm. Acid-catalysis in the presence of phloroglucinol indicated that the wine tannin contained 45% w/w known proanthocyanidin material. The wine tannins gave a complicated mass spectrum featuring a large number of intense signals derived from proanthocyanidins and, to a much lesser extent, a series of ions corresponding to masses consistent with direct condensation products of an anthocyanin with proanthocyanidins. The degree of polymerisation by mass spectrometry was detected to be as high as an octamer composed of an anthocyanin combined with a proanthocyanidin heptamer. Based upon current evidence, the anthocyanin was proposed to be bound from the C-6/8 or C-4 positions of the anthocyanin to the respective electrophilic or nucleophilic centre of the proanthocyanidins. Only the species with the C-6/8 position of the anthocyanin linked with the proantbocyanidins seemed to be pigmented. Under acid-catalysed conditions, an increase in malvidin-3-glucoside was observed, suggesting that the nonpigmented form is possibly converted to the pigmented flavylium form. This is the first mass spectrometric evidence confirming the existence of pigmented polymers with an extension to octamers in wine.