Malvidin, HPLC analysis

Anthocyanins are usually represented as the red flavylium cations (Figure 5.1, left). However, this form is predominant only in very acidic solvents (pH < 2) such as those used for HPLC analysis. In mildly acidic media, the flavylium cations undergo proton transfer and hydration reactions, respectively, generating the quinonoidal base and the hemiketal syn carbinol) form (Figure 5.1, right) that can tautomerize to the chalcone. Thus, at wine pH, malvidin 3-glucoside occurs mostly as the colorless hemiketal (75%), the red flavylium cation, yellow chalcone, and blue quinonoidal base being only minor species. 

Polymeric fractions were obtained from wines, seed and skin extracts by fractionation on a Toyopearl HW-40 column as described by Souquet et al (4). Two aliquots of the fractions containing polymeric material were t en to dryness under vacuum. The first one was used to determine proanthocyanidin composition by thiolysis followed with HPLC analysis (17). The other one was dissolved in MeOH acidified with 2% HCl and used to estimate the concentration of total polymeric polyphenols and polymeric pigments by measuring the absorbance, respectively at 280 nm and 530 nm. Absorbance data were converted to equivalent epicatechin and equivalent malvidin-3-glucoside, respectively, using the extinction coefficients determined for each compounds under similar conditions. 

Reactions taking place in a model solution containing malvidin 3-0-glucoside, (-)-epicatechin and acetaldehyde was explored by HPLC/DAD and HPLC/ESI-MS analysis 29). Acetaldehyde is a product of yeast metabolism but may also result from ethanol oxidation or from decarboxylation of pyruvic acid during fermentation of grapes (43). 

Fraction A obtained from the elution of a two-year-old Port wine through Toyopearl gel column with 20% aqueous ethanol was found to be mainly comprised of eight pigments which structural identities were ascertained by LC-MS (Table 1). The number attributed to each compoimd in Table 1 corresponds to the elution order by HPLC in the condition described herein. The three major pigments detected were isolated by semi-preparative HPLC and their structures were fully elucidated by mass spectrometry and NMR. Based on their UV-visible characteristics and structural analysis, these pigments were found to correspond to the pyruvic acid adducts of malvidin 3-glucoside (3A), malvidin... 

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