Aplication of pre-column derivatization with 2,3 naphtalenedialdehyde and RP-HPLC-FL for the analysis of glutathione and its precursor G-glutamyl-cysteine in wines and model wines supplemented with oenological inactive preparations

Resumen del póster presentado al 28th International Symposium on Chromatography celebrado en Valencia (España) del 12 al 16 de septiembre de 2010.Glutathione (GSH) is a thiol containing tripeptide (L-γ-glutamyl-L-cysteinyglycine) that can be present in musts and wines. Its importance for wine quality is associated to its strong antioxidant properties helping in the stabilisation of some aroma components and in reducing the browning of musts and wines. Due to its antioxidant activity, in recent years different Inactive Dry Yeast Preparations (IDYs) rich in GSH are being employed in winemaking in order to prevent wine oxidation. However, there are not scientific evidences about the amounts of biological GSH from IDYs that can be released into the wines. Therefore, this study aimed to develop a HPLC methodology to determine reduced GSH, oxidized GSH (GSSG) and its precursor γ-glutamylcysteine (γ-GC). This methodology was applied to quantify GSH and related compounds in model wines supplemented with 8 IDYs. Finally, the evolution of GSH during the production and shelf-life of a wine supplemented with a GSH enriched IDY has been performed. Two RP-HPLC-FL methods were developed to determine GSH, GSSG and γ-GC by adapting several protocols from the literature. Total GSH and γ-GC were determined by using a precolumn derivatization with 2,3-naphtalenedialdehyde (NDA) and borate buffer pH 9.2 containing 1% of ethanethiol. Determination of reduced GSH was carried out in similar conditions, with the exception of borate buffer pH 9.2, that was prepared containing dithiothreitol 0.5 mM instead of ethanethiol. The GSSG concentration was obtained subtracting the reduced GSH level from the total GSH. To know the release of GSH from IDY preparations, eight different types of IDYs supplied by three different manufactures were used. IDY preparations were added into the model wines at the same dose recommended by manufacturers (0.3 g/L). In addition, two types of rose wines, a control wine and an IDY wine (supplemented with an GSH rich IDY preparation) were industrially manufactured. The method showed good analytical characteristics for the quantification of γ-GC, GSH and GSSH. For instance, the method showed a reproducibility above 6% (n=25) and a linear range between 0-20 mg/L with a R2=0.9982 for the determination of total GSH. The results showed that those IDYs recommended to avoid wine oxidation (therefore including glutathione in their composition) released different amounts of GSH and γ-GC depending on the type of IDY (on the provider type). In addition, other types of IDY without specific applications to act as antioxidants, also released small amounts of GSH, likely coming from the yeast cytoplasm. Variations in the evolution of the GSH during the winemaking and shelf life of rose wines produced with the GSH enriched IDY was also found, which could have important implications for the aroma and colour of the wine.Peer Reviewe