Comprehensive profiling of glycosidic aroma precursors in grapes via semi-preparative LC fractionation, GC-MS, and UHPLC-MS analysis

Abstract

Glycosidically bound aroma compounds are the most abundant non-volatile precursors and play a key role in wine aroma formation [1]. Early methodologies for studying these molecules relied on aglycone release through acid or enzymatic hydrolysis, followed by GC-MS analysis. Although such approaches effectively assess the overall aromatic potential of winemaking grapes, they are not suitable for elucidating the structures of the intact glycosides. Advances in UHPLC-MS have enabled the direct determination of aroma precursors [2]; however, analysis remains challenging due to the lack of commercial standards, the structural diversity of precursors linked to the same aroma, and their typically low concentrations. This study aims to identify as many aroma precursors as possible using a fractionation approach involving a sequence of semi-preparative chromatographies, first by size exclusion and then by normal phase. This method is designed to reduce the high complexity of grape samples prior to UHPLC-MS analysis, thereby facilitating precursor identification. For this purpose, 27 phenolic aromatic fractions (PAFs) from Garnacha grapes were prepared [3] (15 from D.O. Campo de Borja and 12 from D.O.Ca. Rioja). An equal mixture of these PAFs was subjected to the fractionation strategy, yielding 96 fractions, 92 of which released aroma upon hydrolysis. These fractions were analysed using SPME-GC-MS to identify the location of the precursors of some relevant varietal aromas, including terpenes, norisoprenoids, phenols, vanillins, and cinnamates. Aromatic molecules of interest were found in hydrolysates from 55 of these fractions; therefore, the corresponding non-hydrolysed fractions were analysed using UHPLC-MS to detect potential precursors. Identification was based on three criteria: a) the expected molecular mass of the precursor, b) the presence of MS/MS fragments consistent with its proposed structure, and c) the correlation between the precursor signal in UHPLC-MS and the corresponding aroma signal in GC-MS. A total of 185 precursors were identified, 98 of which had not been previously reported. Of these, 75 precursors were confirmed based on all three identification criteria. Most of the precursors were disaccharides (94) and trisaccharides (57), with a smaller proportion of monosaccharides (34). These included precursors of 83 terpenes, 30 phenols, 22 cinnamic acid derivatives, 21 norisoprenoids, 19 vanillin-related compounds, 6 ethyl leucate and 4 furaneol.

References

1. Liu, J., Zhu, X.-L., Ullah, N., and Tao, Y.-S. (2017). Aroma Glycosides in Grapes and Wine. Journal of Food Science, 82(2), 248-259.

2. Caffrey, A., Lerno, L., Zweigenbaum, J., and Ebeler, S. E. (2020). Direct Analysis of Glycosidic Aroma Precursors Containing Multiple Aglycone Classes in Vitis vinifera Berries. Journal of Agricultural and Food Chemistry, 68(12), 3817-3833.

3. Alegre, Y., Arias-Pérez, I., Hernández-Orte, P., & Ferreira, V. (2020). Development of a new strategy for studying the aroma potential of winemaking grapes through the accelerated hydrolysis of phenolic and aromatic fractions (PAFs). Food Research International, 127, 108728.