Advanced structural characterization of condensed tannin evolution markers in long-aged red wines under different closure oxygen permeabilities.

Understanding the long-term evolution of condensed tannins (CTs) in wine remains challenging due to the molecular complexity of aged wines. In this study, an advanced analytical workflow combining tannin-fraction isolation, thioglycolysis, UHPLC-UHRMS with AcquireX™ data-dependent acquisition, and Feature-Based Molecular Networking (FBMN) driven by the t-distributed stochastic neighbor embedding (t-SNE) algorithm was applied for the first time to wines. The studied wines are authentic red wines aged for 17 years. This exceptionally rare sample set, stored under four closures of differing oxygen permeability, provided a unique opportunity to assess the applicability of this workflow for the in-depth structural characterization of condensed tannin evolution markers in a complex wine matrix. Using this approach, 139 evolution markers were annotated. Among these, 122 markers were assigned to levels 2-3, while 17 markers were assigned to level 4. Overall, 72 of the 139 annotated markers were previously unreported, including 55 markers within the levels 2-3 group and 17 within the levels 3-4 group. These markers arose from interactions between tannins and anthocyanins, pyranoanthocyanins, and aromatic aldehydes with high oxidation levels of up to 5. Exploratory univariate (ANOVA) and multivariate statistical methods including Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) revealed correlative trends between marker abundance and closure oxygen permeability within this specific sample set. Overall, this work demonstrates the power of this transferable analytical workflow for comprehensive structural characterization of condensed tannin evolution markers in complex matrix form long-aged red wines and provides new molecular insights into their chemical evolution.