Volatilomic Differentiation of Protected‐Origin Italian Balsamic Vinegars by HS‐SPME‐GC×GC‐TOFMS

Balsamic vinegars produced under Protected Designation of Origin (PDO) and Protected Geographical Indication (PGI) schemes differ substantially in raw materials, processing intensity, and aging regimes. Although these differences are expected to shape their volatile composition, a comprehensive chemical characterization capable of reliably discriminating the two denominations remains analytically challenging. In this study, headspace solid-phase microextraction combined with comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (HS-SPME-GC×GC-TOFMS) was used to characterize PDO and PGI balsamic vinegars and to evaluate the feasibility of volatilomic differentiation between the investigated products. Extraction conditions were comparatively assessed using a pooled quality-control sample by evaluating two SPME fiber coatings (PDMS/DVB and DVB/CAR/PDMS) and a high ionic-strength condition (40% w/v NaCl); the DVB/CAR/PDMS fiber combined with salting-out provided the highest analytical response and was selected for subsequent analyses. After peak detection and alignment, 1397 features were initially detected and reduced to 227 after blank and artifact filtering. Mann-Whitney U testing with false discovery rate correction identified 100 statistically significant features, of which 67 were putatively identified. Multivariate analysis revealed a clear separation between PDO and PGI samples: analyzed PDO vinegars were enriched in furanoids, lactones, esters, aldehydes, acids, phenols, ketones, and alcohols, whereas PGI samples showed comparatively higher levels of terpenoids. These chemical trends align with the technological differences between the two production systems. Mapping volatile features to literature-reported odor descriptors suggested that the higher abundance of furanoids, lactones, and related compounds in the PDO samples may contribute to sweeter, caramel-like, creamy, balsamic, woody, and floral nuances. Overall, the integration of HS-SPME-GC×GC-TOFMS with statistical filtering and multivariate analysis demonstrates the feasibility of a volatilomic analytical workflow capable of differentiating selected PDO and PGI products, and establishes a methodological framework for future large-scale studies on authenticity and quality control.