Polyfunctional thiols are key contributors to wine aroma due to their extremely low odor thresholds, and their quantitative analysis remains challenging as a result of their ultra-trace concentrations and high reactivity. This work presents the first method based on Orbitrap highresolution mass spectrometry (HRMS) in parallel reaction monitoring (PRM) mode for quantifying thiols at ng/L levels in wine. Thiols in wine were derivatized using 4,4'-dithiodipyridine and isolated by liquid-liquid extraction. This protocol allowed rapid sample preparation with minimum labor input and low consumable expenses. Instrumental analysis was conducted using ultra-highperformance liquid chromatography (UHPLC) coupled to quadrupole Orbitrap HRMS in PRM mode. Twenty thiol analytes, including literature-known, recently identified, and novel thiols were selected and validated by the optimized method in three wine matrices. The overall analytical performances demonstrated by this method were equivalent, and in most cases, greater than many previously developed GC–MS or LC–MS methods. The validated method was applied to analyze a selection of wines in which 12 thiols were quantified.< Leer menos