Identification and characterisation of thiolated polysulfides in must and wine using online SPE UHPLC-HRMS

3-Mercaptohexanol and 4-methyl-4-mercaptopentanone are volatile thiols with a low perception threshold and are found in relatively low concentrations in several types of wines, e.g. Sauvignon blanc. They contribute positively to the flavour of a wine when in their free form, but they can oxidise, especially in the presence of Cu²⁺ and sulfur residues originating from pesticide treatments on the grapes. This condensation reaction results in the formation of polysulfides, which during storage can cause the release of H₂S: a compound known to give rise to off-flavour in wine. The formation of these polysulfides has been proposed to originate from cysteinyl and glutathionyl S-conjugate precursors, but they have not yet been characterised. In this work, a method using ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry and online solid-phase extraction was designed and optimised for detection of these S-conjugates. The method allowed the detection of 21 suspected symmetrical and asymmetrical S-conjugates in thiol-supplemented aqueous solutions, of which 17 were also recovered in supplemented synthetic musts and wines. Moreover, the proposed method was used to investigate polysulfide formation upon addition of two different types of sulfur. Differences in formation of S-conjugates were evident in the synthetic samples: a higher relative abundance was observed upon addition of wettable sulfur compared to washed sulfur. For the commercial wines, differences in polysulfide formation were minor and merely related to the differences between the wines and not to the type of sulfur added.