Chiral mass spectrometry probe-based simultaneous analysis of thiol compounds in beer and urinary metabolism after consumption

A method for analyzing DL-thiol compounds in beer is established by NCS-OTPP. • The content of DL-thiol compounds are used to distinguish light and wheat beers. • The fitting curve reflect metabolic status of thiol compounds after drinking beer. • Provides novel methods for evaluating the quality of fermented foods. Thiol compounds are pivotal determinants of beer flavor and quality. However, conventional analytical approaches only enable the quantification of total racemic thiols, failing to achieve enantiomeric separation and targeted detection, which severely restricts the in-depth exploration of thiol-related flavor characteristics and biological activities. Herein, we developed and validated a UHPLC-HRMS strategy based on a self-synthesized chiral mass spectrometry probe, ( R )-(5-(3-isothiocyanatopyrrolidin-1-yl)-5-oxopentyl) triphenylphosphonium (NCS-OTPP), for the simultaneous enantioseparation and quantitative analysis of four chiral thiols (glutathione (GSH), homocysteine (Hcy), cysteine (Cys), N -acetylcysteine (NAC)) and γ-glutamylcysteine (γ-GluCys) in beer. Benefiting from the integrated advantages of permanent charge enhancement, specific chiral recognition, and thiol-targeted derivatization of NCS-OTPP, all diastereomeric derivatives were well baseline-separated on a conventional C18 reversed-phase column with resolution ranging from 1.52 to 1.71. The established method exhibits excellent analytical performance, including limits of detection of 2.50–14.40 fmol, intra-and inter-day RSDs of 1.45%–10.47%, and matrix-spiked recoveries of 98.55%–105.87%. Furthermore, PCA analysis based on the enantiomeric profiles of ten commercial beer samples (five wheat beers and five light beers) achieved precise classification of beer types, with L-Cys and D-Hcy confirmed as the core differential characteristic markers. In vivo metabolic monitoring revealed that urinary thiol enantiomers from healthy volunteers after beer ingestion presented a dynamic trend of “rapid surge-gradual decline” within 30–60 min. To demonstrate the rapid absorption, hepatic antioxidant metabolism and renal clearance characteristics of exogenous thiol compounds. Overall, this innovative chiral derivatization-MS platform provides a reliable technical support for beer flavor evaluation, quality traceability, and food bioactivity research, and offers a novel and feasible workflow for in-depth in vivo analysis of chiral thiol metabolites. Download: Download high-res image (132KB) Download: Download full-size image