Simultaneous Determination and Enantiomeric Separation of Five DL-Thiol Compounds Using UHPLC-HRMS With Chiral Derivatization in Sake: Dynamic Monitoring of Human Urine After Drinking

Abstract

Sake, a fermented alcoholic beverage made from rice and Aspergillus oryzae through microbial fermentation, contains thiol compounds that are the primary source of its distinctive sweet aroma. However, to date, there has been no report on the detection of chiral thiol compounds in sake. This study introduces a novel UHPLC-HRMS method utilizing the (R)-(5-(3-isothiocyanatopyrrolidin-1-yl)-5-oxopentyl) triphenylphosphonium (NCS-OTPP) chiral mass spectrometry probe for the simultaneous detection of five DL-thiol compounds in sake. Separation was achieved using a YMC Triart C18 column (2.0 × 150 mm, 1.9 μm), employing an isocratic elution method to isolate DD/LL-GSH, γ-L-Glu-L-Cys, DL-Cys, DL-Hcy, and DL-Ac-Cys. The method demonstrated excellent linearity (R² ≥ 0.9992), intraday precision (0.43%–13.18%), and an average recovery rate of 91.62%–110.40%. A comparative analysis of DL-thiol compound content in seven different types of sake from various countries and manufacturers revealed the presence of five chiral thiol compounds—LL-GSH, γ-L-Glu-L-Cys, DL-Cys, DL-Hcy, and L-Ac-Cys—across Japanese and Korean sake, with L-Cys being the most abundant and D-Ac-Cys the least. Notably, γ-L-Glu-L-Cys was detected only in two types of Korean sake and absent in Japanese sake. Additionally, the study investigated the metabolic kinetics of chiral thiol compounds in human urinary specimens after alcohol ingestion, constructing a metabolic fitting curve. The peak concentrations of DL-Cys, DL-Hcy, and LL-GSH were attained 15 min following consumption, with slow clearance, returning to baseline at 60 min. The metabolic fitting curve effectively captures the dynamic changes in urinary metabolism. This research presents a new method for detecting chiral thiol compounds in sake and monitoring urinary metabolism after alcohol consumption.