Simultaneous chiral analysis of four DL-thiol compounds in human saliva by Ultra-high-performance liquid chromatography high-resolution mass spectrometry: Differential evaluation of oxidative stress between smokers and non-smokers

Smoking is detrimental to health. However, the potential of dynamic changes in chiral thiol compounds in human saliva, both before and after smoking, as indicators of oxidative stress in vivo remains unexplored. A novel ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC HRMS) method was developed for the simultaneous separation of four DL-thiol compounds in human saliva, utilizing a new ( R )-(5-(3-isothiocyanatopyrrolidin-1-yl)-5-oxopentyl (NCS-OTPP) pre-column derivatization approach. Chromatographic separation was carried out using a YMC Triart C18 column (2.0 × 150 mm, 1.9 µm), with isocratic elution of 0.1 % formic acid aqueous solution and 0.1 % formic acid methanol-acetonitrile (7:3, v/v ) at a ratio of 65:35. Separation of DL-glutathione (GSH), DL-cysteine (Cys), DL-homocysteine (Hcy), and γ-glutamylcysteine (γ-Glu-Cys) achieved resolution degrees of 1.60–1.89, with complete separation. Linear calibration was observed (R 2 ≥ 0.9992) within a range of 0.5–20 µM, with detection limits spanning 2.50–14.40 fmol. The intraday and interday precisions ranged from 0.33 % to 9.64 %, while average spiked recoveries were 92.15–106.22 %. This method was successfully applied to assess the four DL-thiol compounds in the saliva of 10 smokers and 10 non-smokers at different time points. Results revealed significantly higher concentrations of the DL-thiol compounds in the saliva of smokers compared to non-smokers, with significant differences for l-GSH, DL-Cys, and l-Hcy ( p < 0.05), returning to baseline levels within 15 min. Dynamic monitoring curves before and after smoking exhibited consistent trends with the fitted curves. This study introduces a new method for monitoring dynamic changes in DL-thiol compounds in saliva and offers insight into potential subhealth indicators linked to abnormal chiral thiol metabolism in vivo.