Simultaneous determination of DL-cysteine, DL-homocysteine, and glutathione in saliva and urine by UHPLC-Q-Orbitrap HRMS: Application to studies of oxidative stress

A high-sensitivity and -selectivity mass spectrometry derivatization reagent, ( R )-(5-(3-isothiocyanatopyrrolidin-1-yl)-5-oxopentyl) triphenylphosphonium (NCS-OTPP), was developed for the enantiomeric separation of chiral thiol compounds as prospectively important diagnostic markers for oxidative stress-related diseases. Complete separation of GSH, DL-Cys, and DL-Hcy was achieved. The parent ions of all derivatives had a fragment of m/z 473.18 and a structure of m/z 75.95 (R-S = C-S-R’), conducive to qualitative and quantitative analysis. Good linear relationships were obtained for all analytes ( R 2 ≥ 0.9995). The intra-day and inter-day precision were 0.82–5.16 % and 1.02–4.18 % in saliva, and 0.81–3.45 % and 0.99–6.47 % in urine, with mean recoveries of 83.31–105.66 % and 84.09–101.11 %, respectively. The limit of detection ( S/N = 3) was 19.20–57.60 nM. Free and total GSH, DL-Cys, and DL-Hcy were detected simultaneously in saliva and urine from 10 volunteers in the normal, stressed, and stable states by UHPLC-Q-Orbitrap HRMS . The thiol compounds were quantitatively related to oxidative stress state changes.