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

• Published in: Journal of pharmaceutical and biomedical analysis 2021-03, Vol.196, p.113939-113939, Article 113939
• Main Authors: Ma, Qingkun, Qi, Chao, Li, Xi-Ling, Shi, Qing, Xu, Chun-Yan, Jin, Toufeng, Min, Jun Zhe
• Format: Article
• Language: English
• Subjects: Chiral resolution; DL-cysteine; DL-homocysteine; NCS-OTPP; Oxidative stress; Thiol; UHPLC-Q-Orbitrap HRMS
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2021.113939

[Display omitted] •A highly sensitive UHPLC-HRMS method was provided for the simultaneous analysis of GSH, DL-Cys and DL-Hcy.•D-Hcy was determined in human urine for the first time.•Fitted curves for ratios of D/L-Cys and D/L-Hcy were a convenient and effective method to evaluate 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 (R2≥ 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.