Simultaneous Determination of Glutathione, Cysteine, Homocysteine, and Cysteinylglycine in Biological Fluids by Ion-Pairing High-Performance Liquid Chromatography Coupled with Precolumn Derivatization

Biologically active low-molecular-mass thiols, mainly including glutathione (GSH), cysteine (Cys), homocysteine (Hcy), and cysteinylglycine (Cys-Gly), are important physiological components in biological fluids, and their analytical methods have gained continuous attention over recent years. We developed and validated a novel HPLC method for the quantification of GSH, Cys, Hcy, and Cys-Gly in human plasma, urine, and saliva using 4-chloro-3,5-dinitrobenzotrifluoride as the derivatization reagent. Analyses were linear from 0.15 to 500 μM with the coefficient regression range of 0.9987–0.9994. Detection limits ranged from 0.04 to 0.08 μM (S/N = 3). The developed method was applied to quantification of four thiols in human biological fluids collected from five donors with the concentration range of 2.50–124.25 μM, 0–72.81 μM, and 0–4.25 μM for plasma, urine, and saliva, respectively. The present method seemed to be an attractive choice for the determination of thiols in plasma, urine, and saliva.