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 mu M with the coefficient regression range of 0.9987-0.9994. Detection limits ranged from 0.04 to 0.08 mu M (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 mu M, 0-72.81 mu M, and 0-4.25 mu M 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.