A new chromatographic method for the determination of cysteine, glutathione, homocysteine and Nɛ-homocysteinyllysine isopeptide in human plasma

Cysteine and glutathione can be applied as therapeutic targets in civilization diseases such as diabetes mellitus and cancers. On the other hand, an elevated concentration of homocysteine, and its metabolites such as homocysteine thiolactone and Nɛ-homocysteinyllysine result in health problems and has been indicated as an independent risk factor for cardiovascular disease and accelerated atherosclerosis. This work describes the first simplified HPLC-UV method that allows simultaneous determination of Nɛ-homocysteinyllysine isopeptide, cysteine, glutathione and homocysteine in human plasma. The assay is based on reversed-phase high performance liquid chromatography with UV detection and simultaneous reduction of disulfide bound with tris(2-carboxyethyl)phosphine and the selective pre-column derivatization of the thiol group with 1-benzyl-2-chloropyridinium bromide. Linearities of the detector responses for plasma samples were observed in ranges: 0.1-10.0 nmol/mL for Nɛ-homocysteinyllysine, 2.0-60.0 nmol/mL for glutathione and homocysteine, 20.0-600.0 nmol/mL for cysteine. The proposed method reduces the number of steps, shortens the total time of sample preparation, and limits the amount of single-use polypropylene laboratory materials.