Ion-pair high-performance liquid chromatography of cysteine and metabolically related compounds in the form of their S-pyridinium derivatives

Abstract

A procedure was developed for converting cysteine, glutathione, homocysteine, acetylocysteine, N-(2-mercaptopropionyl)glycine and its metabolise 2-mercaptopropionic acid into their S-pyridinium derivatives for determination by paired-ion reversed-phase high-performance liquid chromatography. The thiol compounds were derivatized with 2-chloro-1-methylpyridinium iodide within a few minutes at room temperature. The thiol group reacted smoothly with the reagent in the pH 8.2 buffer to form an S-pyridinium derivative showing strong UV absorption with a maximum at 314 nm. The reaction mixture was injected directly into a chromatograph without purification and detected spectrophotometrically at 314 nm. The six thiols in the pmol range were separated and determined in a single run on an octadecyl-bonded silica column under isocratic conditions using 0.175 M citrate buffer containing 10 mmol/l sodium octanesulphonate (pH 2.8), acetonitrile and methanol (82:6:12, v/v/v) as the mobile phase. Linear calibration graphs were obtained for concentrations of the thiols between 1 and 50 μmol/l. The detection limits ranged from 0.75 pmol for acetylocysteine to 2.1 pmol for 2-mercaptopropionic acid and the relative standard deviations were equal to or better than 9.0% at the 1 μmol/l thiol level and 0.86% at the 50 μmol/l level. Optimum derivatization reaction conditions and HPLC separation conditions were elucidated.