High-performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide, and related thiols and disulfides

A rapid and sensitive high-performance liquid chromatography method for determination of nanomole levels of glutathione, glutathione disulfide, cysteine glutathione-mixed disulfide and 20 related sulfur-containing amino acids or their derivatives has been described. The procedure is based upon the initial formation of S-carboxymethyl derivatives of free thiols with iodoacetic acid followed by conversion of free amino groups to 2,4-dinitrophenyl derivatives by reaction with 1-fluoro-2,4-dinitrobenzene. Chromatography of the reaction mixture without sample isolation is on a 3-aminopropylsilane derivatized silica column and elution with a sodium or ammonium acetate gradient in a water-methanol-acetic acid solvent at pH 4.5. Determination of nanomole levels of glutathione, glutathione disulfide, and cysteine glutathione-mixed disulfide in biological samples is described.