Determination of glutathione, cysteine and N-acetylcysteine in rabbit eye tissues using high-performance liquid chromatography and post-column derivatization with 5,5'-dithiobis(2-nitrobenzoic acid).

A high-performance liquid chromatographic method to determine glutathione, cysteine and N-acetylcysteine in rabbit retina, vitreous and lens has been developed. The thiols are separated using a 25 x 0.46-cm octadecylsilane column with 0.5 M phosphate buffer, pH 3, as mobile phase. The detection, at 412 nm, involves a post-column derivatization with 5,5-dithiobis(2-nitrobenzoic acid) in presence of cationic micelles of hexadecyltrimethylammonium bromide that enhances the sensitivity. The detection limits are 0.21, 0.92 and 0.61 mumol/g wet sample for glutathione, cysteine and N-acetylcysteine, respectively.