Analysis of Glutathione in Biological Samples by HPLC Involving Pre-Column Derivatization with o-Phthalaldehyde.

Abstract

Glutathione (GSH) forms conjugates with polyamines in prokaryotes and eukaryotes. There is also evidence suggesting cross-talk between GSH and polyamines to regulate cellular homeostasis and function, particularly under the conditions of oxidative stress. Because of its versatile roles in cell metabolism and function, a number of high performance liquid chromatography (HPLC) methods have been developed for glutathione analysis. Here, we describe our rapid and sensitive method for the analysis of GSH and the oxidized form of glutathione (GS-SG) in animal tissues and cells by HPLC involving pre-column derivatization with o-phthalaldehyde (OPA). OPA reacts very rapidly (within 1min) with S-carboxymethyl-glutathione at room temperatures (e.g.,20-25°C) in an autosampler, and their derivatives are immediately injected into the HPLC column without any need for extraction. This method requires two simple steps (a total of 15min) before samples are loaded into the autosampler: (a) the conversion of GS-SG into GSH by 2-mercaptoethanol; and (b) the oxidation of GSH by iodoacetic acid to yield S-carboxymethyl-glutathione. The autosampler is programmed to mix S-carboxymethyl-glutathione with OPA for 1min to generate a highly fluorescent derivative for HPLC separation and detection (excitation wavelength 340nm and emission wavelength 450nm). The detection limit for GSH and GS-SG is 15pmol/ml or 375fmol/injection. The total time for chromatographic separation (including column regeneration) is 16min for each sample. Our routine HPLC technique is applicable for analyses of cysteine and cystine, as well as polyamines and GSH-polyamine conjugates in biological samples.