Determination of total glutathione in cell lysates by high performance liquid chromatography with O-phthalaldehyde precolumn derivatization in the presence of tris(2-carboxyethyl)-phosphine

A very sensitive HPLC method for the determination of total glutathione (GS H) and gamma -glutamyl-cysteine (gamma -Glu-Cys) content in cell lysates is presented. It is based on a precolumn derivatization with o-phthalaldehyde (OPA) using tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent. Sepa ration of the peptide derivatives is carried out by reversed-phase chromato graphy on a C18 column, followed by spectrofluorimetric detection. The fluo rescence response of GSH and gamma -Glu-Cys derivatives is linear over a ra nge of 0.05 to 100 muM and 1 to 25 muM, respectively, with good precision. The detection limits approach 26 fmol and 60 fmol on column, respectively. In contrast the detection limit for GSH in the presence of DTT, which is of ten used as the reducing agent, is 250 fmol GSH on column. The results for the GSH concentration of cell lysates are in agreement with a radiotracer m ethod based on metabolic labeling of GSH with [S-35]-Cys.