Assay of γ-glutamylcysteine synthetase activity in Plasmodium berghei by liquid chromatography with electrochemical detection

This work describes a high-performance liquid chromatography (HPLC) method to determine gamma -glutamylcysteine (gamma -GC). the intermediate product of glutathione biosynthesis. Separation relies on isocratic reversed-phase chromatography using a Symmetry C18 HPLC column, particle size 5 mum, 4.6 x 250 mm i.d. The mobile phase is methanol-dibasic sodium phosphate (pH 6.6; 2.8 mM) (10.90, v/v) at the flow-rate of 0.5 ml;min and detection is operated electrochemically ( + 200 and + 5 50 mV) with a pre-column derivatisation reaction using ortho - phthalaldehyde (OPA) as reagent. Under these conditions the calibration range of gamma -GC was 0.3-10 mug/ml; the limit of quantification was 0.3 mug/ml; accuracy, expressed as % Bias, was < 10 and precision (%CV) was < 6. The proposed HPLC assay was used to quantitate the gamma -glutamylcysteine produced by the gamma -glutamylcysteine synthetase of the rodent malaria parasite Plasmodium berghei in an in vitro enzymatic assay. (C) 2001 Elsevier Science B.V. All rights reserved.