Assay of gamma-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 microm, 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 +550 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 microg/ml; the limit of quantification was 0.3 microg/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.