Simultaneous high-performance capillary electrophoresis analysis of the reduced and oxidised forms of ascorbate and glutathione

We describe here a procedure for the simultaneous analysis of the oxidised and reduced forms of the major cellular hydrophillic antioxidants, ascorbic acid (vitamin C) and glutathione (gamma-L-glutamyl-L-cysteinylglycine), by high-performance capillary electrophoresis. Separations are performed in uncoated fused-silica capillaries using 200 mmol/l borate pH 9.0, containing 20% (v/v) acetonitrile as the background electrolyte with fixed-wavelength UV absorbance detection at 185 nm. The influence of pH, organic solvent and other additives on the resolution of these compounds is described and we show that the optimised protocol is capable of simultaneously resolving other thiol components including, N-acetylcysteine and methyl-S-glutathione. The method is suitable for the analysis of these antioxidants in Arabidopsis and Nicotiana leaf tissue and is compatible with the use of the high ionic strength, acidic extraction solvents which are necessary to quench the redox equilibria of these labile components.