Jorge Mendoza Paula Soto Inés Ahumada Tatiana Garrido Departamento de Química Inorgánica y Analítica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, Chile Determination of oxidized and reduced glutathione, by capillary zone electrophoresis, in Brassica juncea plants treated with copper and cadmium A rapid method of capillary zone electrophoresis is described to determine the oxi- dized (GSSG) and reduced (GSH) form of glutathione in plant tissue. In order to sepa- rate both analytes in a fused-silica capillary, the pH and composition of the electrolyte solution were optimized. The electrolyte composition was 100 mmol/L, borate 25 mmol/LTris, and 0.2% w/v metaphosphoric acid (MPA), pH 8.2. Some instrumental conditions used to run the samples were hydrostatic injection for 30 s, 30 kV applied voltage, and UV detection (185 nm) at 257C. Linearity and useful range obtained for the calibration curves were optimum, with correlation coefficients about 0.999 in the 0–120 mmol/L range. The migration time was highly reproducible, less than 5 min being afforded to run a sample. Electrolyte buffer and samples required a careful pH control for optimal separation of both analytes. This aspect constitutes a critical analytical step when acids are used in the procedure for sample preparation. Simultaneous analysis of GSH and GSSG may provide a useful tool for comparative studies of plants in order to select those species with a potential capacity for detoxification from toxic elements or those appearing promising from phytoremediation for these elements. Keywords: Capillary zone electrophoresis / Heavy metals / Oxidized glutathione / Phytotoxicity / Reduced glutathione DOI 10.1002/elps.200305759 1 Introduction Heavy-metal phytotoxicity may trigger a variety of adap- tative responses in plants detoxification by chelation being a fairly common mechanism [1]. Glutathione per- forms several roles in plants, such as storage and trans- port of reduced sulfur, as a precursor in protein and nucleic acid synthesis, and as a modulator of the activity of several enzymes [2]. In addition, glutathione is acknowledged as one of the precursors in the synthesis of phytochelatins, which are polypeptides rich in thiol groups, capable of binding heavy metals for their sequestration. Qualitative and quantitative analysis of glutathione may provide useful information about plant response to high concentrations of heavy metals in the environment [3]. Detailed and updated reviews of differ- ent methods for quantitative analysis of reduced (GSH) and oxidized (GSSG) forms of glutathione have recently been published [4, 5]. The most commonly used meth- ods involve HPLC associated to photometric and fluori- metric detection; however, in many cases, several pre- treatments are required before injection of the sample into the column. In this respect, electrochemical detec- tion makes these previous steps unnecessary and offers higher specificity to the analysis of redox reactive com- pounds. In the last years, some methods have been developed using capillary electrophoresis (CE) technique which allow to determine simultaneously both forms of glu- tathione [6–11]. In this respect, CE has some advanta- ges compared with traditional methods, namely good reproducibility, simplicity of procedure, short time of analysis, low injection volume, and low cost of analyses [12]. Some authors have described mainly the deter- mination of reduced species [13–15] or that of both ana- lytes, GSH and GSSG, in animal fluids or tissues [6, 8–10]. Studies on plant samples are, however, scarce [11, 16]. Borate buffer is the most widely used background elec- trolyte (BGE), while phosphate [16, 17], acetate [6] and bicarbonate [14] are used to a lesser extent. In some cases, these electrolytes have been modified in order to optimize separation, migration time [18] or to stack the sample directly in the capillary [19, 20]. BGEs with photometric detector have been mostly used, however, Correspondence: Dr. Jorge Mendoza, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Olivos 1007, Casilla 233, Santiago, Chile E-mail: jmendoza@ciq.uchile.cl Fax: 156-2-7370567 Abbreviations: AAS, atomic absorption spectroscopy; GSH, reduced glutathione; GSSG, oxidized glutathione; MPA, meta- phosphoric acid 890 Electrophoresis 2004, 25, 890–896  2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim