Development of an amperometric assay for the determination of reduced glutathione, using glutathione peroxidase and screen‐printed carbon electrodes chemically modified with cobalt phthalocyanine

The systematic characterization and optimization of bovine erythrocyte glutathione peroxidase is described, together with its application in an assay employing amperometry in stirred solutions for the determination of endogenous levels of reduced glutathione (GSH). Initial studies employed UV spectroscopy, cyclic voltammetry, and amperometry in stirred solutions (to construct hydrodynamic voltammograms) for several investigations including the selection of a suitable hydroperoxide second substrate, the investigation of solution conditions on the enzyme reaction rate, and reconstituted enzyme stability. Subsequently, amperometry in stirred solutions was used to monitor the enzyme-catalyzed rate of GSH oxidation and to construct calibration plots over the concentration range 10 to 50 μM (corresponding to normal circulating GSH levels after the envisaged sample preparation step consisting of a simple twenty fold dilution of whole blood). Enzyme selectivity studies were performed using several potential physiological interferents and selected pharmaceuticals that may be encountered in the circulation of patients being treated for rheumatoid arthritis. The coefficient of variation for the enzyme linked amperometric assay was 7.28% (n = 5) for solutions containing 40 μM GSH.