Simultaneous determination of alpha-tocopherol and alpha-tocopherolquinone by high-performance liquid chromatography...

A simple, selective and highly sensitive assay method for the simultaneous determination of alpha-tocopherol and alpha-tocopherolquinone in plasma or erythrocyte membrane by high-performance liquid chromatography (HPLC) with a series of multiple coulometric working electrodes (CWE) was investigated. For good separation of alpha-tocopherol and alpha-tocopherolquinone, an MC MEDICAL C18 reversed-phase column and a mobile phase consisting of 96% methanol [methanol-HPLC-grade distilled water (96:4, v/v)] with 40 mM sodium perchlorate were used. Also, selective, highly sensitive and simultaneous detection of these substances was performed in redox mode using a series of four CWE. In this detection mode, the first, second and third CWE were set at -0.45 V for pre-reaction and to prevent interference, the fourth CWE was used as an electrode for actual measurement with its potential set at +0.40 V against a palladium reference electrode. The detection limits were 50-100 pg. Excellent chromatograms of alpha-tocopherol and alpha-tocopherolquinone were obtained within 8 min. The usefulness of reversed-phase HPLC with the redox detection mode was confirmed by application to the determination of the concentrations of alpha-tocopherol and alpha-tocopherolquinone in a crude ethanol-hexane extract of rat plasma or erythrocyte membrane. These findings suggest that reversed-phase HPLC with the redox detection mode using a series of four CWE is applicable to study the preventive effect of alpha-tocopherol on lipid peroxidation.