SIMULTANEOUS DETERMINATION OF ALPHA-TOCOPHEROL AND ALPHA-TOCOPHEROLQUINONE BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND COULOMETRIC DETECTION IN THE REDOX MODE

A simple, selective and highly sensitive assay method for the simultan eous determination of alpha-tocopherol and alpha-tocopherolquinone in plasma or erythrocyte membrane by high-performance liquid chromatograp hy (HPLC) with a series of multiple coulometric working electrodes (CW E) was investigated. For good separation of alpha-tocopherol and alpha -tocopherolquinone, an MC MEDICAL C-18 reversed-phase column and a mob ile phase consisting of 96% methanol [methanol-HPLC-grade distilled wa ter (96:4, v/v)] with 40 mM sodium perchlorate were used. Also, select ive, highly sensitive and simultaneous detection of these substances w as performed in redox mode using a series of four CWE. In this detecti on mode, the first, second and third CWE were set at -0.45 V for pre-r eaction and to prevent interference, the fourth CWE was used as an ele ctrode for actual measurement with its potential set at +0.40 V agains t a palladium reference electrode. The detection limits were 50-100 pg . Excellent chromatograms of alpha-tocopherol and alpha-tocopherolquin one were obtained within 8 min. The usefulness of reversed-phase HPLC with the redox detection mode was confirmed by application to the dete rmination of the concentrations of alpha-tocopherol and alpha-tocopher olquinone in a crude ethanol-hexane extract of rat plasma or erythrocy te 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.