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 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.
Takeda
H
H
Department of Pharmacology, Tokyo Medical College, Japan.
Shibuya
T
T
Yanagawa
K
K
Kanoh
H
H
Takasaki
M
M
D000818:Animals; D000975:Antioxidants / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D004563:Electrochemistry; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000378:metabolism; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley; D014810:Vitamin E / Q000031:analogs & derivatives* / Q000032:analysis / Q000097:blood
D000975:Antioxidants; D014810:Vitamin E; C002421:tocopherylquinone