Determination of alpha-tocopherol and alpha-tocopherylquinone in rat tissues and plasma by high-performance liquid chromatography with electrochemical detection.
Alpha-tocopherol and alpha-tocopherylquinone in rat tissues and plasma were determined simultaneously by using high-performance liquid chromatography-electrochemical detection (HPLC-ED) with dual electrodes in the series mode. Biological samples were saponified in the presence of a mixture of butylated hydroxytoluene, ascorbic acid, and pyrogallol and then extracted with hexane. The compounds were separated on a C18 column using a mobile phase containing 95% methanol and 0.05 M sodium perchlorate as the supporting electrolyte. After HPLC separation, alpha-tocopherylquinone was first reduced at an upstream electrode at -500 mV Both alpha-tocopherol and the reduction product of alpha-tocopherylquinone were then oxidized downstream at +600 mV. Only the downstream electrode current was monitored for the determination. Linearity of the standard curves was obtained over the range 5-30 pmol for alpha-tocopherol and alpha-tocopherylquinone. Minimum detectable quantities (S/N of 3) were 0.25 pmol for alpha-tocopherol and 0.31 pmol for alpha-tocopherylquinone. The method was applied to analysis of the contents of alpha-tocopherol and alpha-tocopherylquinone in rat tissues and plasma. By hyperoxia, the content of alpha-tocopherol was decreased remarkably in lung, and in contrast, the contents of alpha-tocopherylquinone were increased in all tissues studied with the exception of plasma, though the content of alpha-tocopherylquinone in normal rats is quite small. The technique is particularly useful in the quantitation of the oxidation of alpha-tocopherol in biological samples.
Kanazawa
H
H
Kyoritsu College of Pharmacy, Tokyo, Japan. kanazawa-hd@kyoritsu-ph.ac.jp
Miyata
C
C
Nagata
Y
Y
Urano
S
S
Matsushima
Y
Y
D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004563:Electrochemistry; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D014018:Tissue Distribution; D014810:Vitamin E / Q000031:analogs & derivatives* / Q000032:analysis* / Q000097:blood
D014810:Vitamin E; C002421:tocopherylquinone