Quantitative determination of vitamin E and oxidized and reduced coenzyme Q by high-performance liquid chromatography with in-line ultraviolet and electrochemical detection

Abstract

We describe a sensitive quantitative high-performance liquid chromatographic (HPLC) assay for the simultaneous determination of vitamin E isomers (α, γ- and δ-tocopherol) oxidized coenzyme Q species (ubiquinone 9, ubiquinone 10) and reduced coenzyme Q homologues (ubiquinol 9, ubiquinol 10) in various tissues, including blood and plasma. The compounds of interest are quantitatively extracted with a fast one-step lipid extraction procedure and subjected to HPLC without further purification. The extract is separated on a reversed-phase column and the eluted compounds are monitored by sequential UV and electrochemical detection. Ubiquinones are detected at their 275 nm absorbance maximum, by the UV detector, whereas tocopherols and ubiquinols are monitored by the electrochemical detector with high sensitivity and selectivity. The method can detect as little as 1 pmol of the individual ubiquinones. Detection limits for tocopherols and ubiquinols are at least two orders of magnitude lower.