Automated high-performance liquid chromatographic method with precolumn reduction for the determination of ubiquinol and ubiquinone in human plasma.

We developed a gradient HPLC method with automated precolumn reduction for direct electrochemical detection of ubiquinol-10 (CoQ10H2) and total coenzyme Q10 (TQ10) in human plasma. The concentration of ubiquinone-10 (CoQ10) was calculated by subtraction of CoQ10H2 from TQ10. Preparation of reducing agent and precolumn reduction was performed by a programmable auto-injector. The two mobile phases used were: A, 100% of methanol containing 50 mM sodium perchlorate and 10 mM perchloric acid; and B, a mixture of ethanol and tert.-butanol (80:20, v/v). Sample preparation was simply a deproteinisation process with 10-fold ethanol. A good linear relationship was obtained for CoQ10H2 concentration from 0.1 to 3 micromol/l. The detection limit was 2.5 nmol/l with an injection volume of 20 microl. The analytical recovery and reproduciblity were generally >90%. To validate the method, 18 freshly collected plasma samples of normal healthy subjects were analysed. The mean ratio of CoQ10H2/CoQ10 was 93:7. The proposed method is sensitive, reliable and can be used for clinical investigation.