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 (CoQ(10)H(2)) and total co enzyme Q(10) (TQ(10)) in human plasma. The concentration of ubiquinone-10 ( CoQ(10)) was calculated by subtraction of CoQ(10)H(2) from TQ(10). Preparat ion of reducing agent and precolumn reduction was performed by a programmab le auto-injector. The two mobile phases used were: A, 100% of methanol cont aining 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 CoQ(10)H(2) concentration from 0.1 to 3 mu mol/l. The dete ction limit was 2.5 nmol/l with an injection volume of 20 mu l. The analyti cal recovery and reproducibility were generally >90%. To validate the metho d, 18 freshly collected plasma samples of normal healthy subjects were anal ysed. The mean ratio of CoQ(10)H(2)/CoQ(10) was 93:7. The proposed method i s sensitive, reliable and can be used for clinical investigation. (C) 1999 Elsevier Science B.V. All rights reserved.