Determination of in plasma samples by dual-electrode amperometric detection and liquid chromatography

Co-Q10 is a lipid-soluble benzoquinone that is an important factor in free radical scavenging, mitochondrial membrane stability and ATP synthesis. Dietary Co-Q10 is a powerful antioxidant that has been useful in lessening the damage associated with ischemia-reperfusion injuries and aiding in the recovery of myocardial function after myocardial infarction. However, the role of dietary Co-Q10 in oxidative damage and repair is not well understood. Previous LC-EC methods have used packed carbon bed electrodes with high overpotentials that were sufficient to oxidize and reduce several biological compounds, thereby decreasing the selectivity that can be achieved with EC detection. Thin-layer cell dual electrode detection enables monitoring of reduced and oxidized forms of Co-Q10 simultaneously and selectively. The oxidation (+0.45 V vs. Ag/AgCl) and reduction (-0.4 V vs. Ag/AgCl) electrode potentials were optimized to oxidize and reduce the electroactive quinone moiety. The reduced form of Co-Q10 was prepared from the commercially available oxidized form using a Jones reductor. Confirmation of its formation was determined using the current ratios of the peak and half wave potentials from previously generated hydrodynamic voltammograms, using the oxidized form with electrodes in a series configuration. This analytical system was successfully applied to determine basal concentrations of oxidized (510 nM) and reduced (500 nM) Co-Q10 in human plasma. Peak identity of oxidized and reduced Co-Q10 was confirmed by two orthogonal methods: by the current ratios at +0.45 V and +0.25 V and -0.4 V and -0.2 V (vs. Ag/AgCl) as well as by retention time. Detection limits were determined to be 5 nM, with a linear range of three orders of magnitude.