Determination of Ubiquinone in Blood by High-Performance Liquid Chromatography with Post-Column Fluorescence Derivatization Using 2-Cyanoacetamide

It was shown that ubiquinone (CoQ10) and ubiquinol (CoQ10H2) produce fluorescence products under alkaline conditions when reacted with 2-cyanoacetamide. The reaction mixture from CoQ10 gave fluorescence with excitation and emission maximum wavelengths at 442 nm and 549 nm, respectively. This reaction was considered to proceed via Craven’s reaction. Moreover, 2-cyanoacetamide was shown to be a useful reagent for high-performance liquid chromatography (HPLC) with post-column fluorescence derivatization of CoQ10 and CoQ10H2 in blood. CoQ10 showed a linear response in the range of 0.32–1276 ng, and the detection limit (S/N = 3) was 0.16 ng. Moreover, the sample pretreatment by deproteinization and extraction of CoQ10 and CoQ10H2 from plasma using 1-propanol with potassium formate was effective for excellent separation of CoQ10 and CoQ10H2 from other fluorescent substances in the blood. This simple and rapid pretreatment was considered to minimize the oxidation of CoQ10H2. On the other hand, CoQ10 and CoQ10H2 in plasma samples obtained by finger prick were detected, as in venous blood obtained by venipuncture. Our method involving the simple and rapid collection of plasma by finger prick and sample pretreatment is thought to be applicable for the determination of CoQ10H2/total CoQ10 ratio as a biomarker of oxidative stress.