Determination of ubiquinone in blood by high-performance liquid chromatography with post-column fluorescence derivatization using 2-cyanoacetamide.

It was shown that ubiquinone (CoQ(10)) and ubiquinol (CoQ(10)H(2)) produce fluorescence products under alkaline conditions when reacted with 2-cyanoacetamide. The reaction mixture from CoQ(10) 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 CoQ(10) and CoQ(10)H(2) in blood. CoQ(10) 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 CoQ(10) and CoQ(10)H(2) from plasma using 1-propanol with potassium formate was effective for excellent separation of CoQ(10) and CoQ(10)H(2) from other fluorescent substances in the blood. This simple and rapid pretreatment was considered to minimize the oxidation of CoQ(10)H(2). On the other hand, CoQ(10) and CoQ(10)H(2) 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 CoQ(10)H(2)/total CoQ(10) ratio as a biomarker of oxidative stress.