Simultaneous determination of adenosine and its metabolites by capillary electrophoresis as a rapid monitoring tool for 5′-nucleotidase activity

A simple and rapid capillary electrophoretic method was developed for the simultaneous determination of micro-molar adenosine, hypoxanthine and inosine in enzyme assays without using radioactive labeled substrates. Prior to electrophoretic separation, addition of acetonitrile and sodium chloride to the assay solution and brief centrifugation are recommended for the purpose of sample cleanup and sample stacking. Under the optimal condition, the good separation with high efficiency was achieved in 6 min. Using deoxyadenylate as an internal standard, the linear range of the method was 5–200 μM, and the concentration limits of detection of adenosine, hypoxanthine and inosine were 2.2, 3.6 and 1.4 μM, respectively. Application of the proposed method was demonstrated by the activity assay of 5′-nucleotidase from Hep G2 cells.