Simultaneous determination of allopurinol and oxypurinol by liquid chromatography using immobilized xanthine oxidase with electrochemical detection

A novel liquid chromatographic method using an immobilized xanthine oxidase reactor and an electrochemical detector was developed for the simultaneous determination of allopurinol and oxypurinol in rat plasma, intestinal wash and bile. Xanthine oxidase was immobilized on 5-microns aldehyde silica (prepacked into a 2 mm x 10 mm cartridge) in a simple procedure. Allopurinol eluted from an analytical column was converted to oxypurinol in the enzyme reactor with the eluent as the reaction medium and detected with high selectivity using an amperometric detector with a glassy carbon electrode at the applied potential of +0.85 V. High specificity of the enzymatic reaction combined with selectivity of the electrochemical detection eliminated the need for an extensive sample preparation. The assay was linear in the range 15-500 ng/ml of rat plasma, intestinal wash and bile with a low limit of detection of 10 pg on-column (signal-to-noise ratio = 4) for both allopurinol and oxypurinol.