Fluorometric determination of hypoxanthine and xanthine in biological fluids by high-performance liquid chromatography using enzyme reactors.

A selective and sensitive assay of hypoxanthine and xanthine in biological fluids by high-performance liquid chromatography coupled with immobilized-enzyme reactors was developed. The separations were achieved by reversed-phase liquid chromatography. Hydrogen peroxide produced from hypoxanthine and xanthine by immobilized xanthine oxidase was determined fluorometrically using immobilized peroxidase and p-hydroxyphenylacetic acid. Immobilized enzymes were prepared by intermolecular cross-linking to controlled-pore glass. Assay of allopurinol was also possible by the present method. The method was applied to serum and urine. The detection limits of hypoxanthine and xanthine were approximately 50 and 120 pg per injection, respectively.