Simultaneous determination of purine metabolites in ovine urine and blood plasma by high-performance liquid chromatography

Abstract

High-performance liquid chromatographic methods for directly determining products of nucleic acid and purine catabolism in urine and blood plasma of sheep were developed. Urine and plasma samples were diluted 1:3 with deionized water. Fractionation and quantification of allantoin, uric acid, hypoxanthine and xanthine were performed using two Nova-Pak C18 columns (300 x 3.9 mm, Waters). Binary gradient programs and UV detection were used for purine metabolite analysis. Satisfactory fractionation of all analytes in urine and plasma was obtained in less than 19 and 21 min, respectively. The average recoveries of standard compounds added to the assayed samples were ∼100%. The low coefficient of variation (1-2%) as well as the low detection limits (0.09-0.36 nmol) indicate satisfactory precision, reproducibility and sensitivity of the proposed methods. These chromatographic methods are suitable for routine quantification of purine metabolites in a large number of samples.