Quantification of allantoin, uric acid, xanthine and hypoxanthine in ovine urine by high-performance liquid chromatography and photodiode array detection

Abstract

A HPLC method for the determination of allantoin, uric acid, hypoxanthine and xanthine (purine metabolites) in ovine urine without the disadvantages inherent in derivatization is described. After dilution 1:6 with water, urine samples were injected onto the column. Separation and quantification of purine metabolites was achieved using two Nova-Pak C 18 columns (4 μm, 250×4.6 mm, Waters). A binary gradient program and UV detection were used for purine metabolites analysis. Clear resolution of purine metabolites was obtained in less than 15 min. Allantoin, uric acid, hypoxanthine and xanthine in the effluent were monitored at 225, 284, 250 and 267 nm, respectively. The average recoveries of purine metabolite standards added to urine samples were satisfactory (100.2–102.9%). The low coefficients of variation (0.29–0.73%) as well as the low detection (0.16–0.70 nmol) and quantification (0.52–2.32 nmol) limits indicate satisfactory precision, reproducibility and sensitivity of the proposed method. This procedure is suitable for routine quantification of purine metabolites in a large number of urine samples.