Simultaneous determination of guanine, uric acid, hypoxanthine and xanthine in human plasma by reversed-phase high-performance liquid chromatography with amperometric detection

Abstract

A reversed-phase high-performance liquid chromatographic method with amperometric detection is described for the separation and quantification of uric acid, guanine, hypoxanthine and xanthine. The isocratic separation of a standard mixture of the compounds was achieved in 5 min on a Spherisorb 5 C₁₈ reversed-phase column, with a mobile phase of NaH₂PO₄(300 mmol dm^–3, pH 3.0)–methanol–acetonitrile–tetrahydrofuran (97.8 + 0.5 + 1.5 + 0.2). Uric acid, guanine, hypoxanthine and xanthine were completely separated, with detection limits in the range 2–20 pmol per injection. The effect of pH and the composition of the mobile phase on the separation are described. The hydrodynamic voltammograms of these compounds were recorded at a glassy carbon electrode. The linear range of the calibration graph for each compound was: uric acid, 1–5000 µmol dm^–3; guanine, 0.5–2000 µmol dm^–3; hypoxanthine, 0.1–5000 µmol dm^–3 and xanthine, 0.5–5000 µmol dm^–3. The within- and between-day precision was good. The uric acid and hypoxanthine content in human plasma was measured using the proposed method. Good recoveries of uric acid (97.9–103%), hypoxanthine (98.0–99.2%), guanine (96.0–98.3%) and xanthine (96.0–102%) were obtained from human plasma. The results of electrochemical detection were in good agreement with those of UV detection.