Determination of Xanthine and Uric Acid in Xanthinuric Urine and Extracellular Fluid of Porcine Endothelial Cells of the Pulmonary Artery by High Performance Liquid Chromatography

A reverse phase high performance liquid chromatography method with ultraviolet light detection was developed to determine the concentrations of xanthine and uric acid in the extracellular fluid of endothelial cells of porcine pulmonary arteries and in normal and xanthinuric urine. Normal urine samples were collected randomly and filtered through a 0.45 ?m nylon filter before analysis. Xanthine and uric acid concentrations were determined by a constant volume standard addition method. The mobile phase was 20 mM KH2PO4 at pH 5.1 and the detection wavelength 270 nm (xanthine) and 293 nm (uric acid). The injection volume was 20.0 ?L and attenuation was 0.01 absorbance unit full scale (AUFS). Retention times for xanthine and uric acid standards were respectively 13 ? 0.1 minutes and 7.0 ? 0.1 minutes. The linear correlation coefficient for xanthine and uric acid working curves were 0.995 and 0.998 respectively. The linear dynamic range, at the low concentration limits of xanthine and uric acid, was 5 ?M to 40 ?M, and 2 ?M to 20 ?M, respectively. The sensitivity of xanthine in 31 mM Na2HPO4/NaH2PO4 at pH 7.4 was 0.08 AU/?M, whilst that of uric acid in the same physiological buffer was 0.21 AU/?M. The limit of detection (LOD) for xanthine and uric acid were respectively 5.1 ?M, (S/N = 2) and 1.6 ?M (S/N = 3). Xanthine in xanthinuric urine was 2.8 ? 0.1 mM; while uric acid in normal urine was 5.7 ? 0.1 mM. In the extracellular fluid, the oxypurine peaks were identified as uric acid, hypoxanthine and xanthine. ( en )