Analysis of normal and modified nucleosides in urine by capillary electrophoresis

Modified nucleosides excreted in urine have been studied as potential diagnostic markers for cancer and AIDS, and as indicators for the whole-body turnover of RNA. Until now reversed-phase (RP) HPLC and, to some extent, immunoassays are the preferred analytical methods for urinary nucleosides. A new capillary electrophoretic method for the analysis of normal and modified nucleosides in urine has been developed and optimized in our laboratory. The separation of nucleosides extracted from normal human urine on phenyl boronic acid affinity chromatography columns was performed in uncoated 565 mm (500 mm to detection window) x 50 mu m i.d. capillary tubing using a 300 mM SDS - 25 mM berate - 50 mM phosphate buffer (pH 6.7), a 45-s load, a voltage of 7.5 kV (41 mu A) and UV detection at 260 and 210 nm. The average recovery of the nucleosides was 91 %. The calibration curves were linear over all physiological and pathophysiological concentration ranges and the limits of detection were at micromolar levels. Reproducibility of migration times were better than 1 % (coefficient of variation, CV), and the reproducibilities of the determined concentrations were better than 5 % for standards and 6-15 % for extracted urine. The developed method was used to quantify 15 normal and modifled nucleosides in 25 normal urines to establish reference ranges. The analysis time was less than 45 min.