Determination of urinary nucleosides by direct injection and coupled-column high-performance liquid chromatography

A coupled-column liquid chromatographic method for the direct analysis of 14 urinary nucleosides is described. Efficient on-line clean-up and concentration of 14 nucleosides from urine samples were obtained by using a boronic acid-substituted silica column (40 mm x 4.0 mm I.D.) as the first column (Col-1) and a Hypersil ODS2 column (250 mm x 4.6 mm I.D.) as the second column (Col-2). The mobile phases applied consisted of 0.25 mol/L ammonium acetate (pH 8.5) on Col-1, and of 25 mmol/L potassium dihydrogen phosphate (pH 4.5) on Col-2, respectively. Determination of urinary nucleosides was performed on Col-2 column by using a linear gradient elution comprising 25 mmol/L potassium dihydrogen phosphate (pH 4.5) and methanol-water (60:40, v/v) with UV detection at 260 nm. Urinary nucleosides analysis can be carried out by this procedure in 50 min requiring only pH adjustment and the protein precipitation by centrifugation of urine samples. Calibration plots of 14 standard nucleosides showed excellent linearity (r > 0.995) and the limits of detection were at micromolar levels. Both of intra- and inter-day precisions of the method were better than 6.6% for direct determination of 14 nucleosides. The validated method was applied to quantify 14 nucleosides in 20 normal urines to establish reference ranges.