ISOLATION AND IDENTIFICATION OF URINARY NUCLEOSIDES - APPLICATIONS OF HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHODS TO THE SYNTHESIS OF 5'-DEOXYXANTHOSINE AND THE SIMULTANEOUS DETERMINATION OF 5,6-DIHYDROURIDINE AND PSEUDOURIDINE

Modified nucleosides from pooled normal human urine were extracted using a boronate affinity gel column and fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC). The major constituents in each of the 30 RP-HPLC fractions were determined by gas chromatography-mass spectrometry of the trimethylsilyl derivatives of the fractions. The same RP-HPLC method was used in the synthesis of 5′-deoxyxanthosine from authentic 5′-deoxyadenosine. In addition, the simultaneous determination of urinary 5,6-dihydrouridine (D) and pseudouridine (Ψ) was carried out by RP-HPLC using two ODS columns in series. The level of D in pooled normal urine was 4.87 nmol/μmol creatinine. The RP-HPLC method was applied to the measurement of D and Ψ levels in urine collected before and after surgery from four patients with gastrointestinal cancer. A large decline in both nucleoside levels in urines after surgery was observed in three of the four cancer patients. © 1990.