Reversed-phase liquid chromatography of 5-fluoro-uracil nucleosides and nucleotides in the presence of quaternary ammonium ions

Abstract

Reversed-phase liquid chromatography is used for the separation of 5-fluoro-uracil, its deoxyribo- and ribonucleosides and nucleotides. The bases and nucleosides are easily separated from their naturally occurring analogues on an octadecyl silica column eluted with 2·10 −2 M KH 2PO 4 (pH 5.0) containing 5% (v/v) of methanol. This system can be applied to the measurement of 5-fluoro-2′-deoxyuridine serum levels down to 0.1 μg/ml. Addition of small amounts (10 −3 M) of tetrabutylammonium phosphate to the eluent results in a large retention increase for the nucleotides, while the capacity ratios of the bases and nucleosides remain unchanged. The influence of the tetrabutylammonium phosphate and ammonium phosphate concentrations and pH of the eluent on the various k′ values was investigated. Evidence is presented indicating that the quaternary ammonium compound is adsorbed onto the octadecyl silica surface; nucleotides are probably retained as adsorbed tetrabutylammonium ion-pairs.