Determination of 5-fluorouracil and its main metabolites in plasma by high-performance liquid chromatography

A relatively simple and sensitive ion-pair high-performance liquid chromatographic method has been developed for measuring the anticancer drug fluorouracil and its main metabolites, fluorouridine, fluorodeoxyuridine and fluorodeoxyuridine monophosphate in human plasma. A reversed-phase C18 column (150 mm x 2 mm I.D.) and ultraviolet detection (280 nm) were used. The influence of the tetrabutylammonium phosphate and monopotassium phosphate concentrations and pH of the mobile phase on the various k' values was investigated. The optimal k' values obtained for the four compounds ranged between 0.7 and 5.9. The optimized eluent was (10(-4) M tetrabutylammonium phosphate plus 2.10(-2) M potassium dihydrogen phosphate, pH 5.9)-methanol (95.5:4.5, v/v). The flow-rate was 0.3 ml/min. The procedure for plasma preparation included solvent extraction using ethyl acetate-methanol (80:20) followed by elution on C18 Sep-Pak cartridges to separate the four compounds from constituents normally occurring in plasma. The detection limit of the assay was 2 ng/ml (5-fluorouracil), 10 ng/ml (5-fluorouridine), 10 ng/ml (5-fluoro-2'-deoxyuridine) and 50 ng/ml (5-fluoro-2'-deoxyuridine 5'-monophosphate).