Improved high-performance liquid chromatographic assay for the quantification of 5-bromo-2'-deoxyuridine and 5-bromouracil in plasma.

A sensitive and specific procedure using high-performance liquid chromatography (HPLC) was developed for the quantification of 5-bromo-2'-deoxyuridine (BUdR) and 5-bromouracil (BU) in plasma. BUdR and BU were first extracted with a mixture of ethyl acetate and 2-propanol from plasma presaturated with solid ammonium sulfate. Following evaporation of the organic extract, the remaining residue was reconstituted in saturated ammonium sulfate solution, washed with a mixture of n-pentane-methylene chloride and re-extracted with the original solvent mixture. The organic extract was evaporated, reconstituted in mobile phase and chromatographed on a regular-bore ODS HPLC column using ultraviolet absorbance detection. The BUdR and BU quantification limits were both 0.1 microM, the mean intra-assay coefficients of variation were 5.0 and 5.6%, respectively, and the mean inter-assay coefficients of variation were 5.4 and 10.7%, respectively. This method was used to determine steady-state femoral arterial and hepatic venous plasma concentrations of BUdR and BU in a patient receiving a continuous intravenous infusion of BUdR (20 mg/kg per day).