Analysis of 5-fluorouracil in plasma by precolumn derivatization with 4-bromomethyl-7-methoxycoumarin, followed by multi-dimensional high-performance liquid chromatography.

An assay for 5-fluorouracil (5-FU) has been developed that utilizes a double extraction with ethyl acetate, followed by precolumn derivatization with 4-bromo-methyl-7-methoxycoumarin. The reaction mixture was quenched with 5% acetic acid, extracted with hexane, and analyzed by multi-dimensional high-performance liquid chromatography. Derivatized 5-FU was injected into a cyanopropyl column and a heart cut containing the analyte was then switched to an octadecyl column and quantitated by fluorescence detection. The assay had a limit of detection of 0.5 ng 5-FU/ml plasma and was linear to 20 micrograms/ml. It was shown to be free of interferences from the other anticancer agents commonly used in combination with 5-FU. This assay should have the sensitivity needed to measure the low levels that occur after low-dose, continuous infusion of 5-FU.