Simultaneous Determination of Dihydrofluorouracil and 5-Fluorouracil in Plasma by High-Performance Liquid Chromatography

Dihydrofluorouracil (FUH2) is the product of the first rate-limiting step in catabolism of 5-fluorouracil (5-FU), catalyzed by the enzyme dihydropyrimidine dehydrogenase. In humans, more than 80% of administered 5-FU is degraded through this catabolic pathway. The ability to measure FUH2and 5-FU simultaneously may provide an index of the extent to which 5-FU is catabolized. A sensitive and efficient extraction and HPLC method has been developed for simultaneous measurement of FUH2and 5-FU in patients’ plasma. Trichloroacetic acid precipitation of plasma proteins was followed by extraction into ethyl acetate, evaporation under nitrogen, and reconstitution in phosphate buffer. The extract was analyzed by isocratic chromatography using a C18reversed-phase column with uv detection at 268 nm (5-FU) and 220 nm (FUH2). The detection limit is 0.005 nmol on column for aqueous standards or 0.20 μmin 1 ml of plasma standards for both compounds. This method can be applied to pharmacokinetic studies of 5-FU in patients and may be useful as a means of assessing the activity of dihydropyrimidine dehydrogenase.