Administration of
5-fluorouracil (5-FU) may be associated with severe toxicities in patients who are deficient of
dihydropyrimidine dehydrogenase (DPD) activity. For this reason, a sensitive HPLC method for the analysis of
5-FU and 5-fluoro-5,6-dihydrouracil (5-FDHU) was developed in the present study for the determination of DPD activity in nucleated cells of peripheral blood and pharmacokinetic analysis of
5-FU and 5-FDHU in humans.
5-FU and 5-FDHU were extracted from biologic matrices by adding
sodium acetate,
sodium sulfate, and
diethyl ether/
propanol. Dried samples were reconstituted in a mobile phase (KH2PO4 35 mmol/L, pH 4.0), isocratically eluted with a Hypersil C18 stationary phase (25 cm x 4.6 mm, 10 microm), and detected by a diode array detector (measurement and reference wavelengths, 215 and 360 nm, respectively).
5-Fluorocytosine (internal standard), 5-FDHU, and
5-FU were eluted within 13 minutes of the injection without interferences. Recoveries ranged between 81% to 85% for all compounds, and the method proved to be linear, with a coefficient of linearity of 0.999. The limits of detection and quantification were 3.2 and 16 ng/mL, respectively, and the within-day and between-day CV were less than 10% for both
5-FU and 5-FDHU. The present assay proved to be sufficiently sensitive and specific to evaluate cellular DPD activity and measure
5-FU and 5-FDHU plasma concentrations in
cancer patients, thus allowing therapeutic
5-FU monitoring in patients and identification of DPD-deficient subjects at major risk of severe toxicities.