title: Improved analysis of 5-Fluorouracil and 5,6-dihydro-5-Fluorouracil by HPLC with diode array detection for determination of cellular dihydropyrimidine dehydrogenase activity and pharmacokinetic profiling.
abstract: 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.
authors.affiliation: Division of Pharmacology and Chemotherapy, Department of Oncology, Transplants and Advanced Technologies in Medicine, University of Pisa, 56126 Pisa, Italy. a.dipaolo@ao-pisa.toscana.it
authors.forename: Antonello
authors.initials: A
authors.lastname: Di Paolo
authors.forename: Romano
authors.initials: R
authors.lastname: Danesi
authors.forename: Laura
authors.initials: L
authors.lastname: Ciofi
authors.forename: Francesca
authors.initials: F
authors.lastname: Vannozzi
authors.forename: Guido
authors.initials: G
authors.lastname: Bocci
authors.forename: Marianna
authors.initials: M
authors.lastname: Lastella
authors.forename: Federica
authors.initials: F
authors.lastname: Amatori
authors.forename: Bianca Maria
authors.initials: BM
authors.lastname: Martelloni
authors.forename: Toni
authors.initials: T
authors.lastname: Ibrahim
authors.forename: Dino
authors.initials: D
authors.lastname: Amadori
authors.forename: Alfredo
authors.initials: A
authors.lastname: Falcone
authors.forename: Mario
authors.initials: M
authors.lastname: Del Tacca
mesh_terms: D000368:Aged; D000964:Antimetabolites, Antineoplastic / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D042943:Dihydrouracil Dehydrogenase (NADP) / Q000378:metabolism*; D016903:Drug Monitoring; D005260:Female; D005472:Fluorouracil / Q000097:blood* / Q000493:pharmacokinetics; D006801:Humans; D008297:Male; D008875:Middle Aged; D014498:Uracil / Q000031:analogs & derivatives* / Q000097:blood
chemical_list: D000964:Antimetabolites, Antineoplastic; C007419:dihydrouracil; D014498:Uracil; D042943:Dihydrouracil Dehydrogenase (NADP); D005472:Fluorouracil