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.
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.
Di Paolo
Antonello
A
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
Danesi
Romano
R
Ciofi
Laura
L
Vannozzi
Francesca
F
Bocci
Guido
G
Lastella
Marianna
M
Amatori
Federica
F
Martelloni
Bianca Maria
BM
Ibrahim
Toni
T
Amadori
Dino
D
Falcone
Alfredo
A
Del Tacca
Mario
M
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
D000964:Antimetabolites, Antineoplastic; C007419:dihydrouracil; D014498:Uracil; D042943:Dihydrouracil Dehydrogenase (NADP); D005472:Fluorouracil