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

Antonello Di Paolo, Romano Danesi, Laura Ciofi, Francesca Vannozzi, Guido Bocci, Marianna Lastella, Federica Amatori, Bianca Maria Martelloni, Toni Ibrahim, Dino Amadori, Alfredo Falcone, Mario Del Tacca

Research output: Contribution to journalArticlepeer-review

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 × 4.6 mm, 10 μm), 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.

Original languageEnglish
Pages (from-to)362-368
Number of pages7
JournalTherapeutic Drug Monitoring
Volume27
Issue number3
DOIs
Publication statusPublished - Jun 2005

Keywords

  • 5-fluorouracil
  • Dihydropyrimidine dehydrogenase
  • Diode array detector
  • Pharmacokinetics
  • Therapeutic drug monitoring
  • Toxicity

ASJC Scopus subject areas

  • Toxicology
  • Health, Toxicology and Mutagenesis
  • Pharmacology
  • Biochemistry
  • Biochemistry, Genetics and Molecular Biology(all)
  • Pharmacology (medical)
  • Public Health, Environmental and Occupational Health

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