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

Ther Drug Monit. 2005 Jun;27(3):362-8. doi: 10.1097/01.ftd.0000162016.11148.1b.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aged
  • Antimetabolites, Antineoplastic / blood*
  • Chromatography, High Pressure Liquid
  • Dihydrouracil Dehydrogenase (NADP) / metabolism*
  • Drug Monitoring
  • Female
  • Fluorouracil / blood*
  • Fluorouracil / pharmacokinetics
  • Humans
  • Male
  • Middle Aged
  • Uracil / analogs & derivatives*
  • Uracil / blood


  • Antimetabolites, Antineoplastic
  • dihydrouracil
  • Uracil
  • Dihydrouracil Dehydrogenase (NADP)
  • Fluorouracil