A simple and rapid high-performance liquid chromatographic (HPLC) method for 5-fluorouracil (5-FU) assay in plasma and possible detection of patients with impaired dihydropyrimidine dehydrogenase (DPD) activity

J Clin Pharm Ther. 2004 Aug;29(4):307-15. doi: 10.1111/j.1365-2710.2004.00569.x.


Background: Dihydropyrimidine dehydrogenase (DPD) gene polymorphism may lead to severe toxicity with 5-fluorouracil (5-FU), a major anticancer drug extensively used in clinical oncology. Drug monitoring combined with early detection of patients at risk would enable timely dose adaptation so as to maintain drug concentrations within a therapeutic window. However, the best method to identify such patients remains to be determined.

Objective: The aim of this study was to develop a rapid and simple high-performance liquid chromatographic (HPLC) method for estimating uracil/dihydrouracil (U/UH2) ratio in plasma, as an index of DPD status, and for assaying 5-FU as part of drug level monitoring.

Method: Assay of 5-FU, and U/UH2 detection were performed on a HPLC system equipped with UV detector. Analytes were separated at room temperature using a 5 microm particles, 25 cm RP-18 X-Terra column. The mobile-phase consisted of a KH(2)PO(4) salt solution (0.05 m) + 0.1% triethylamine (TEA) pumped at 0.4 mL/min. Detection of 5-FU and 5-bromouracil were performed at 254 nm; U and UH2 elution was monitored at 210 nm.

Results: The method was sensitive and specific for assaying 5-FU within the 5-500 ng/mL concentration range, which covers exposure levels currently met in clinical practice. The method was simple, and relatively cheap, and rapid, with an analytical run time of about 30 min. Data from a patient with 5-FU toxicity suggest that the method was capable of identifying DPD metabolic phenotype in cancer patients, based on measurement of plasma U/UH2 ratio.

Conclusion: The method described should be suitable both for detecting patients at high risk of 5-FU toxicity, and for drug level monitoring during chemotherapy.

Publication types

  • Case Reports

MeSH terms

  • Antimetabolites, Antineoplastic / adverse effects
  • Antimetabolites, Antineoplastic / blood*
  • Antimetabolites, Antineoplastic / pharmacokinetics
  • Antineoplastic Agents / metabolism*
  • Chromatography, High Pressure Liquid / methods*
  • Dihydrouracil Dehydrogenase (NADP) / genetics
  • Dihydrouracil Dehydrogenase (NADP) / metabolism*
  • Female
  • Fluorouracil / adverse effects
  • Fluorouracil / blood*
  • Fluorouracil / pharmacokinetics
  • Humans
  • Middle Aged
  • Polymorphism, Genetic
  • Uracil / analogs & derivatives*
  • Uracil / blood*


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