Abstract：5-Fluorouracil (5-FU) has a broad spectrum of anti-tumor activity, widely applied to the treatment of cancers. However, it is necessary to determine the plasma concentration of 5-FU in clinical practice due to its narrow therapeutic index. Therefore, a simple, economic and sensitive high-performance liquid chromatography (HPLC) method was developed and validated for the determination of 5-FU in human plasma. Ethyl acetate was chosen as extraction reagent. Chromatographic separation was performed on a Diamonsil C 18 column (250 mm × 4.6 mm i.d., 5 μm) with the mobile phase consisting of methanol and 20 mmol/L ammonium formate using a linear gradient elution at a flow rate of 0.8 mL/min. 5-FU and 5-bromouracil (5-BU) were detected by UV detector at 265 nm. The calibration curve was linear over the concentration range of 5-500 ng/mL and the correlation coefficient was not less than 0.992 6 for all calibration curves. The intra-and inter-day precisions were less than 10.5% and 4.3%, respectively, and the accuracy was within ±3.7%. The recovery at all concentration levels was 80.1±8.6%. 5-FU was stable under possible conditions of storing and handling. This method is proved applicable to therapeutic drug monitoring and pharmacokinetic studies of 5-FU in human. Keywords：5-fluorouracil(5-FU); high-performance liquid chromatography (HPLC); human plasma 5-Fluorouracil (5-FU, Fig.1) is still a mainstay in the treatment of various cancers including head and neck cancer, gastrointestinal tract cancer and breast cancer since being introduced into practice in the 1950s [1] . In addition, several prodrugs of 5-FU, such as capecitabine and tegafur, were developed. The action mechanism of 5-FU is that 5-FU is converted into 5-fluoro-20-deoxyuridine monophosphate, and then irreversibly inhibits activity of thymidylate synthase to result in decreased intracellular concentrations of deoxythymidine monophosphate and DNA damage [2] . However, 5-FU, as activated metabolite of many derivatives and novel formulations, has the disadvantage of a low and unpredictable bioavailability (between 28% and 100%) [3] . Thus, 5-FU plasma concentrations have been correlated with toxicity and efficacy. Furthermore, therapeutic drug monitoring has been reported to result in an improved response/toxicity balance, and hence pharmacokinetic studies of 5-FU in human are more important and necessary. (a) 5-FU (b) 5-Bromouracil
