Determination of 5-fluorouracil in serum microsamples by a high-performance liquid chromatographic method and its application to pharmacokinetic studies in rats

A high-performance liquid chromatographic method is described for the quantitative determination of 5-fluorouracil (5FU) in only 30 microL of serum. A reversed-phase C(18) column is used for the separation and analysis of 5FU. The mobile phase consists of methanol-20mmol/L phosphate buffer, pH 6.80 (5:95, v/v). The calibration curve for 5FU is linear over the range of 0.05-10 microg/mL. The extraction recoveries of 5FU and p-aminobenzoic acid (as an internal standard) are > 91.28% and 81.98%, respectively. The intra-day and inter-day coefficients of variation of 5FU are less than 6.29% and 9.57% at four different concentrations, respectively. The method is simple, sensitive, and reliable. It is superior to previous methods in that the sample volume needed is relatively small (just 30 microL of serum). Therefore, this method can be utilized to determine 5FU in rats for study in various fields, especially in pharmacokinetics, controlled-release, and combination therapy with 5FU, etc. In this study, it is successfully applied to pharmacokinetic studies of 5FU after administrations of an intravenous bolus dose (25 mg/kg) and two oral doses (25 and 75 mg/kg) in rats.