Determination of 5-fluorouracil in microvolumes of human plasma by solvent extraction and high-performance liquid chromatography

In the present study, a new reversed-phase HPLC method has been developed and validated for the quantitative determination of 5-fluorouracil (5-FU) in human plasma using only 100-μl samples. The sample extraction and clean-up procedure involved a simple liquid–liquid extraction after addition of 5-chlorouracil (5-CU), used as internal standard, with 5 ml ethyl acetate. Chromatographic separations were performed on an Inertsil ODS-3 column (250×4.6 mm ID; 5 μM particle size), eluted with a mobile phase composed of acidified water (pH 2.0). The column effluent was monitored by UV absorption measurement at a wavelength of 266 nm. The calibration curves were constructed over a range of 0.20–50.0 μM and were fitted by weighted (1/x) linear regression analysis using the ratio of peak heights of 5-FU and 5-CU versus concentrations of the nominal standards. Extraction recoveries over the total range averaged 92 and 93% for 5-FU and 5-CU, respectively. The lower limit of quantitation was established at 0.20 μM (26 ng/ml), with within-run and between-run precisions of 4.2 and 7.0%, respectively, and an average accuracy of 109.3%. The within-run and between-run precisions at four tested concentrations analyzed in quintuplicate over a time period of four days were <1.4 and <4.4%, respectively. The accuracy at the tested concentrations ranged from 98.4 to 102.3%. Compared to previously described validated analytical methods for 5-FU, our present assay provides equivalent to superior sensitivity using only microvolumes of sample.