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

In the present study, a new reversed-phase HPLC method has been developed a nd validated for the quantitative determination of 5-fluorouracil (5-FU) in human plasma using only 100-mu l 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 mi ethyl acetate, C hromatographic separations were performed on an Inertsil ODS-3 column (250X 4.6 mm ID; 5 mu M particle size), eluted with a mobile phase composed of ac idified water (pH 2.0). The column effluent was monitored by UV absorption measurement at a wavelength of 266 nm, The calibration curves were construc ted over a range of 0.20-50.0 mu M and were fitted by weighted (1/x) Linear regression analysis using the ratio of peak heights of 5-FU and 5-CU versu s concentrations of the nominal standards. Extraction recoveries over the t otal range averaged 92 and 93% for 5-FU and 5-CU, respectively. The lower l imit of quantitation was established at 0.20 mu M (similar to 26 ng/ml), wi th 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 a t four tested concentrations analyzed in quintuplicate over a time period o f four days were <1.4 and <4.4%, respectively. The accuracy at the tested c oncentrations ranged from 98.4 to 102.3%. Compared to previously described validated analytical methods for 5-FU, our present assay provides equivalen t to superior sensitivity using only microvolumes of sample. (C) 1999 Elsev ier Science B.V. All rights reserved.