Rapid and sensitive high-performance liquid chromatographic analysis of halogenopyrimidines in plasma

Recent studies have stressed the need for individual adjustment of 5-fluorouracil (5-FU) dosage. Most of the techniques previously reported are not well adapted to routine application. We describe a sensitive, selective and simple HPLC technique under isocratic conditions for the quantitation of 5-FU and other halogenopyrimidines. The proportion of reagents and internal standard were optimised to allow the use of minitubes, particularly adapted to large series of plasma assays. High extraction yield, 75% for 5-FU and 90% for 5-bromouracil and 5-chlorouracil, was obtained using 1.2 ml isopropanol–ethyl acetate (15:85, v/v) following precipitation of plasma proteins with 300 mg ammonium sulfate. The mobile phase was 0.01 M phosphate buffer (pH 3.0). Uracil and 5-fluorouracil were fully resolved with Spherisorb ODS2 column. The limits of quantitation and detection in human plasma were 6 ng ml⁻¹ and 3 ng ml⁻¹, respectively, for all compounds studied. The total analysis time required for each run was 25 min. Final results could be given within 90 min of blood sampling. At least 50 plasma samples could be analysed per day. This method has been successfully used for monitoring 5-FU-based treatments.