DERIVATIZATION OF 5-FLUOROURACIL WITH 4-BROMOMETHYL-7-METHOXYCOUMARINFOR DETERMINATION BY LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY

A robust new analytical method has been developed for the determinatio n of 5-fluorouracil (5-FU) in human plasma samples using high-performa nce liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The m ethod is based on a liquid-liquid extraction procedure, precolumn deri vatization, reversed-phase HPLC separation, and detection using atmosp heric pressure chemical ionization and selected reaction monitoring. T he derivatization agent used was 4-bromomethyl-7-methoxycoumarin. The internal standard for the assay procedure was a stable isotope labeled analog of 5-FU. The lower limit of quantitation was 1.0 ng/mL using 5 00 mu L aliquots of plasma. Sample throughput on the mass spectrometer was approximately 17 samples/h (3.5 min/sample). The method was fully validated. The recovery of 5-FU averaged 76.1%. The accuracy of the a ssay, assessed from quality control samples, ranged from 99.1% to 104. 3% (% theoretical). The overall interassay precision (% RSD) was 2.7%, and the intraassay precision (% RSD) ranged from 1.5% to 3.9%. The de rivatized samples were found to be stable under sample analysis condit ions and during refrigerator storage. The method was specific for the determination of 5-FU. (C) 1998 American Society for Mass Spectrometry .