Derivatization of 5-fluorouracil with 4-bromomethyl-7-methoxycoumarin for determination by liquid chromatography-mass spectrometry.
A robust new analytical method has been developed for the determination of 5-fluorouracil (5-FU) in human plasma samples using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The method is based on a liquid-liquid extraction procedure, precolumn derivatization, reversed-phase HPLC separation, and detection using atmospheric pressure chemical ionization and selected reaction monitoring. The 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 500 microL 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 assay, 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 derivatized samples were found to be stable under sample analysis conditions and during refrigerator storage. The method was specific for the determination of 5-FU.
Wang
K
K
Department of Drug Metabolism and Pharmacokinetics, Hoffmann-La Roche, Inc., Nutley, New Jersey 07110, USA. ka.wang@roche.com
Nano
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M
Mulligan
T
T
Bush
E D
ED
Edom
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RW
D000963:Antimetabolites / Q000097:blood* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D005472:Fluorouracil / Q000031:analogs & derivatives / Q000097:blood* / Q000737:chemistry; D006801:Humans; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D011786:Quality Control; D014468:Umbelliferones
D000963:Antimetabolites; D007202:Indicators and Reagents; D014468:Umbelliferones; C000317:4-bromomethyl-7-methoxycoumarin; D005472:Fluorouracil