title: Development of an LC-MS/MS assay for the quantitative determination of the intracellular 5-fluorouracil nucleotides responsible for the anticancer effect of 5-fluorouracil.
abstract: 5-Fluorouracil (5-FU) and its oral prodrug capecitabine are among the most widely used chemotherapeutics. For cytotoxic activity, 5-FU requires cellular uptake and intracellular metabolic activation. Three intracellular formed metabolites are responsible for the antineoplastic effect of 5-FU: 5-fluorouridine 5'-triphosphate (FUTP), 5-fluoro-2'-deoxyuridine 5'-triphosphate (FdUTP) and 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). In this paper, we describe the development of an LC-MS/MS assay for quantification of these active 5-FU nucleotides in peripheral blood mononuclear cells (PBMCs). Because the intracellular 5-FU nucleotide concentrations were very low, maximization of the release from the cell matrix and minimization of interference were critical factors. Therefore, a series of experiments was performed to select the best method for cell lysis and nucleotide extraction. Chromatography was optimized to obtain separation from endogenous nucleotides, and the effect of different cell numbers was examined. The assay was validated for the following concentration ranges in PBMC lysate: 0.488-19.9 nM for FUTP, 1.66-67.7 nM for FdUTP and 0.748-30.7 nM for FdUMP. Accuracies were between -2.2 and 7.0% deviation for all analytes, and the coefficient of variation values were ≤ 4.9%. The assay was successfully applied to quantify 5-FU nucleotides in PBMC samples from patients treated with capecitabine and patients receiving 5-FU intravenously. FUTP amounts up to 3054 fmol/10(6) PBMCs and FdUMP levels up to 169 fmol/10(6) PBMCs were measured. The FdUTP concentrations were below the lower limit of quantification. To our knowledge, this is the first time that 5-FU nucleotides were quantified in cells from patients treated with 5-FU or capecitabine without using a radiolabel.
authors: Department of Pharmacy & Pharmacology, Antoni van Leeuwenhoek Hospital - The Netherlands Cancer Institute, Louwesweg 6, 1066 EC Amsterdam, The Netherlands. Electronic address: ellen.derissen@slz.nl.
authors: Ellen J B
authors: EJ
authors: Derissen
authors: Department of Pharmacy & Pharmacology, Antoni van Leeuwenhoek Hospital - The Netherlands Cancer Institute, Louwesweg 6, 1066 EC Amsterdam, The Netherlands.
authors: Michel J X
authors: MJ
authors: Hillebrand
authors: Department of Pharmacy & Pharmacology, Antoni van Leeuwenhoek Hospital - The Netherlands Cancer Institute, Louwesweg 6, 1066 EC Amsterdam, The Netherlands.
authors: Hilde
authors: H
authors: Rosing
authors: Department of Clinical Pharmacology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands; Science Faculty, Utrecht Institute for Pharmaceutical Sciences (UIPS), Division of Pharmaco-epidemiology & Clinical Pharmacology, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands.
authors: Jan H M
authors: JH
authors: Schellens
authors: Department of Pharmacy & Pharmacology, Antoni van Leeuwenhoek Hospital - The Netherlands Cancer Institute, Louwesweg 6, 1066 EC Amsterdam, The Netherlands; Science Faculty, Utrecht Institute for Pharmaceutical Sciences (UIPS), Division of Pharmaco-epidemiology & Clinical Pharmacology, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands.
authors: Jos H
authors: JH
authors: Beijnen
mesh_terms: D000970:Antineoplastic Agents / Q000378:metabolism* / Q000493:pharmacokinetics; D001692:Biological Transport; D001711:Biotransformation; D002138:Calibration; D002853:Chromatography, Liquid / Q000592:standards; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D016903:Drug Monitoring / Q000379:methods* / Q000592:standards; D005467:Floxuridine / Q000031:analogs & derivatives / Q000378:metabolism; D005468:Fluorodeoxyuridylate / Q000378:metabolism; D005472:Fluorouracil / Q000378:metabolism* / Q000493:pharmacokinetics; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D016014:Linear Models; D012015:Reference Standards; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000592:standards; D014544:Uridine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D003856:Deoxyuracil Nucleotides; C522804:fluorodeoxyuridine triphosphate; D005467:Floxuridine; D005468:Fluorodeoxyuridylate; C025635:5-fluorouridine 5'-triphosphate; D005472:Fluorouracil; D014544:Uridine Triphosphate
keywords: 5-Fluorodeoxyuridine monophosphate (FdUMP); 5-Fluorodeoxyuridine triphosphate (FdUTP); 5-Fluorouracil; 5-Fluorouridine triphosphate (FUTP); LC–MS/MS