title: Simultaneous quantification of 5-FU, 5-FUrd, 5-FdUrd, 5-FdUMP, dUMP and TMP in cultured cell models by LC-MS/MS.
abstract: To specifically quantify several metabolites of 5-fluorouracil (5-FU) and two endogenous monophosphate nucleotides, we developed an original method based on a liquid chromatography-tandem mass spectrometry (LC-MS/MS). This assay allowed the determination of: (i) the intracellular production of 5-fluoro-2'-deoxyuridine-5'-monophosphate (5-FdUMP) from 5-FU or 5-fluoro-2'-deoxyuridine (5-FdUrd), (ii) the impact of 5-FdUMP concentration on the intracellular 2'-deoxyuridine-5'-monophosphate (dUMP)/thymidine-5'-monophosphate (TMP) ratio, and (iii) the secretion extent of 5-FdUMP and 5-FU from human cultured cells by ABC transporters. Under our experimental conditions, cells were incubated with 5-FU or 5-FUrd. Then, cellular proteins were precipitated by methanol. This procedure provided high extraction recovery. In addition, to measure 5-FU and 5-FdUMP secretion from cells, we carried out quantification of these molecules in culture medium. Media were either directly injected (5-FU) or underwent a solid phase extraction using Oasis Wax extraction cartridge (5-FdUMP). Separation of analytes was performed on a dC18 Atlantis 3.5microm, (100mmx2.1mm i.d) column with isocratic mode using ammonium formate buffer/methanol/water (5/5/90, v/v) as mobile phase. The run time did not exceed 6.2min. The analytes were ionized in an electrospray interface under negative ion mode. We validated the method over a range of 2.5-150ngmL(-1) according to the compounds. Intra- and inter-assay variability was lower than 10% over seven days. All compounds were stable in cells or in culture medium when samples were stored at -20 degrees C for at least two weeks, and after three freeze-thaw cycles. No matrix effect was observed in both media.
authors.affiliation: Hospices Civils de Lyon, F-69495, Pierre Bénite, France.
authors.forename: Delphine
authors.initials: D
authors.lastname: Carli
authors.forename: Mylène
authors.initials: M
authors.lastname: Honorat
authors.forename: Sabine
authors.initials: S
authors.lastname: Cohen
authors.forename: Mehdi
authors.initials: M
authors.lastname: Megherbi
authors.forename: Bruno
authors.initials: B
authors.lastname: Vignal
authors.forename: Charles
authors.initials: C
authors.lastname: Dumontet
authors.forename: Léa
authors.initials: L
authors.lastname: Payen
authors.forename: Jérôme
authors.initials: J
authors.lastname: Guitton
mesh_terms: D002460:Cell Line; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods*; D003856:Deoxyuracil Nucleotides / Q000032:analysis / Q000378:metabolism; D004355:Drug Stability; D005467:Floxuridine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000378:metabolism; D005468:Fluorodeoxyuridylate / Q000032:analysis* / Q000378:metabolism; D005472:Fluorouracil / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013938:Thymidine Monophosphate / Q000032:analysis / Q000378:metabolism
chemical_list: D003856:Deoxyuracil Nucleotides; D005467:Floxuridine; D005468:Fluorodeoxyuridylate; D013938:Thymidine Monophosphate; C007267:2'-deoxyuridylic acid; D005472:Fluorouracil