Journal of Chromatography B 2009-01-01

Simultaneous quantification of 5-FU, 5-FUrd, 5-FdUrd, 5-FdUMP, dUMP and TMP in cultured cell models by LC-MS/MS

Delphine Carli, Mylène Honorat, Sabine Cohen, Mehdi Megherbi, Bruno Vignal, Charles Dumontet, Léa Payen, Jérôme Guitton, Delphine Carli, Mylène Honorat, Sabine Cohen, Mehdi Megherbi, Bruno Vignal, Charles Dumontet, Léa Payen, Jérôme Guitton

Index: J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 877 , 2937-2944, (2009)

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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 3.5 μm, (100 mm × 2.1 mm 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 6.2 min. The analytes were ionized in an electrospray interface under negative ion mode. We validated the method over a range of 2.5–150 ng mL −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 °C for at least two weeks, and after three freeze-thaw cycles. No matrix effect was observed in both media.