Simultaneous quantification of 5-FU, 5-FUrd, 5-FdUrd, 5-FdUMP, dUMP and TMP in cultured cell models by LC-MS/MS
| Autor: | Mylène Honorat, Bruno Vignal, Charles Dumontet, Jérôme Guitton, Léa Payen, Delphine Carli, Mehdi Megherbi, Sabine Cohen |
|---|---|
| Rok vydání: | 2009 |
| Předmět: |
Electrospray
Clinical Biochemistry Tandem mass spectrometry Sensitivity and Specificity Biochemistry High-performance liquid chromatography Cell Line Analytical Chemistry chemistry.chemical_compound Drug Stability Tandem Mass Spectrometry Cell Line Tumor Fluorodeoxyuridylate Thymidine Monophosphate Ammonium formate Humans Solid phase extraction Thymidine monophosphate Chromatography Solid Phase Extraction Extraction (chemistry) Reproducibility of Results Cell Biology General Medicine chemistry Fluorouracil Deoxyuracil Nucleotides Floxuridine Quantitative analysis (chemistry) Chromatography Liquid |
| Zdroj: | Journal of Chromatography B. 877:2937-2944 |
| ISSN: | 1570-0232 |
| DOI: | 10.1016/j.jchromb.2009.07.004 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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