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: 0.488–19.9nM for FUTP, 1.66–67.7nM for FdUTP and 0.748–30.7nM 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 3054fmol/106 PBMCs and FdUMP levels up to 169fmol/106 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.