Quantification of endogenous nucleotides is of interest for investigation of numerous cellular biochemical processes, such as energy metabolism and signal transduction, and may also be applied in cancer and antiretroviral therapies in which nucleoside analogues are used. For these purposes we developed and validated a sensitive and high accuracy ion-pair liquid chromatography tandem mass spectrometry (IP LC-MS/MS) method for simultaneous quantification of eight endogenous nucleotides (ATP, CTP, GTP, UTP, dATP, dCTP, dGTP, dTTP) and 2',2'-difluoro-2'-deoxycytidine triphosphate (dFdCTP), an intracellular metabolite of the nucleoside analogue gemcitabine. The assay was validated using 200μL aliquots of peripheral blood mononuclear cell (20×10(6)cells/ml, 4×10(6)cells) extracts, pretreated with activated charcoal and spiked with unlabeled nucleotides, deoxynucleotides and dFdCTP. Analytes were extracted by simple precipitation with cold 60% methanol containing isotope labeled internal standards and separated on a porous graphitic carbon column. For method validation, the concentration ranges were: 0.125-20.8pmol injected for deoxynucleotides, 0.25-312.5pmol injected for dFdCTP and 5-3200pmol injected for nucleotides. The highest coefficients of variation (CV) were 12.1% for within run assay and 11.4% for between run assay, both representing the precision at the lowest analyte concentrations. The method was applied to monitor dFdCTP and changes in endogenous nucleotides in patients who were receiving gemcitabine infusions.
Keywords: Gemcitabine; LC–MS/MS; Nucleotides; PBMC; dFdCTP.
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