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Background  Nucleoside triphosphates participate in fundamental cellular processes as building blocks of DNA and RNA, energy carriers, and cofactors in enzymatic reactions, and their balance is tightly regulated. Here, we established a simultaneous and absolute quantification method for eight nucleoside triphosphates using liquid chromatography–triple quadrupole tandem mass spectrometry and hydrophilic interaction chromatography. Our method was successfully applied to the extract of human acute myeloid leukemia Molm-13 cells.    Results  Levels of ribonucleoside triphosphates (2.07 × 10 8 –2.29 × 10 9  molecules/cell) in Molm-13 cells were two orders of magnitude higher than those of deoxyribonucleoside triphosphates (1.72 × 10 6 –1.40 × 10 7  molecules/cell). Exposure of Molm-13 cells for 24 h to thymidine, a nucleotide imbalance inducer, increased the levels of cellular dTTP, dGTP, and dATP and decreased only dCTP, resulting in significant inhibition of cell proliferation.    Conclusion  Our quantification method for nucleoside triphosphates revealed the quantitative relationship between the arrest of cell proliferation and the imbalance of nucleoside triphosphates in thymidine-treated Molm-13 cells. Owing to the short run time (15 min/run), broad adaptability, and throughput performance, we believe that our method is a powerful tool for not only genetic and molecular biology research but also for studying the mechanism of genotoxic compounds and anti-cancer or anti-virus drugs, drug screening, clinical studies, and other fields.

Simultaneous and absolute quantification of nucleoside triphosphates using liquid chromatography–triple quadrupole tandem mass spectrometry