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

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 × 108 -2.29 × 109 molecules/cell) in Molm-13 cells were two orders of magnitude higher than those of deoxyribonucleoside triphosphates (1.72 × 106 -1.40 × 107 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.