Simultaneous analysis of eight nucleoside triphosphates in cell lines by liquid chromatography coupled with tandem...

In this study, we developed a new method for the simultaneous determination of eight endogenous ribonucleoside triphosphates and deoxyribonucleoside triphosphates based on a combination of a selective sample preparation and an ion-pair liquid chromatography-electrospray tandem mass spectrometry. The sample preparation was based on a protein precipitation coupled with a solid phase extraction using a weak-anion-exchange cartridge. The analytical separation of the nucleotides was achieved on a porous graphitic carbon stationary phase with a binary elution gradient program employing ion-pairing reagents (diethylamine and hexylamine) and organic eluent (methanol). The triple quadrupole mass spectrometer operated in both negative and positive multiple reaction monitoring modes. The calibration assay used the stable isotope labelled analogs of each compounds as standard. Standard calibrations were from 0.25 to 10pmol injected according to deoxyribonucleotides and from 12.5 to 3000pmol injected according to ribonucleotides. The within-run precision of the assay was less than 14.5% and the between-run precision was less than 12.4% for each analytes. Assay accuracy was in the range of 92.3-107.6%. This method allows the determination of NTP and dNTP pools from lysats of several cell lines or peripheral blood mononuclear cell from patient. Assays were performed with different preparation of cells to confirm the quality and the relevance of the described method.