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Quantitation of endogenous nucleoside triphosphates and nucleosides in human cells by liquid chromatography tandem mass spectrometry

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Abstract

Nucleosides and nucleoside triphosphates are the building blocks of nucleic acids and important bioactive metabolites, existing in all living cells. In the present study, two liquid chromatography tandem mass spectrometry methods were developed to quantify both groups of compounds from the same sample with a shared extraction procedure. After a simple protein precipitation with methanol, the nucleosides were separated with reversed phase chromatography on an Atlantis T3 column while for the separation of the nucleoside triphosphates, an anion exchange column (BioBasic AX) was used. No addition of ion pair reagent was required. A 5500 QTrap was used as analyzer, operating as triple quadrupole. The analytical method for the nucleoside triphosphates has been validated according to the guidelines of the US Food and Drug Administration. The lower limit of quantification values were determined as 10 pg on column (0.5 ng/mL in the injection solution) for deoxyadenosine triphosphate and deoxyguanosine triphosphate, 20 pg (1 ng/mL) for deoxycytidine triphosphate and thymidine triphosphate, 100 pg (5 ng/mL) for cytidine triphosphate and guanosine triphosphate, and 500 pg (25 ng/mL) for adenosine triphosphate und uridine triphosphate respectively. This methodology has been applied to the quantitation of nucleosides and nucleoside triphosphates in primary human CD4 T lymphocytes and macrophages. As expected, the concentrations for ribonucleosides and ribonucleoside triphophates were considerably higher than those obtained for the deoxy derivatives. Upon T cell receptor activation, the levels of all analytes, with the notable exceptions of deoxyadenosine triphosphate and deoxyguanosine triphosphate, were found to be elevated in CD4 T cells.

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Acknowledgments

The authors thank Dr. Andreas Weigert from the Institute of Biochemistry I of the Goethe University Frankfurt for providing the macrophages. This work was supported by the Else Kroener Fresenius Foundation (Translational Research Innovation Pharma, TRIP) and the LOEWE program from the state of Hesse (Translational Medicine and Pharmacology, TMP). Furthermore, it was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG) to O.T.K. (KE 742/5-1).

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Authors and Affiliations

  1. pharmazentrum frankfurt/ZAFES, Institute of Clinical Pharmacology, Goethe University, Theodor-Stern-Kai 7, 60590, Frankfurt, Germany

    Dominique Thomas, Gerd Geisslinger & Nerea Ferreirós

  2. Institute of Medical Virology, Goethe University, Theodor-Stern-Kai 7, 60590, Frankfurt, Germany

    Nikolas Herold & Oliver T. Keppler

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  1. Dominique Thomas
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  2. Nikolas Herold
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  3. Oliver T. Keppler
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  5. Nerea Ferreirós
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Correspondence to Nerea Ferreirós.

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Thomas, D., Herold, N., Keppler, O.T. et al. Quantitation of endogenous nucleoside triphosphates and nucleosides in human cells by liquid chromatography tandem mass spectrometry. Anal Bioanal Chem 407, 3693–3704 (2015). https://doi.org/10.1007/s00216-015-8588-3

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  • DOI: https://doi.org/10.1007/s00216-015-8588-3

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