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Analytical and Bioanalytical Chemistry

, Volume 398, Issue 5, pp 2071–2080 | Cite as

Tandem mass spectrometry identification and LC–MS quantification of intact cytokinin nucleotides in K-562 human leukemia cells

  • Tibor Béres
  • Marek Zatloukal
  • Jiří Voller
  • Percy Niemann
  • Marie Christin Gahsche
  • Petr Tarkowski
  • Ondřej Novák
  • Jan Hanuš
  • Miroslav Strnad
  • Karel DoležalEmail author
Original Paper

Abstract

We describe here a new reversed-phase high-performance liquid chromatography with mass spectrometry detection method for quantifying intact cytokinin nucleotides in human K-562 leukemia cells. Tandem mass spectrometry was used to identify the intracellular metabolites (cytokinin monophosphorylated, diphosphorylated, and triphosphorylated nucleotides) in riboside-treated cells. For the protein precipitation and sample preparation, a trichloroacetic acid extraction method is used. Samples are then back-extracted with diethyl ether, lyophilized, reconstituted, and injected into the LC system. Analytes were quantified in negative selected ion monitoring mode using a single quadrupole mass spectrometer. The method was validated in terms of retention time stabilities, limits of detection, linearity, recovery, and analytical accuracy. The developed method was linear in the range of 1–1,000 pmol for all studied compounds. The limits of detection for the analytes vary from 0.2 to 0.6 pmol.

Keywords

Cytokinins Nucleotides HPLC Mass spectrometry K-562 

Notes

Acknowledgements

This work was supported by the Czech Ministry of Education (MSM 6198959216, 1M06030) and by the Grant Agency of the Czech Republic (522/08/H003, 522/08/0920).

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Tibor Béres
    • 1
  • Marek Zatloukal
    • 1
  • Jiří Voller
    • 1
  • Percy Niemann
    • 3
  • Marie Christin Gahsche
    • 3
  • Petr Tarkowski
    • 1
    • 2
  • Ondřej Novák
    • 1
  • Jan Hanuš
    • 4
  • Miroslav Strnad
    • 1
  • Karel Doležal
    • 1
    Email author
  1. 1.Laboratory of Growth RegulatorsIEB AS CR and Palacký UniversityOlomoucCzech Republic
  2. 2.Department of Biochemistry, Faculty of SciencePalacký UniversityOlomoucCzech Republic
  3. 3.BIOLOG Life Science InstituteBremenGermany
  4. 4.Institute of Experimental Botany AS CRPrague 4Czech Republic

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