Tandem mass spectrometry identification and LC–MS quantification of intact cytokinin nucleotides in K-562 human leukemia cells
- 370 Downloads
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.
KeywordsCytokinins Nucleotides HPLC Mass spectrometry K-562
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).
- 5.Ishii Y, Hori Y, Sakai S, Honma Y (2002) Control of differentiation and apoptosis of human myeloid leukemia cells by cytokinins and cytokinin nucleosides, plant redifferentiation-inducing hormones. Cell Growth Differ 13:19–26Google Scholar
- 7.Lin BB, Hurley MC, Fox IH (1988) Regulation of adenosine kinase by adenosine-analogs. Mol Pharmacol 34:501–505Google Scholar
- 10.Dervieux T, Meyer G, Barham R, Matsutani M, Barry M, Boulieu R, Neri B, Seidman E (2005) Liquid chromatography–tandem mass spectrometry analysis of erythrocyte thiopurine nucleotides and effect of thiopurine methyltransferase gene variants on these metabolites in patients receiving azathioprine/6-mercaptopurine therapy. Clin Chem 51:2074–2084CrossRefGoogle Scholar
- 21.Metzler DE (2001) Biochemistry: the chemical reactions of living cells. Harcourt/Academic, San DiegoGoogle Scholar
- 25.Crauste C, Lefebvre I, Hovaneissian M, Puy JY, Roy B, Peyrottes S, Cohen S, Guitton J, Dumontet C, Perigaud C (2009) Development of a sensitive and selective LC/MS/MS method for the simultaneous determination of intracellular 1-beta-d-arabinofuranosylcytosine triphosphate (araCTP), cytidine triphosphate (CTP) and deoxycytidine triphosphate (dCTP) in a human follicular lymphoma cell line. J Chromatogr B 877:1417–1425CrossRefGoogle Scholar
- 26.Pruvost A, Becher F, Bardouille P, Guerrero C, Creminon C, Delfraissy JF, Goujard C, Grassi J, Benech H (2001) Direct determination of phosphorylated intracellular anabolites of stavudine (d4T) by liquid chromatography tandem mass spectrometry. Rapid Commun Mass Spectrom 15:1401–1408CrossRefGoogle Scholar
- 27.Asakawa Y, Tokida N, Ozawa C, Ishiba M, Tagaya O, Asakawa N (2008) Suppression effects of carbonate on the interaction between stainless steel and phosphate groups of phosphate compounds in high-performance liquid chromatography and electrospray ionization mass spectrometry. J Chromatogr A 1198:80–86CrossRefGoogle Scholar