title: Tandem mass spectrometry identification and LC-MS quantification of intact cytokinin nucleotides in K-562 human leukemia cells.
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.
authors.affiliation: Laboratory of Growth Regulators, IEB AS CR and Palacký University, Šlechtitelů 11, 78371 Olomouc, Czech Republic.
authors.forename: Tibor
authors.initials: T
authors.lastname: Béres
authors.forename: Marek
authors.initials: M
authors.lastname: Zatloukal
authors.forename: Jiří
authors.initials: J
authors.lastname: Voller
authors.forename: Percy
authors.initials: P
authors.lastname: Niemann
authors.forename: Marie Christin
authors.initials: MC
authors.lastname: Gahsche
authors.forename: Petr
authors.initials: P
authors.lastname: Tarkowski
authors.forename: Ondřej
authors.initials: O
authors.lastname: Novák
authors.forename: Jan
authors.initials: J
authors.lastname: Hanuš
authors.forename: Miroslav
authors.initials: M
authors.lastname: Strnad
authors.forename: Karel
authors.initials: K
authors.lastname: Doležal
mesh_terms: D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D003583:Cytokinins / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry; D015394:Molecular Structure; D009711:Nucleotides / Q000032:analysis* / Q000737:chemistry; D053719:Tandem Mass Spectrometry
chemical_list: D003583:Cytokinins; D009711:Nucleotides