title: Measurement of 2',3'-cyclic nucleotides by liquid chromatography-tandem mass spectrometry in cells.
abstract: Recently, the occurrence of 2',3'-cyclic nucleoside monophosphates (2',3'-cNMPs) in addition to 3',5'-cNMPs in mammalian tissues was reported. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the measurement of four 2',3'-cyclic nucleotides, i.e., 2',3'-cAMP, 2',3'-cCMP, 2',3'-cGMP, 2',3'-cUMP, in cell samples. Chromatographic separation was achieved using a Zorbax eclipse XCB-C18 (50 mm×4.6 mm; 1.8 μm column; Agilent) connected to a QTRAP5500 system (AB Sciex) operating in positive ionization mode. Calibration curves were constructed in the range 0.41 fmol/μL to 1666.6 fmol/μL for 2',3'-cAMP, 2',3'-cCMP, and 2',3'-cGMP, and 3.3-1666.6 fmol/μL for 2',3'-cUMP, respectively, showing squared correlation coefficients >0.9992. Accuracy and inter- and intra-day precision lay within the required ranges of <20% for LLOQ and <15% for higher concentration levels. The method was applied to the analysis of nucleotides in two different cell lines (Hek293T and HuT-78).
authors.affiliation: Institute of Pharmacology, Hannover Medical School, Hannover, Germany; Research Core Unit Metabolomics, Hannover Medical School, Hannover, Germany. Electronic address: baehre.heike@mh-hannover.de.
authors.forename: Heike
authors.initials: H
authors.lastname: Bähre
authors.affiliation: Research Core Unit Metabolomics, Hannover Medical School, Hannover, Germany.
authors.forename: Volkhard
authors.initials: V
authors.lastname: Kaever
mesh_terms: D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D057230:Limit of Detection; D009712:Nucleotides, Cyclic / Q000032:analysis*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D009712:Nucleotides, Cyclic
keywords: 2′,3′-Cyclic nucleotide monophosphates; Cells; Liquid chromatography–tandem mass spectrometry