Measurement of 2',3'-cyclic nucleotides by liquid chromatography-tandem mass spectrometry in cells

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).