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).