采用液相色谱-串联质谱法测定细胞中的2',3'-环核苷酸。
Measurement of 2',3'-cyclic nucleotides by liquid chromatography-tandem mass spectrometry in cells.
作者信息
Bähre Heike, Kaever Volkhard
机构信息
Institute of Pharmacology, Hannover Medical School, Hannover, Germany; Research Core Unit Metabolomics, Hannover Medical School, Hannover, Germany.
Research Core Unit Metabolomics, Hannover Medical School, Hannover, Germany.
出版信息
J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Aug 1;964:208-11. doi: 10.1016/j.jchromb.2014.02.046. Epub 2014 Mar 12.
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).
最近，有报道称哺乳动物组织中除了存在3',5'-环核苷酸单磷酸（3',5'-cNMPs）外，还出现了2',3'-环核苷酸单磷酸（2',3'-cNMPs）。我们开发了一种液相色谱-串联质谱（LC-MS/MS）方法，用于测定细胞样品中的四种2',3'-环核苷酸，即2',3'-cAMP、2',3'-cCMP、2',3'-cGMP、2',3'-cUMP。使用连接到以正离子模式运行的QTRAP5500系统（AB Sciex）的Zorbax eclipse XCB-C18（50 mm×4.6 mm；1.8 μm柱；安捷伦）实现色谱分离。2',3'-cAMP、2',3'-cCMP和2',3'-cGMP的校准曲线分别在0.41 fmol/μL至1666.6 fmol/μL范围内构建，2',3'-cUMP的校准曲线在3.3 - 1666.6 fmol/μL范围内构建，平方相关系数>0.9992。对于定量下限（LLOQ），准确度以及日内和日间精密度在<20%的要求范围内，对于更高浓度水平，在<15%的要求范围内。该方法应用于两种不同细胞系（Hek293T和HuT-78）中核苷酸的分析。
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