Simultaneous determination of multiple intracellular metabolites in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle by liquid chromatography-mass spectrometry
Simultaneous determination of multiple intracellular metabolites in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle by liquid chromatography-mass spectrometry
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DOI：
10.1016/j.chroma.2007.02.034
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发表时间：
2007-04-20
影响因子：
4.1
通讯作者：
Oldiges, Marco
中科院分区：
文献类型：
--
作者：
Luo, Bing;Groenke, Karsten;Oldiges, Marco
建立了一种高选择性、高灵敏度的液相色谱-串联四极杆质谱（LC-MS/MS）鉴定和定量细胞内参与中心碳代谢（包括糖酵解、磷酸戊糖途径和三羧酸循环）的代谢产物的方法。在移动的相中使用挥发性离子对改性剂三丁基乙酸铵（TBAA），在普通C18反相柱上同时分离了磷酸糖、核苷酸和羧酸等29种带负电荷的化合物。方法验证结果显示，根据DIN（德国标准化研究所）32645计算的检测限（LOD）大多低于60 nM，仅丙酮酸和苹果酸除外。校准曲线主要在三个数量级上显示出良好的线性，相关系数R-2 > 0.9982。该LC-MS/MS方法已成功应用于大肠杆菌细胞提取物中代谢产物的测定。大多数细胞内代谢物在检测范围内，细胞提取物样品的测量值的相对标准偏差小于5.65%（n = 5）。(c)2007 Elsevier B. V.保留所有权利。
A highly selective and sensitive method for identification and quantification of intracellular metabolites involved in central carbon metabolism (including glycolysis, pentose phosphate pathway and tricarboxylic acid cycle) by means of liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS) was developed. The volatile ion pair modifier tributylammonium acetate (TBAA) was employed in the mobile phase for simultaneously separation of 29 negatively charged compounds including sugar phosphates, nucleotides, and carboxylic acids on a common C 18 reversed-phase column. Method validation results displayed that limits of detection (LODs) calculated according to DIN (German Institute for Standardization) 32645 are mostly below 60 nM, only with the exception of pyruvate and malate. The calibration curves showed excellent linearity mainly over three orders of magnitude with correlation coefficients R-2 > 0.9982. This LC-MS/MS method was successfully applied to determine these metabolites in cell extracts of Escherichia coli. Most of the intracellular metabolites were found within the detection range and the relative standard deviations of the measurements were smaller than 5.65% (n = 5) for a cell extract sample. (c) 2007 Elsevier B.V. All rights reserved.