In vitro stable isotope labeling for discovery of novel metabolites by liquid chromatography-mass spectrometry: Confirmation of gamma-tocopherol metabolism in human A549 cell.
In vitro stable isotope labeling for discovery of novel metabolites by liquid chromatography-mass spectrometry: Confirmation of gamma-tocopherol metabolism in human A549 cell.
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DOI：
10.1016/j.chroma.2009.12.002
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发表时间：
2010-01-29
期刊：
影响因子：
--
通讯作者：
Adamec J
中科院分区：
文献类型：
--
作者：
Yang WC;Regnier FE;Jiang Q;Adamec J
建立了一种从母体化合物中发现新的分解代谢产物的通用方法，并在人A549细胞中对γ-生育酚的代谢进行了验证。该方法基于体外稳定同位素标记代谢物的LC-MS分析，并假设母体化合物及其代谢物具有共同的官能团，可以通过充分记录的试剂进行衍生化。在该方法中，将两等份提取的代谢物分别用同位素编码（重）和非同位素编码（轻）形式的衍生试剂衍生化，以1：1的比例混合，并使用LC-MS进行分析。然后通过测定色谱共洗脱的同位素异构体对（MS双峰）（具有相似的峰强度和对应于重和轻形式衍生试剂之间质量差的质量差），轻松识别具有共同官能团的代谢物。通过使用N-甲基-烟酸N-羟基琥珀酰亚胺酯（C1-NANHS）和N-甲基-d3-烟酸N-羟基琥珀酰亚胺酯（C1-d3-NANHS）衍生试剂鉴定人A549细胞培养基中γ-生育酚催化剂的产物，证明和验证了该方法的可行性。总共鉴定了4种γ-生育酚代谢物，包括9 '-COOH、11'-COOH、13 '-COOH和13'-OH。此外，所开发的LC-MS方法也可用于γ-生育酚和其他形式的维生素E相关化合物的快速和灵敏的定量分析。
A general approach for discovering novel catabolic metabolites from a parent biocompound was developed and validated on metabolism of γ-tocopherol in human A549 cell. Method is based on LC-MS analysis of in vitro stable isotope labeled metabolites and assumes that a parent compound and its metabolites share a common functional group that can be derivatized by well-documented reagents. In this method, two equal aliquots of extracted metabolites are separately derivatized with isotope-coded (heavy) and non-isotope-coded (light) form of derivatizing reagent, mixed at 1:1 ratio and analyzed using LC-MS. The metabolites with common functional group are then easily recognized by determination of a chromatographically co-eluted pair of isotopomers (MS doublet peaks) with similar peak intensities and mass difference corresponding to the mass difference between heavy and light form of derivatization reagent. The feasibility of this approach was demonstrated and validated by identification of products of γ-tocopherol catabolism in human A549 cell culture media using N-methyl-nicotinic acid N-hydroxysuccinimide ester (C1-NANHS) and N-methyl-d3-nicotinic acid N-hydroxysuccinimide ester (C1-d3-NANHS) derivatizing reagent. Overall four γ-tocopherol metabolites were identified including 9'-COOH, 11'-COOH, 13'-COOH and 13'-OH. In addition, the developed LC-MS method can also be used for the fast and sensitive quantitative analysis of γ-tocopherol and other forms of vitamin E related compounds.
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影响因子：
7.4
作者：
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