Separation, identification and quantitation of ceramides in human cancer cells by liquid chromatography-electrospray ionization tandem mass spectrometry
Separation, identification and quantitation of ceramides in human cancer cells by liquid chromatography-electrospray ionization tandem mass spectrometry
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DOI：
10.1016/s0021-9673(01)01422-4
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发表时间：
2002-03-08
影响因子：
4.1
通讯作者：
Crommen, J
中科院分区：
文献类型：
--
作者：
Fillet, M;Van Heugen, JC;Crommen, J
神经酰胺是重要的细胞内第二信使，在细胞生长、分化和程序性死亡的调节中发挥作用。对这些第二信使的定性和定量分析需要灵敏和特异的分析方法来检测单个神经酰胺物种的内源水平并区分它们。在C-18键合硅胶色谱柱上，用液-质联用法分离了9种人工合成神经酰胺。以水-乙腈-异丙醇为流动相，采用梯度洗脱方式洗脱。通过对阳离子电喷雾产生的离子进行反应监测来检测它们。对神经酰胺进行的碰撞诱导裂解产生了m/z为264的特征产物离子，使得多反应监测(MRM)非常适合于各种神经酰胺的定量测量。在对提取步骤进行优化后，所提出的方法能够识别和定量人类癌细胞产生的不同神经酰胺种类。该方法可用于在纳克水平上定量的C-16、C-28和C-20神经酰胺。验证结果表明，在线性、准确度和精密度方面都有较好的结果。(C)2002 Elsevier Science B.V.保留所有权利。
Ceramides are important intracellular second messengers that play a role in the regulation of cell growth, differentiation and programmed cell death. Qualitative and quantitative analysis of these second messengers requires sensitive and specific analytical methods to detect endogenous levels of individual ceramide species and to differentiate between them. Nine synthetic ceramides were separated by liquid chromatography coupled to tandem mass spectrometry on a C-18 bonded silica column. The lipids were eluted in gradient elution mode using a mixture of water, acetonitrile and 2-propanol as mobile phase. They were detected by reaction monitoring performed on positive ion electrospray generated ions. Collision-induced fragmentations conducted on ceramides produced a well characteristic product ion at m/z 264, making multiple reaction monitoring (MRM) well suited for various ceramides quantitative measurements. After optimization of the extraction step, the proposed methodology was able to identify and quantify different ceramide species issued from human cancer cells. The method could be validated for C-16, C-28, and C-20 ceramides, quantified at the nanogram level. The validation exhibits good results with respect to linearity, accuracy and precision. (C) 2002 Elsevier Science B.V. All rights reserved.