Simultaneous quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry
Simultaneous quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry
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DOI：
10.1016/j.ymeth.2006.05.004
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发表时间：
2006-06-01
期刊：
影响因子：
4.8
通讯作者：
Bielawska, Alicja
中科院分区：
文献类型：
--
作者：
Bielawski, Jacek;Szulc, Zdzislaw M.;Bielawska, Alicja
近年来，人们对神经酰胺（Cer）、鞘氨醇（Sph）和鞘氨醇1-磷酸（Sph- 1p）等新型生物活性鞘脂（SPLs）进行了大量研究，这就需要开发一种准确、用户友好的方法来分析和定量这些分子的内源性水平。ESI/MS/MS方法提供了一种用于检测和监测生物材料中SPL水平和组成变化的通用工具。同时ESI/MS/MS分析sphingoid碱（SBs）， sphingoid碱1-磷酸（SB-IPs）， Cers和sphingomyelins （SMs）在Thermo Finnigan TSQ 7000三重四极杆质谱仪上进行多反应监测（MRM）正电离模式。生物材料（细胞，组织或生理液体）用内标（ISs）强化，提取到单相中性有机溶剂系统中，并通过Surveyor/TSQ 7000 LC/MS系统进行分析。SPLs的定性分析是通过对特定类型SPLs的共同片段离子特征的母离子扫描进行的。定量分析是基于用已知量的目标合成标准物和等量的is对人工矩阵进行峰化所产生的校准曲线。校准曲线是通过使用线性回归模型绘制分析物与各自IS的峰面积比与浓度的关系来构建的。这种可靠的分析方法可以确定各种生物材料中内源性鞘脂（ESPLs）的组成，并达到1 pmol或更低的检测限。这种方法和相关的方法已经定义了个体生物活性SPLs不同亚种的代谢和功能中意想不到的专门化和特异性。(c) 2006爱思唯尔公司版权所有。
There has been a recent explosion in research concerning novel bioactive sphingolipids (SPLs) such as ceramide (Cer), sphingosine (Sph) and sphingosine 1-phosphate (Sph-1P) that necessitates development of accurate and user-friendly methodology for analyzing and quantitating the endogenous levels of these molecules. ESI/MS/MS methodology provides a universal tool used for detecting and monitoring changes in SPL levels and composition from biological materials. Simultaneous ESI/MS/MS analysis of sphingoid bases (SBs), sphingoid base 1-phosphates (SB-IPs), Cers and sphingomyelins (SMs) is performed on a Thermo Finnigan TSQ 7000 triple quadrupole mass spectrometer operating in a multiple reaction monitoring (MRM) positive ionization mode. Biological materials (cells, tissues or physiological fluids) are fortified with internal standards (ISs), extracted into a one-phase neutral organic solvent system, and analyzed by a Surveyor/TSQ 7000 LC/MS system. Qualitative analysis of SPLs is performed by a Parent Ion scan of a common fragment ion characteristic for a particular class of SPLs. Quantitative analysis is based on calibration curves generated by spiking an artificial matrix with known amounts of target synthetic standards and an equal amount of IS. The calibration curves are constructed by plotting the peak area ratios of analyte to the respective IS against concentration using a linear regression model. This robust analytical procedure can determine the composition of endogenous sphingolipids (ESPLs) in varied biological materials and achieve a detection limit at 1 pmol or lower level. This and related methodology are already defining unexpected specialization and specificity in the metabolism and function of distinct subspecies of individual bioactive SPLs. (c) 2006 Elsevier Inc. All rights reserved.