Normal phase liquid chromatography coupled to quadrupole time of flight atmospheric pressure chemical ionization mass spectrometry for separation, detection and mass spectrometric profiling of neutral sphingolipids and cholesterol
Normal phase liquid chromatography coupled to quadrupole time of flight atmospheric pressure chemical ionization mass spectrometry for separation, detection and mass spectrometric profiling of neutral sphingolipids and cholesterol
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DOI：
10.1016/j.jchromb.2009.07.008
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发表时间：
2009-10-01
影响因子：
3
通讯作者：
Sandhoff, Konrad
中科院分区：
文献类型：
--
作者：
Farwanah, Hany;Wirtz, Jennifer;Sandhoff, Konrad
许多脂质组学方法专注于研究鞘脂代谢的各个方面。特别强调中性鞘脂和胆固醇及其相互作用。这种兴趣归因于这些脂质在一系列严重疾病（包括各种鞘脂病）中发生改变的事实。高效薄层色谱（HPTLC）已成为一种广泛使用的脂质分析技术。然而，质谱分析是不可替代的，以获得有关脂质类内的各种分子种类的概述。在这项工作中，我们已经开发了一种灵敏的方法，基于梯度正相高效液相色谱（HPLC）耦合四极杆飞行时间（QTOF）大气压化学电离质谱（APCI-MS）在正模式下，这是第一次能够分离，在线检测和质谱分析多种中性鞘脂，包括神经酰胺，葡萄糖神经酰胺，乳糖神经酰胺，在不到15分钟的时间内即可分离出三己糖神经酰胺、四己糖神经酰胺、鞘磷脂以及胆固醇。所提出的HPLC/APCI-MS方法的一个重要优势是，分离模式模拟了通过多阶段开发优化的HPTLC方法获得的分离模式。因此，先前通过HPTLC分离和定量的脂质类可以通过HPLC/APCI-MS根据其质谱图容易地筛选。此外，所选的电离条件使得能够在源内片段化，从而提供有用的结构信息。应用HPLC/APCI-MS和优化的HPTLC方法分析人成纤维细胞中的上述脂质。这种方法主要针对基于遗传修饰或药物治疗进行研究的研究人员，这些研究导致中性鞘脂和胆固醇的生化途径发生变化。此外，它可能对与鞘脂代谢损伤相关的疾病的研究感兴趣。(C)2009 Elsevier B. V.保留所有权利。
Many lipidomic approaches focus on investigating aspects of sphingolipid metabolism. Special emphasis is put on neutral sphingolipids and cholesterol and their interaction. Such an interest is attributed to the fact that those lipids are altered in a series of serious disorders including various sphingolipidoses. High performance thin-layer chromatography (HPTLC) has become a widely used technique for lipid analysis. However, mass spectrometric profiling is irreplaceable for gaining an overview about the various molecular species within a lipid class. In this work we have developed a sensitive method based on a gradient normal phase high performance liquid chromatography (HPLC) coupled to quadrupole time of flight (QTOF) atmospheric pressure chemical ionization mass spectrometry (APCI-MS) in positive mode, which for the first time enables separation, on-line detection, and mass spectrometric profiling of multiple neutral sphingolipids including ceramide, glucosylceramide, lactosylceramide, globotriaosylceramide, globotetraosylceramide, sphingomyelin as well as cholesterol within less than 15 min. An important advantage of the presented HPLC/APCI-MS approach is that the separation pattern emulates the one obtained by an optimized HPTLC method with a multiple stage development. Thus, the lipid classes previously separated and quantified by HPTLC can be easily screened regarding their mass spectrometric profiles by HPLC/APCI-MS. In addition, the selected ionization conditions enable in-source fragmentation providing useful structural information. The methods (HPLC/APCI-MS and the optimized HPTLC) were applied for the analysis of the mentioned lipids in human fibroblasts. This approach is aimed basically at investigators who perform studies based on genetic modifications or treatment with pharmacological agents leading to changes in the biochemical pathways of neutral sphingolipids and cholesterol. In addition, it can be of interest for research on disorders related to impairments of sphingolipid metabolism. (C) 2009 Elsevier B.V. All rights reserved.