Quantification of sphingosine-1-phosphate and related sphingolipids by liquid chromatography coupled to tandem mass spectrometry.
Quantification of sphingosine-1-phosphate and related sphingolipids by liquid chromatography coupled to tandem mass spectrometry.
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DOI：
10.1007/978-1-61779-800-9_3
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发表时间：
2012-01-01
期刊：
影响因子：
--
通讯作者：
Graler, Markus H
中科院分区：
文献类型：
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作者：
Bode, Constantin;Graler, Markus H
与其他方法相比，液相色谱-串联质谱法具有高灵敏度和上级特异性，因此已发展成为检测鞘脂代谢物的首选方法。在这里，我们描述了一种简单而强大的方法，用于检测和定量的鞘氨醇-1-磷酸（S1 P）和相关的鞘脂类生物样品。组织匀浆、细胞、上清液、血浆和全血加标内标物，以说明样品处理期间的材料损失。在酸化条件下用氯仿提取脂质后，蒸发溶剂，将剩余的脂质提取物溶于20% CHCl（3）和80%甲醇中。在鞘脂的反相高效液相色谱阶梯梯度分离和正电喷雾电离后，使用AB Sciex QTrap三重四极杆质谱仪进行检测，该质谱仪在多反应监测中运行。监测S1 P和相关鞘脂的特征碎片离子，随后相对于已知标准浓度的纯化合物进行分析。已知的问题S1 P定量，如携带和HPLC分离不足，进行了讨论。
Liquid chromatography coupled to tandem mass spectrometry has evolved as the method of choice for the detection of sphingolipid metabolites due to its high sensitivity and superior specificity compared to other methodological approaches. Here, we describe a simple and robust method for the detection and quantification of sphingosine-1-phosphate (S1P) and related sphingolipids in biological samples. Tissue homogenates, cells, supernatant, plasma, and whole blood are spiked with an internal standard to account for loss of material during sample handling. After chloroform extraction of lipids under acidified conditions, the solvent is evaporated, and the remaining lipid extracts are dissolved in 20% CHCl(3) and 80% methanol. Following reversed-phase high-performance liquid chromatography step-gradient separation of sphingolipids and positive electrospray ionization, detection is carried out with the AB Sciex QTrap triple-quadrupole mass spectrometer operating in multiple reaction monitoring. Characteristic fragment ions of S1P and related sphingolipids are monitored and subsequently analyzed relative to known standard concentrations of the pure compounds. Known problems of S1P quantification, such as carryover and insufficient HPLC separation, are discussed.