Simultaneous determination of sphingosine and sphingosine 1-phosphate in biological samples by liquid chromatography-tandem mass spectrometry
Simultaneous determination of sphingosine and sphingosine 1-phosphate in biological samples by liquid chromatography-tandem mass spectrometry
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液相色谱-串联质谱法同时测定生物样品中的鞘氨醇和1-磷酸鞘氨醇
DOI：
10.1016/j.jchromb.2011.01.015
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发表时间：
2011-03-01
影响因子：
3
通讯作者：
Huang, Heqing
中科院分区：
文献类型：
--
作者：
Lan, Tian;Bi, Huichang;Huang, Heqing
D-β-鞘氨醇（Sph）及其磷酸化产物D-β-鞘氨醇1-磷酸（S1 P）是介导许多细胞过程的鞘脂。Sph/S1 P水平的不平衡导致许多疾病。鉴于这两种相反的信号分子的相互转化，同时检查它们的水平是必要的。在本研究中，我们开发了一种快速，灵敏的液相色谱-串联质谱（LC-MS/MS）方法，同时定量的Sph和SIP的生物样品中使用C17-Sph和C17-S1 P作为内标物的水平。通过一步甲醇诱导的蛋白质沉淀，在选择的反应监测模式下使用正电喷雾电离对每个样品进行LC-MS/MS分析。以甲醇-0.1%甲酸（95：5，v/v）为移动的相，流速为0.2 mL/min，在1-100 ng/mL和0.1 ~ 10 ng/mL范围内，Sph和SIPs的线性关系良好，相关系数r（2）均大于0.997。Sph和Si P的定量下限（LLOQ）分别为1 ng/mL和0.1 ng/mL。所有质控样品的批内和批间精密度均小于15%。方法的回收率为76.36- 89.84%。该方法用于同时测定用肿瘤坏死因子-α（TNF-α）和N，N-二甲基鞘氨醇（DMS）处理的小鼠肾脏、人血浆和HEK 293细胞中的Sph和S1 P水平。用TNF-α处理的细胞中S1 P水平增加，而用DMS处理的细胞中S1 P水平降低。结果表明，LC-MS/MS方法快速、灵敏、特异、可靠，可同时测定生物样品中的Sph和SIP。(C)2011 Elsevier B. V.保留所有权利。
D-erythro-sphingosine (Sph) and its phosphorylated product, D-erythro-sphingosine 1-phosphate (S1P) are sphingolipids mediating numerous cellular processes. Imbalance of Sph/S1P levels contributes to many diseases. Given the interconversion of these two opposing signaling molecules, it is essential to examine their levels simultaneously. In the present study, we developed a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to simultaneously quantify the levels of Sph and SIP in biological samples using C17-Sph and C17-S1P as internal standards. With one step of methanol-induced protein precipitation, each sample was subjected to LC-MS/MS analysis using positive electrospray ionization under selected reaction monitoring mode. The running time was within 4 min with a simple mobile phase consisting of methanol-0.1% formic acid (95:5, v/v) at a flow rate of 0.2 mL/min. Standard curves were linear over ranges of 1-100 ng/mL for Sph and 0.1-10 ng/mL for SI P with correlation coefficient (r(2)) greater than 0.997. The lower limit of quantifications (LLOQs) were 1 ng/mL for Sph and 0.1 ng/mL for Si P. The intra-batch and inter-batch precision was less than 15% for all quality control samples. The recoveries of the method were found to be 76.36-89.84%. The method was applied to simultaneously determine the Sph and S1P levels in mouse kidney, human plasma, and HEK 293 cells treated with tumor necrosis factor-alpha (TNF-alpha) and N,N-dimethylsphingosine (DMS). The S1P levels increased in cells treated with TNF-alpha whereas decreased in cells treated with DMS. These results indicated that this new LC-MS/MS method was rapid, sensitive, specific and reliable to quantify Sph and SIP levels in biological samples simultaneously. (C) 2011 Elsevier B.V. All rights reserved.