液相串联质谱法同时定量检测生物样本中鞘脂类化合物

鞘脂类中的主要活性分子鞘氨醇1-磷酸（S1P），可通过介导细胞增殖、分化和迁移等发挥广泛的生物学功能|同时,S1P可在相关酶的作用下转变为其它形式.本文旨在建立一种简便的鞘脂类的制备方法，并结合液相串联质谱（LC-MS/MS）快速检测生物样本中纳克水平的鞘脂类化合物. 采用甲醇沉淀或经典的脂质提取方法获得鞘脂类化合物，再采用LC-MS/MS在多反应监测模式（MRM）下进行定量分析. 实验结果表明，甲醇沉淀法可简便快捷地获得血浆或脂蛋白中鞘氨醇类化合物; S1P、二氢鞘氨醇（DH-S1P）和鞘氨醇（SPH）的定量限分别为110.5、215.6和44.3 pg;人血浆中S1P、DH-S1P和SPH的含量分别为257.8±49.4 nmol/L、93.5±17.3 nmol/L和44.6± 7.4 nmol/L, 鞘脂类化合物在人血浆脂蛋白上的分布存在显著性差异|在ApoE-/-小鼠血浆中S1P、DH-S1P和SPH的含量分别为590.1±78.2 nmol/L、197.8±60.6 nmol/L和35.4±16.7 nmol/L|每1×106个人脐静脉内皮细胞（EA.hy926）中，S1P和SPH的含量分别为103.7±21.8 pg和16.3±5.3 ng.甲醇沉淀法结合LC-MS/MS可简便快捷地对血浆和脂蛋白中的鞘脂类化合物进行定量检测. 该方法特别适用于大量生物样本中鞘脂类的快速定量分析.

Simultaneous Measurement of Sphingolipid in Biological Samples by Liquid Chromatography Tandem Mass Spectrometry

As one of the most important bioactive sphingolipid metabolites, sphingosine 1-phosphate (S1P) exerts various biological functions by regulating cell proliferation, differentiation and migration. S1P could be transformed to other sphingolipid metabolites by several related enzymes. The aim of this study is to establish a simplified preparation method for sphingolipid metabolites in biological sample, and to quantify them by liquid chromatography tandem mass spectrometry (LC-MS/MS). Methanol precipitation together with ultrasonic extraction was used to prepare sphingolipid metabolites in plasma or lipoprotein, then the supernatant was quantified with multiple reaction monitoring (MRM) mode by LC-MS/MS after centrifugation. Results showed that methanol preparation was effective and time-saving. The limitation of quantification of S1P, DH-S1P and SPH were 110.5, 215.6 and 44.3 pg, respectively; The amounts of S1P, DH-S1P and SPH in human plasma was 257.8±49.4 nmol/L, 93.5±17.3 nmol/L and 44.6±7.4 nmol/L, respectively. The levels of sphingolipid metabolites exhibited significant difference in lipoproteins. The amounts of S1P, DH-S1P and SPH in apoE-/-mouse plasma were 590.1±78.2 nmol/L, 197.8±60.6 nmol/L and 35.4±16.7 nmol/L, respectively; The amounts of S1P and SPH were 103.7±21.8 pg and 16.3±5.3 ng per 106 EA.hy926 cells, respectively. In summary, methanol precipitation together with LC-MS/MS could effectively quantify sphingolipid metabolites in plasma or lipoprotein. This method is suitable for quantitative analysis of a great deal of biological samples at one time.