Laboratory of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, 132 Wai Huan Dong Road, Guangzhou Higher Education Mega Center, Guangzhou 510006, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Mar 1;879(7-8):520-6. doi: 10.1016/j.jchromb.2011.01.015. Epub 2011 Jan 18.
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 S1P 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 S1P with correlation coefficient (r²) greater than 0.997. The lower limit of quantifications (LLOQs) were 1 ng/mL for Sph and 0.1 ng/mL for S1P. 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-α (TNF-α) and N,N-dimethylsphingosine (DMS). The S1P levels increased in cells treated with TNF-α 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 S1P levels in biological samples simultaneously.
D-erythro-鞘氨醇（Sph）及其磷酸化产物 D-erythro-鞘氨醇 1-磷酸（S1P）是介导许多细胞过程的鞘脂类。Sph/S1P 水平的失衡与许多疾病有关。鉴于这两种相反信号分子的相互转化，同时检测它们的水平至关重要。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，使用 C17-Sph 和 C17-S1P 作为内标，同时定量生物样品中的 Sph 和 S1P 水平。通过一步甲醇诱导的蛋白质沉淀，每个样品在正电喷雾电离下，采用选择反应监测模式，在 4 分钟内完成 LC-MS/MS 分析。流动相由甲醇-0.1%甲酸（95:5，v/v）组成，流速为 0.2 mL/min，运行时间在 4 分钟内。Sph 的标准曲线在 1-100ng/mL 范围内呈线性，相关系数（r²）大于 0.997。S1P 的定量下限（LLOQ）为 1ng/mL。所有质控样品的批内和批间精密度均小于 15%。该方法的回收率为 76.36-89.84%。该方法应用于同时测定 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 Sph 和 S1P 的水平。TNF-α 处理的细胞中 S1P 水平升高，而 DMS 处理的细胞中 S1P 水平降低。这些结果表明，该新的 LC-MS/MS 方法快速、灵敏、特异、可靠，可同时定量生物样品中的 Sph 和 S1P 水平。
