CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
J Chromatogr A. 2013 Dec 13;1320:103-10. doi: 10.1016/j.chroma.2013.10.064. Epub 2013 Oct 26.
Sphingolipids are a family of bioactive molecules with high structural diversity and complexity. They not only serve as integral components of cellular membrane, but also play pivotal roles in signaling and other cellular events. It is desirable for the development of sensitive, robust and structural-specific analytical approaches enabling rapid determination of as many sphingolipid species as possible. Herein we present an analytical method for large-scaled profiling of sphigolipids in human serum, which consisted of an improved extraction protocol using tert-butyl methyl ether combined with mild alkaline hydrolysis, and an ultra high performance reversed-phase liquid chromatography-dynamic multiple reaction monitoring-mass spectrometric (RPLC-dynamic MRM-MS) method. In total 84 endogenous sphingolipid species covering six subcategories (i.e. free sphingoid base, dihydroceramide, ceramide, hexosylceramide, lactosylceramide, and sphingomyelin), were separated and quantified in a single run within 10min. A broad linear range over 2.5-4 orders of magnitude (r(2)>0.99), a limit of detection of 0.01-0.17pmol/mL, and a limit of quantitation of 0.02-0.42pmol/mL were obtained for each subcategory. Average recovery of each subcategory was within 85.6-95.6%. Median values of coefficient of variation (CV) of all detected 84 sphingolipids were 3.9% and 6.8% for intraday and interday precision, respectively. This method was exemplarily applied in a study regarding dysregulated sphingolipid homeostasis in hepatocellular carcinoma. The establishment of this method provides a useful tool for serum-based high throughput screening of sphingolipid biomarkers and mechanism investigation of sphingolipid metabolic regulation in human disease.
鞘脂是一类具有高度结构多样性和复杂性的生物活性分子。它们不仅作为细胞膜的组成部分，而且在信号转导和其他细胞事件中发挥关键作用。因此，需要开发灵敏、稳健且具有结构特异性的分析方法，以实现尽可能多地快速测定鞘脂种类。本文提出了一种用于人血清中鞘脂大规模分析的方法，该方法包括使用叔丁基甲基醚进行改进的提取方案，与温和的碱性水解相结合，以及超高效反相液相色谱-动态多重反应监测-质谱（RPLC-dynamic MRM-MS）方法。总共分离和定量了 84 种内源性鞘脂，涵盖了六个亚类（即游离鞘氨醇碱基、二氢神经酰胺、神经酰胺、己糖神经酰胺、乳糖神经酰胺和神经鞘磷脂），在 10 分钟内可以一次运行。每种亚类均具有较宽的线性范围（2.5-4 个数量级，r(2)>0.99），检测限为 0.01-0.17pmol/mL，定量限为 0.02-0.42pmol/mL。每个亚类的平均回收率在 85.6-95.6%之间。所有检测到的 84 种鞘脂的日内和日间精密度的变异系数（CV）中位数分别为 3.9%和 6.8%。该方法已成功应用于肝细胞癌中鞘脂稳态失调的研究。该方法的建立为基于血清的高通量筛选鞘脂生物标志物以及研究人类疾病中鞘脂代谢调控机制提供了有用的工具。
