Center for Human Nutrition University of Texas Southwestern Medical Center, Dallas, TX 75390
Department of Molecular Genetics University of Texas Southwestern Medical Center, Dallas, TX 75390.
J Lipid Res. 2019 Mar;60(3):694-706. doi: 10.1194/jlr.D090795. Epub 2019 Jan 4.
An unbiased sample preparation free of interferents (i.e., competing analytes, detergents, plastics) is critical to any lipid MS workflow. Here we present a novel three-phase lipid extraction (3PLE) technique using a single-step liquid-liquid extraction (LLE) that allows both extraction and fractionation of lipids by polarity. 3PLE is composed of one aqueous and two organic phases. The upper organic phase is enriched in neutral lipids (triacylglycerols and cholesteryl esters), while the middle organic phase contains the major glycerophospholipids. Thin-layer chromatography, radioactive labeling, and MS were used to confirm lipid partitioning. 3PLE efficiency was demonstrated for bovine liver, human pooled plasma, mouse liver, mouse brain, and mouse white adipose tissue. Compared with the gold-standard Bligh/Dyer LLE, 3PLE showed significant advantages. For direct-infusion workflows, there was a decrease in ion suppression with a corresponding increased number of lipid species identified. For LC/MS workflows, increased signal intensities were observed for lower-abundance lipid species such as phosphatidic acid and phosphatidylserine. 3PLE also proved to be a valuable tool for fatty acid profiling by GC/MS, allowing for the separate identification of neutral and polar fatty acids.
无偏的样本制备，无干扰物（即竞争分析物、清洁剂、塑料），这对任何脂质 MS 工作流程都至关重要。在这里，我们提出了一种新的三相脂质提取（3PLE）技术，该技术使用一步液-液萃取（LLE），通过极性对脂质进行同时提取和分级。3PLE 由一个水相和两个有机相组成。上层有机相富含中性脂质（三酰基甘油和胆固醇酯），而中层有机相含有主要的甘油磷脂。薄层层析、放射性标记和 MS 用于确认脂质分配。3PLE 的效率已在牛肝、人混合血浆、鼠肝、鼠脑和鼠白色脂肪组织中得到证明。与黄金标准的 Bligh/Dyer LLE 相比，3PLE 具有明显的优势。对于直接注入工作流程，离子抑制减少，鉴定的脂质种类数量相应增加。对于 LC/MS 工作流程，观察到低丰度脂质种类（如磷脂酸和磷脂酰丝氨酸）的信号强度增加。3PLE 还被证明是 GC/MS 脂肪酸分析的有用工具，允许分别鉴定中性和极性脂肪酸。
