Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, United States.
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, United States.
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 May 15;1145:122099. doi: 10.1016/j.jchromb.2020.122099. Epub 2020 Apr 3.
Three normal phase HPLC methods were produced to separate lipid classes on a PVA-Sil stationary phase including: 9 polar lipids (method 1); 13 combined polar and neutral lipids (method 2); and a combined method that further separates the neutral lipids into 2-4 subclasses based on the presence of fatty acids containing a polar functional group (e.g. hydroxyl) for a total of 20 lipid classes and subclasses separated in a single run (method 3). Polar lipids separated include: the phosphoglycerolipids PG, PE, PI, PS, PC and LPC; the galactoglycerolipids MGDG and DGDG; and a sulfoglycerolipid SQDG. Neutral lipids include TAG, DAG, and MAG classes and sub-classes containing 0-3, 0-2, and 0-1 hydroxy fatty acids, respectively. The hexane/isopropanol/methanol/aqueous system separates polar lipids without the use of chloroform such that it is suitable for radioactivity analysis by in-line flow scintillation counting. Each method was optimized using the natural lipid standards comprised of diverse molecular species that were detected by ELSD. All molecular species of each lipid class eluted together as single peak detected by ELSD. The methods were demonstrated to be suitable for resolving lipid extracts from animal, microbial, and plant sources as well as application to C based metabolic tracing of lipid metabolism in leaves and seeds.
三种正相 HPLC 方法被开发用于在 PVA-Sil 固定相上分离脂质类别，包括：9 种极性脂质（方法 1）；13 种组合的极性和中性脂质（方法 2）；以及一种组合方法，根据含有极性官能团（如羟基）的脂肪酸的存在进一步将中性脂质分离成 2-4 个亚类，总共分离出 20 种脂质类别和亚类在单个运行中（方法 3）。分离出的极性脂质包括：磷酸甘油酯 PG、PE、PI、PS、PC 和 LPC；半乳糖甘油酯 MGDG 和 DGDG；以及硫酸甘油酯 SQDG。中性脂质包括 TAG、DAG 和 MAG 类别以及分别含有 0-3、0-2 和 0-1 个羟基脂肪酸的亚类。该正己烷/异丙醇/甲醇/水体系无需使用氯仿即可分离极性脂质，因此适合在线流动闪烁计数进行放射性分析。每种方法都使用由不同分子物种组成的天然脂质标准进行了优化，这些标准通过蒸发光散射检测（ELSD）进行检测。每种脂质类别的所有分子物种都作为 ELSD 检测到的单个峰一起洗脱。该方法被证明适用于分离动物、微生物和植物来源的脂质提取物，以及应用于 C 基代谢示踪脂质代谢在叶片和种子中的应用。
