Normal phase HPLC method for combined separation of both polar and neutral lipid classes with application to lipid metabolic flux
Normal phase HPLC method for combined separation of both polar and neutral lipid classes with application to lipid metabolic flux
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DOI：
10.1016/j.jchromb.2020.122099
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发表时间：
2020-05-15
影响因子：
3
通讯作者：
Bates, Philip D.
中科院分区：
文献类型：
--
作者：
Kotapati, Hari Kiran;Bates, Philip D.
制备了三种正相HPLC方法以在PVA-Sil固定相上分离脂质类，包括：（方法1）; 13种极性和中性脂质组合（方法2）;以及基于含有极性官能团的脂肪酸的存在将中性脂质进一步分离成2-4个亚类的组合方法对于在单次运行中分离的总共20种脂质类别和亚类，测定脂质（例如羟基）（方法3）。分离的极性脂质包括：磷酸甘油脂PG、PE、PI、PS、PC和LPC;半乳糖甘油脂MGDG和DGDG;和磺基甘油脂SQDG。中性脂质包括TAG、DAG和MAG类以及分别含有0- 3、0 -2和0-1羟基脂肪酸的亚类。己烷/异丙醇/甲醇/水溶液系统分离极性脂质，无需使用氯仿，因此适用于通过在线流动闪烁计数进行放射性分析。使用由ELSD检测的不同分子种类组成的天然脂质标准品优化每种方法。通过ELSD检测，每种脂质类别的所有分子种类一起洗脱为单峰。该方法被证明是合适的，以解决从动物，微生物和植物来源的脂质提取物，以及应用到基于14 C的代谢示踪叶片和种子中的脂质代谢。
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 14 C based metabolic tracing of lipid metabolism in leaves and seeds.