Schwalbe-Herrmann Miriam, Willmann Jan, Leibfritz Dieter
University of Bremen, Institute of Organic Chemistry, Leobener Strasse NW2C, 28359 Bremen, Germany.
J Chromatogr A. 2010 Aug 6;1217(32):5179-83. doi: 10.1016/j.chroma.2010.05.014. Epub 2010 May 19.
A new isocratic separation method was developed for separation of phospholipid (PL) classes based on a silica hydrophilic interaction liquid chromatography (HILIC) column with electrospray ionization (ESI) mass spectrometric detection. Although HILIC is typically used for polar compounds, also amphiphilic molecules like phospholipids can be separated very well. Compared to normal-phase (NP) chromatography, which is usually used for PL class separation, HILIC has the advantage to use on-line ESI-MS detection because its eluents are ESI compatible. Furthermore, this HILIC method is isocratic and hence less time consuming than most (gradient) NP HPLC methods. A chromatographic baseline separation of a standard mixture containing phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingomyelin (SM) and lysophosphatidylcholine (LPC) was achieved within a total run time of 17 min using a mobile phase consisting of acetonitrile, methanol and ammonium acetate 10 mM. The new method was subsequently tested on phospholipid fractions of a body fluid (human blood plasma) and a tissue extract (swine brain) whereby it achieved nearly the same baseline separation of the PL classes. The detected classes in both cases were PE, PC, SM and LPC.
基于硅胶亲水作用液相色谱（HILIC）柱和电喷雾电离（ESI）质谱检测，开发了一种用于分离磷脂（PL）类别的新型等度分离方法。尽管HILIC通常用于分离极性化合物，但两亲性分子如磷脂也能被很好地分离。与通常用于PL类别分离的正相（NP）色谱相比，HILIC具有可使用在线ESI-MS检测的优势，因为其洗脱液与ESI兼容。此外，这种HILIC方法是等度的，因此比大多数（梯度）NP HPLC方法耗时更少。使用由乙腈、甲醇和10 mM乙酸铵组成的流动相，在17分钟的总运行时间内实现了对含有磷脂酰甘油（PG）、磷脂酰乙醇胺（PE）、磷脂酰胆碱（PC）、鞘磷脂（SM）和溶血磷脂酰胆碱（LPC）的标准混合物的色谱基线分离。随后，该新方法在一种体液（人血浆）和一种组织提取物（猪脑）的磷脂级分上进行了测试，在此过程中它实现了与PL类别几乎相同的基线分离。在这两种情况下检测到的类别都是PE、PC、SM和LPC。
