School of Pharmacy, East China University of Science and Technology, 200237 Shanghai, China.
J Chromatogr A. 2012 Jan 13;1220:26-34. doi: 10.1016/j.chroma.2011.11.034. Epub 2011 Nov 25.
A hydrophilic interaction liquid chromatography (HILIC) - ion trap mass spectrometry method was developed for separation of a wide range of phospholipids. A diol column which is often used with normal phase chromatography was adapted to separate different phospholipid classes in HILIC mode using a mobile phase system consisting of acetonitrile, water, ammonium formate and formic acid. An efficient between-class separation of seven phospholipid classes including phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinostol, phosphatidylcholine, phosphatidylserine, sphingomyelin and lysophosphatidylcholine was successfully achieved within 14 min using a gradient elution which starts with 90% of organic solvent and ends with 70% of organic solvent. 53 mM formic acid (in both organic phase and aqueous phase) and 60mM ammonium formate (only in aqueous phase) were used as mobile phase modifier. The relatively high amount of ammonium formate was essential to obtain well-shaped peaks of each phospholipid class, especially phosphatidylserines; actually, no negative effect due to ammonium formate was observed for electrospray-mass spectrometry detection in real-life samples. Good chromatographic separation between different lipid classes was obtained (Rs, from 0.73 to 4.97) and well-shaped peaks (tailing factor, from 0.98 to 1.20) were obtained. The developed method was fully validated and the satisfactory performance characteristics such as linearity (R(2), 0.990-0.999), retention time stability (RSD<1%), within day repeatability (RSD, 5-13%), between day variation (RSD, 7-14%) and recoveries (99.6-115.5%) indicated the gradient HILIC method was appropriate for profiling of plasma phospholipids. The method was successfully applied to separate phospholipids extracts from human plasma, mouse plasma and rat plasma.
建立了亲水作用色谱（HILIC）-离子阱质谱法，用于分离多种磷脂。通常用于正相色谱的二醇柱被改编为在 HILIC 模式下使用包含乙腈、水、甲酸铵和甲酸的流动相系统分离不同的磷脂类。通过梯度洗脱，在 14 分钟内成功实现了包括磷脂酰甘油、磷脂酰乙醇胺、磷脂酰肌醇、磷脂酰胆碱、磷脂酰丝氨酸、鞘磷脂和溶血磷脂酰胆碱在内的七种磷脂类的高效类间分离，起始时有机相的比例为 90%，结束时有机相的比例为 70%。在有机相和水相均使用 53mM 甲酸（甲酸铵仅在水相中使用）作为流动相调节剂。相对较高量的甲酸铵对于获得每种磷脂类的良好峰形是必不可少的，特别是磷脂酰丝氨酸；实际上，在实际样品的电喷雾质谱检测中，没有观察到甲酸铵的负面影响。获得了不同脂质类之间的良好色谱分离（Rs，从 0.73 到 4.97）和良好的峰形（拖尾因子，从 0.98 到 1.20）。所开发的方法进行了全面验证，表现出令人满意的性能特征，如线性（R²，0.990-0.999）、保留时间稳定性（RSD<1%）、日内重复性（RSD，5-13%）、日间变化（RSD，7-14%）和回收率（99.6-115.5%），表明梯度 HILIC 方法适用于分析血浆磷脂。该方法成功应用于分离人血浆、小鼠血浆和大鼠血浆中的磷脂提取物。
