Sarafian Magali H, Gaudin Mathieu, Lewis Matthew R, Martin Francois-Pierre, Holmes Elaine, Nicholson Jeremy K, Dumas Marc-Emmanuel
Imperial College London , Section of Biomolecular Medicine, Division of Computational Systems Medicine, Department of Surgery and Cancer, Sir Alexander Building, Exhibition Road, South Kensington, London SW7 2AZ, U.K.
Anal Chem. 2014 Jun 17;86(12):5766-74. doi: 10.1021/ac500317c. Epub 2014 Jun 2.
Exploratory or untargeted ultra performance liquid chromatography-mass spectrometry (UPLC-MS) profiling offers an overview of the complex lipid species diversity present in blood plasma. Here, we evaluate and compare eight sample preparation protocols for optimized blood plasma lipid extraction and measurement by UPLC-MS lipid profiling, including four protein precipitation methods (i.e., methanol, acetonitrile, isopropanol, and isopropanol-acetonitrile) and four liquid-liquid extractions (i.e., methanol combined with chloroform, dichloromethane, and methyl-tert butyl ether and isopropanol with hexane). The eight methods were then benchmarked using a set of qualitative and quantitative criteria selected to warrant compliance with high-throughput analytical workflows: protein removal efficiency, selectivity, repeatability, and recovery efficiency of the sample preparation. We found that protein removal was more efficient by precipitation (99%) than extraction (95%). Additionally, isopropanol appeared to be the most straightforward and robust solvent (61.1% of features with coefficient of variation (CV) < 20%) while enabling a broad coverage and recovery of plasma lipid species. These results demonstrate that isopropanol precipitation is an excellent sample preparation procedure for high-throughput untargeted lipid profiling using UPLC-MS. Isopropanol precipitation is not limited to untargeted profiling and could also be of interest for targeted UPLC-MS/MS lipid analysis. Collectively, these data show that lipid profiling greatly benefits from an isopropanol precipitation in terms of simplicity, protein removal efficiency, repeatability, lipid recovery, and coverage.
探索性或非靶向超高效液相色谱-质谱联用（UPLC-MS）分析可概述血浆中复杂脂质种类的多样性。在此，我们评估并比较了八种样品制备方案，用于通过UPLC-MS脂质分析优化血浆脂质提取和测量，包括四种蛋白质沉淀方法（即甲醇、乙腈、异丙醇和异丙醇-乙腈）和四种液-液萃取方法（即甲醇与氯仿、二氯甲烷和甲基叔丁基醚，以及异丙醇与己烷）。然后，使用一套为确保符合高通量分析工作流程而选择的定性和定量标准，对这八种方法进行基准测试：样品制备的蛋白质去除效率、选择性、重复性和回收率。我们发现，沉淀法（99%）去除蛋白质的效率高于萃取法（95%）。此外，异丙醇似乎是最直接、最稳定的溶剂（变异系数（CV）<20%的特征占61.1%），同时能广泛覆盖和回收血浆脂质种类。这些结果表明，异丙醇沉淀法是使用UPLC-MS进行高通量非靶向脂质分析的优秀样品制备方法。异丙醇沉淀法不仅限于非靶向分析，对于靶向UPLC-MS/MS脂质分析也可能具有吸引力。总体而言，这些数据表明，脂质分析在简单性、蛋白质去除效率、重复性、脂质回收率和覆盖范围方面，从异丙醇沉淀法中受益匪浅。
