Objective Set of Criteria for Optimization of Sample Preparation Procedures for Ultra-High Throughput Untargeted Blood Plasma Lipid Profiling by Ultra Performance Liquid Chromatography–Mass Spectrometry

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Abstract

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.

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