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Expanding metabolome coverage to include complex lipids and polar metabolites is essential in the generation of well-founded hypotheses in biological assays. Traditionally, lipid extraction is performed by liquid-liquid extraction using either methyl-tert-butyl ether (MTBE) or chloroform, and polar metabolite extraction using methanol. Here, we evaluated the performance of single-step sample preparation methods for simultaneous extraction of the complex lipidome and polar metabolome from human plasma. The method performance was evaluated using high-coverage Hydrophilic Interaction Liquid Chromatography-ESI coupled to tandem mass spectrometry (HILIC-ESI-MS/MS) methodology targeting a panel of 1159 lipids and 374 polar metabolites. The criteria used for method evaluation comprised protein precipitation efficiency, and relative MS signal abundance and repeatability of detectable lipid and polar metabolites in human plasma. Among the tested methods, the isopropanol (IPA) and 1-butanol:methanol (BUME) mixtures were selected as the best compromises for the simultaneous extraction of complex lipids and polar metabolites, allowing for the detection of 584 lipid species and 116 polar metabolites. The extraction with IPA showed the greatest reproducibility with the highest number of lipid species detected with the coefficient of variation (CV) &lt; 30%. Besides this difference, both IPA and BUME allowed for the high-throughput extraction and reproducible measurement of a large panel of complex lipids and polar metabolites, thus warranting their application in large-scale human population studies.

Single-Step Extraction Coupled with Targeted HILIC-MS/MS Approach for Comprehensive Analysis of Human Plasma Lipidome and Polar Metabolome