A comprehensive evaluation of solid-phase extraction strategies for targeted and untargeted liquid chromatography - mass spectrometry metabolomic analysis.

Sample preparation remains one of the most critical and challenging steps in liquid chromatography - mass spectrometry (LC-MS) metabolomic analysis, as it directly affects metabolite recovery and analytical accuracy and reliability. In this study, we systematically compared solid-phase extraction (SPE) strategies for serum sample preparation in both targeted and untargeted metabolomic approaches. A total of 71 metabolites with diverse structural and polarity characteristics were analyzed using various SPE formats, including dispersive SPE with multiple sorbents and extraction modes, SPE spin columns, SPE pipette tips, and conventional SPE cartridges. Sorbents within the same extraction mode yielded comparable results. Hydrophilic interaction liquid chromatography sorbents demonstrated the highest performance across a wide range of polarities, whereas reversed-phase sorbents favored moderately polar compounds. Ion-exchange sorbents exhibited limited suitability for broad metabolite coverage due to strong pH dependence but improved recovery of ionizable compounds when combined with other sorbents. While different SPE formats showed similar extraction efficiency, their repeatability varied, with spin columns outperforming conventional cartridges. SPE exhibited mitigation of matrix effects in targeted analysis, particularly for highly polar metabolites, compared to protein precipitation (PPT). Although PPT offered higher efficiency in the untargeted workflow, SPE increased feature coverage by up to 50%. Among commercial products, hydrophilic-lipophilic balanced (HLB) sorbents delivered superior efficiency and repeatability, with HLB-packed spin columns providing the most universal and robust performance for LC-MS metabolomic analysis. The results of this systematic evaluation offer practical guidance for selecting appropriate sample preparation strategies tailored to specific analytical goals and target metabolites.