Direct Infusion Acoustic Droplet Ejection Mass Spectrometry: Enabling High-Throughput Shotgun Lipidomics

High-throughput lipidomics is increasingly important for large-scale studies and clinical applications. While shotgun lipidomics enables rapid analysis, it suffers from limitations such as carryover, ion suppression, and limited structural specificity. Acoustic droplet ejection mass spectrometry (ADE-MS) presents a novel approach, enabling touchless nanoliter-scale sample introduction at high speed, precision, and accuracy. Initially designed for single-droplet injection, ADE-MS was adapted for direct infusion with stable signals. In this study, we developed and benchmarked a scalable workflow based on ADE-MS/MS with parallel reaction monitoring (PRM) on a ZenoTOF MS platform implemented in a 384-well format. By optimizing solvent composition, droplet parameters, and MS acquisition settings, the workflow enabled reproducible quantification of over 1000 polar and nonpolar lipid species across 14 subclasses, with low sample consumption and a total run time of approximately five minutes per sample. Applying this method to NIST SRM 1950 plasma, a total of 731 lipid species were quantified. The method demonstrated robust analytical performance in terms of linearity, precision, reproducibility, and recovery across 384-well microplates. Cross-platform comparison with a validated hydrophilic interaction liquid chromatography (HILIC)-MS/MS method using NIST SRM 1950 plasma demonstrated strong agreement (R² > 0.80 for most subclasses) and substantially higher throughput, achieving over 200 lipid identifications per minute and a daily capacity exceeding 280 samples. The applicability of this workflow was demonstrated by identifying 656 differential lipid features associated with progressive lipidomic dysregulation across body mass index categories.