The structural complexity of triacylglycerols (TGs) and the positional specificity of carbon-carbon double bonds (C═C) within fatty acyl (FA) chains present a formidable challenge in structural lipidomics. Traditional tandem mass spectrometry (MS) approaches often lack the capability to distinguish TG isomers, limiting insights into lipid biochemistry and isomer-specific functionality. Here, we introduce an online liquid chromatography (LC) approach that enables isomer-specific TG identification through in-source ozonolysis via ozone electrospray ionization (OzESI) combined with multiple-reaction monitoring (MRM) on a triple quadrupole mass spectrometer. By directly incorporating ozone into the electrospray ionization source, our approach generates diagnostic ozonolysis product ions that localize C═C positions without the need for post-ionization reaction chambers or major instrument modifications. We demonstrate the utility of this method using synthetic TG standards and apply it to real-world samples, including the analysis of canola oil across processing stages. To overcome the challenges of data analytics, automated data processing is achieved via CLAW-OzESI-MRM (Comprehensive Lipidomics Automated Workflow for OzESI-MRMs) which includes an artificially intelligent (AI) agentic framework to streamline structural assignment of TGs with C═C specificity. This robust, accessible platform offers enhanced isomeric resolution for lipidomics research, expanding the analytical capabilities of targeted MS workflows in food, biomedical, and industrial applications.
Randolph et al. (Tue,) studied this question.