Making Ozonolysis Mass Spectrometry More Accessible: A Simple LC-OzESI-MS Workflow for Carbon–Carbon Double Bond Position Elucidation in Lipids and Natural Products

Elucidation of C═C unsaturation sites in biomolecules remains challenging using conventional tandem mass spectrometry techniques. Online ozonolysis inside a mass spectrometer offers a very specific C═C fragmentation strategy; however, adoption of the precursor-selective ozone-induced dissociation has been limited by its low reaction yields and the need for specialized mass spectrometers that can provide higher partial pressure of ozone and/or modifications to enable effective ion trapping. To make the online ozonolysis mass spectrometry more accessible, we present a setup in which ozone is introduced directly into the sheath gas line of a standard heated electrospray ionization source. This minimally modified online ozonolysis setup enables efficient interaction of ozone with electrospray droplets at atmospheric pressure while maintaining normal instrument operation and safety. In combination with reversed-phase liquid chromatography separation, this setup is very effective in annotating C═C positions in unsaturated biomolecules with just MS1 scanning by relying on the coelution of precursors and the ozonolysis product ions. This approach was validated across multiple lipid classes─including fatty acids, glycerophospholipids, sphingolipids, glycerolipids, and cholesteryl esters─as well as selected natural products, in complex mixtures and under both positive and negative ion modes. Reaction yields varied among analytes yet remained sufficient for unambiguous C═C position assignment. This LC-OzESI-MS strategy provides an efficient and easily adoptable platform for high-throughput structural characterization of unsaturated biomolecules, even in complex sample matrices.