A Broadly Accessible Approach to Elucidate the Carbon–Carbon Double Bond Position in Unsaturated Lipids by a Post-column In-Line Photochemical Reaction and Mass Spectrometry

Significant advancements in mass spectrometry in the past few decades have enabled comprehensive proteomics, metabolomics, and lipidomics experiments that can describe complex biological processes. Despite faster scan rates, greater resolution, and higher throughput commonly available for new-generation mass spectrometers, some chemically unsophisticated structural characterizations remain a challenge, such as elucidating the carbon-carbon double bond position in fatty acyl chains. Although extensive strategies have been developed to differentiate double bond positional isomers in lipidomics, typically with chemical derivatizations and specialized instrumentation, a simple and broadly accessible approach is still highly desired. In this study, a postcolumn in-line photochemical reaction initiated within a commonly available photodiode array (PDA) detector was developed to cleave carbon-carbon double bonds and monitor the resulting aldehyde product ions to elucidate double bond positions. A photochemical reaction mechanism was proposed based on observations in isotope tracing studies and supplemental experiments. This approach to lipid characterization has been evaluated with multiple lipid classes to demonstrate its broad applicability for structural elucidation and ease of implementation. The effects of aldehyde product generation efficiency with mobile-phase additives commonly used in lipid profiling methods were evaluated to support the applicability in a global structural lipidomics experiment. This approach has the potential to become a powerful analytical tool, providing widespread access to structural lipidomics and further understanding of lipid biology.