While various mass spectrometric approaches have been applied to lipid analysis, unraveling the extensive structural diversity of lipids remains a significant challenge. Notably, these approaches often fail to differentiate between isomeric lipids�a challenge that is particularly acute for branched-chain fatty acids (FAs) that often share similar (or identical) mass spectra to their straight-chain isomers. Here, we utilize charge-switching strategies that combine ligated magnesium dications with deprotonated fatty acid anions. Subsequent activation of these charge inverted anions yields mass spectra that differentiate anteiso-branched-from straightchain and iso-branched-chain FA isomers with the predictable fragmentation enabling de novo assignment of anteiso branch points. The application of these charge-inversion chemistries in both gasand solution-phase modalities is demonstrated to assign the position of anteiso-methyl branch-points in FAs and, with the aid of liquid chromatography, can be extended to de novo assignment of additional branching sites via predictable fragmentation patterns as methyl branching site(s) move closer to the carboxyl carbon. The gas-phase approach is shown to be compatible with top-down structure elucidation of complex lipids such as phosphatidylcholines, while the integration of solution-phase charge-inversion with reversed phase liquid chromatography enables separation and unambiguous identification of FA structures within isomeric mixtures. Taken together, the presented charge-switching MS-based technique, in combination with liquid chromatography, enables the structural identification of branched-chain FA without the requirement of authentic methyl-branched FA reference standards.

While various mass spectrometric approaches have been applied to lipid analysis, unraveling the extensive structural diversity of lipids remains a significant challenge. Notably, these approaches often fail to differentiate between isomeric lipids - a challenge that is particularly acute for branched-chain fatty acids (FAs) that often share similar (or identical) mass spectra to their straight-chain isomers. Here, we utilize charge-switching strategies that combine ligated magnesium dications with deprotonated fatty acid anions. Subsequent activation of these charge inverted anions yields mass spectra that differentiate anteiso branched- from straight-chain and iso branched-chain FA isomers with the predictable fragmentation enabling de novo assignment of anteiso branch points. Application of these charge-inversion chemistries in both gas- and solution-phase modalities is demonstrated to assign the position of anteiso-methyl branch-points in FAs, and can be extended to de novo assignment of additional branching sites via predictable fragmentation patterns as methyl branching site(s) move closer to the carboxyl carbon. The gas phase approach is shown to be compatible with top-down structure elucidation of complex lipids such as phosphatidylcholines, while integration of solution-phase charge-inversion with reversed phase liquid chromatography enables separation and unambiguous identification of FA structures within isomeric mixtures. Taken together, the presented charge switching MS-based technique, in combination with liquid chromatography enables the structural identification of branched-chain FA without the requirement of authentic methyl branched FA reference standards.