Charge-switch Derivatization of Fatty Acid Esters of Hydroxy Fatty Acids Via Gas-phase Ion/ion Reactions

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of endogenous bioactive lipids with anti-diabetic and anti-inflammatory effects. Identification of FAHFAs is challenging due to both the relatively low abundance of these metabolites in most biological samples and the significant structural diversity arising from the co-occurrence of numerous regioisomers. Ultimately, development of sensitive analytical techniques that enable rapid and unambiguous identification of FAHFAs is integral to understanding their diverse physiological functions in health and disease. While a battery of mass spectrometry (MS) based methods for complex lipid analysis has been developed, FAHFA identification presents specific challenges to conventional approaches. Notably, while the MS product ion spectra of [FAHFA - H]¯ anions afford the assignment of fatty acid (FA) and hydroxy fatty acid (HFA) constituents, FAHFA regioisomers are usually indistinguishable by this approach. Here, we report the development of a novel MS-based technique employing charge inversion ion/ion reactions with tris-phenanthroline magnesium complex dications, Mg(Phen), to selectively and efficiently derivatize [FAHFA - H]¯ anions in the gas phase, yielding fixed-charge cations. Subsequent activation of [FAHFA - H + MgPhen] cations yield product ions that facilitate the assignment of FA and HFA constituents, pinpoints unsaturation sites within the FA moiety, and elucidates ester linkage regiochemistry. Collectively, the presented approach represents a rapid, entirely gas-phase method for near-complete FAHFA structural elucidation and confident isomer discrimination without the requirement for authentic FAHFA standards.