Investigating C[double bond, length as m-dash]C positions and hydroxylation sites in lipids using Paternò-Büchi functionalization mass spectrometry.

Lipid oxidation plays a major role in biochemical processes and nutrition. Structural changes during oxidation can lead to alterations of lipid functions. Rancidification and production of secondary lipid messengers are well-known examples for the impact of oxidation on lipid function. Especially lipids with a high degree of unsaturation are prone to oxidize. In order to investigate structural changes of lipids upon oxidation, we here introduce a photochemical Paternò-Büchi functionalization workflow and subsequent mass spectrometric analysis for analysis of unsaturated, oxidized lipids. Results for hydroxylated fatty acids and triglycerides containing isolated and conjugated C[double bond, length as m-dash]C bonds will be presented making use of 3-acetylpyridine as a photochemically active compound. Photochemical derivatization is performed in nano-electrospray emitter tips in 30 s resulting in the formation of oxetanes without inducing light-triggered oxidation of analytes. Collisional-activation of photoproducts facilitates selective cleavage of oxetane moieties. Resulting fragment ions not only allow the determination of C[double bond, length as m-dash]C bond locations for isolated and conjugated C[double bond, length as m-dash]C bonds but also restrict the site of oxidation. By registering the mass shift in some fragment ions of +15.99 Da due to hydroxylation, the oxidized sections of lipids can be identified. In order to demonstrate its analytical robustness, the method is applied to determine the structural impact of non-selective ambient oxidation on fatty acids, triglycerides and complex triglyceride mixtures obtained from Sacha inchi oil.