Pinpointing Double Bond Positions in Lipids Online Hyphenation of the Paternò Büchi Reaction with Liquid Chromatography-Tandem Mass Spectrometry

Lipids are known for their broad variety of chemical structures, which renders them essential for various biological functions. Moreover, their degree of unsaturation as well as their double bond position play a crucial role, since double bonds have an influence on lipids’ chemical, biochemical and biophysical properties. The full understanding of the lipids’ role in biological processes, however, must yet be uncovered and remains a challenge due to their particularly difficult analysis. Although improvements were made in recent studies, mainly resulting from advances in chromatography and high‑resolution mass spectrometry instrumentation, many applications still suffer from lacking isomeric differentiation. Conventional tandem mass spectrometry (MS/MS) using low-energy collision-induced dissociation (CID) is limited to the determination of the lipid class, the number of carbons and the degree of unsaturation of the fatty acid residues. In order to obtain a more detailed structural elucidation on double bond positional level, further techniques are required, whose development is increasingly discussed in the literature. However, the various current approaches suffer from diverse requirements or limitations. For instance, the mandatory modification of the mass spectrometer or limitations regarding the lipids’ and samples’ complexity hamper the analysis. In this thesis, the development of a novel method for pinpointing double bond positions in lipids is presented. With respect to the known challenges presented by complex biological samples, e.g. the lipids’ structural variety and their different abunclance as weIl as occurring isomenc/isobaric interferences, the work is focused on the hyphenation of high performance liquid chromatography (HPLC)‑MS/MS with an online Paternò Büchi reaction. In the course of the thesis, the present challenges for the elucidation of double bonds in lipids are tackled and the possibilities of the targeted hyphenation are explored. The successful application of the method to complex biological samples is demonstrated by means of three lipid classes that strongly vary in their structural charactenstics and thereby their analytical requirements: diacylglyceroltrimethylhomoserine, sophorolipicis and 3-(3-hydroxyalkanoyloxy) alkanoic acids.