Thesis

Application of high-resolution mass spectrometry with MSn fragmentation in the elucidation of double bond positions in fatty acids following formation of hydroperoxides and hydroxides, and application of derivatisation for improved sensitivity in fatty acid analysis

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Awarding institution
  • University of Strathclyde
Date of award
  • 2023
Thesis identifier
  • T16536
Person Identifier (Local)
  • 201360397
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Department, School or Faculty
Abstract
  • Fatty acids (FAs) are the constitutional component of all lipid classes and the main source of structural diversity of lipids that have a myriad of biochemical functions. These include roles as the fundamental building blocks of complex cell membranes bilayer structures (e.g., glycerophosholipids (GPs)); energy sources (e.g., in triacylglycerols (TAGs)); and cellular signalling (e.g., adipokines and DAG). All these functions are vital to cellular metabolism in living systems. In all these contexts, cellular biochemistry can be profoundly impacted by small differences in FAs molecular structure. Therefore, the identification and quantitation of FAs is a critical and challenging task in bioanalytical chemistry. Many FAs can be found in nature as mixtures of isomeric forms that might differ only in the site(s) of unsaturation. Gas chromatography – mass spectrometry (GC-MS) of fatty acid methyl ester derivatives has been the conventional method of choice for fatty acid analysis. Despite attaining generally effective chromatographic separations of the positional isomeric lipids, disambiguation in assigning the identity of these lipids could not be accomplished because their resulting electron ionisation mass spectra were most often indistinguishable. On the other hand, a relatively novel approach is being increasingly used for FA analysis that makes use of liquid chromatography coupled with electrospray ionisation mass spectrometry (LC-ESI-MS). Derivatisation strategies can further enhance this approach through improving the ionisation efficiency of FAs and/or generating distinct product ions useful for structural elucidation, upon application of collision-induced dissociation (CID). In this project, a novel derivatisation strategy was designed and assessed based on combining LC-MS2 and MRM for several isomers of specific fatty acid oxidation products with the well-established knowledge of chemically induced free radical oxidation that leads to the formation of these isomers which mechanism is not only determined by the number of double bonds and their locations but also on their relative positions to each other. Even though the study of fatty acids hydroperoxides has been proved to generate diagnostic product ions that can be used for pinpointing the position of double bonds, the reduced sensitivity, and the complexity of tandem MS spectra of the hydroperoxides can be limiting factors for the application of this method, especially in complex samples. In contrast, the hydroxides obtained by the reduction of those hydroperoxides exhibited specificity for producing two different types of diagnostic fragment ion species for each isomeric fatty acid hydroxide upon CID via cleavages at the positions allylic to the hydroxyl group. The distinctive mass spectral features, arising from CID, enabled unambiguous assignment of site(s) of unsaturation within FAs. This analytical method offers specific, easily interpreted mass spectrometry data while applying simple low-cost experimental setup without the requirement of any modification of the mass spectrometry instrument. Additionally, this method demonstrated efficiency in the differentiation of regioisomers of MUFAs and PUFAs in a group of natural fixed oils. In chapter two, the suitability of three tagging agents, DPD, DMEE and 1MP in the presence of EDC as an activating agent, for the analysis of long chain fatty acids was investigated. The detection of the FAs reversed-phase liquid chromatography (RPLC) in combination with mass spectrometry (MS) was greatly improved along with chromatographic resolution allowing for easy identification of FAs. Moreover, the possibility of applying EDC independently as a derivatising agent was assessed in principle for the same purpose of improving sensitivity of LC-MS analysis of FAs. The results of the study on EDC as a tagging agent confirmed that this method can offer a one-step highly sensitive and thus very useful derivatisation strategy of FAs that greatly improves their detection identification and quantification by LCMS. Thus, EDC shows potential for the sensitive analysis of bioactive oxidised fatty acids which are found in trace amounts in biological systems. This new toolbox of methods will assist researchers in investigating the composition of the lipidome providing unparalleled fidelity in isomer discrimination.
Advisor / supervisor
  • Gray, Alexander I.
  • Watson, David G., 1952-
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