Synthesis and application of tagging agents in mass spectrometry-based targeted metabolomics studies

Student thesis: Doctoral Thesis

Abstract

Metabolomics is a technique which provides comprehensive metabolite profiling as well as metabolic pathway elucidation and potentially has a wide range of applications for medical research including disease diagnosis and monitoring. In this thesis, some synthetically prepared and existing tagging agents were applied to targeted quantitative analysis of certain metabolites in biological samples. The thesis is divided into six chapters: Chapter one provides a general introduction to metabolomics and to the use of chemical tagging in targeted analysis and briefly looks at the type of instrumentation used in metabolomics profiling. Chapter two, described the synthesis of the tagging agent trimethyl(p-aminophenyl) ammonium chloride (PMTA) and along with aniline and D5-aniline was assessed for suitability for the analysis of hexose sugars. The detection of the sugars by hydrophilic interaction chromatography (HILIC) in combination with mass spectrormetry (MS) was greatly improved along with chromatographic resolution allowing for easy identification of sugar isomers. In Chapter three, analysis of short chain fatty acids utilising N-(-3-Dimethylaminopropyl)-Nethylcarbodiimide hydrochlorice (EDC) and N, N-Dimethyl-p-phenylenediamine (DPD) as derivatizing agents was carried out on urine samples from the [sic].The aim of the study was to evaluate the novel CDTREAT diet in comparison to the standard enteral nutrition (EEN) diet. The study confirmed that the detection and quantification of SCFAs by LCMS was greatly enhanced by derivatisation and showed no difference in the levels of SCFAs between the treatment and control groups which implies that the diets had similar effects on the levels of SCFAs in urine and thus could be deemed equivalent. In Chapter four, an investigation of the effect of ultramarathon exercise on oxylipin metabolites in plasma samples from the University of Kingston was carried out. In this study 2-fluoro-3-methyl pyridine (FMP) was utilised as a catalyst to add choline as a tagging agent which facilitated the sensitive detection and quantification of the oxylipins. These compounds are thought to have biological effects on blood flow. Statistical analysis of the results showed no significant change in the amounts of any of the oxylipins in the study pre and post-exercise.In Chapter five, a study to determine the variation in four monosaccharides from stool samples of a group of IBS suffers plus controls was undertaken using D5-aniline as the tagging agent. The data generated in this study appears on analysis to indicate a clear difference in the levels of some sugars between each of the test groups (UC, CD and healthy controls) which might be a result of alteration in the microbiome in CD. This is significant because it implies that this could be developed as not only a diagnostic tool for IBS it can be used to differentiate between sufferers of CD and UC. In Chapter 6, 2-Hydrazino-1-methylpyridine (HMP), as well as a novel analogue of HMP, MHMP, and its deuterated analogue MHMP-d3 were synthesised. The HMP was passed on to a co-worker who utilised it in a separate study for the analysis of androgens in saliva. It was however not possible to detect any of the tagged steroids utilising MHMP-d3 which may be due to difficulties in obtaining the reagent in high purity.
Date of Award3 Jun 2020
Original languageEnglish
Awarding Institution
  • University Of Strathclyde
SupervisorDavid Watson (Supervisor), Colin Gibson (Supervisor) & Nicholas Rattray (Supervisor)

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