Short-chain fatty acids (SCFAs) and the tricarboxylic acid (TCA) cycle metabolites aresmall hydrophilic compounds that play crucial roles in biological species ranging fromenergy metabolism, immune homeostasis to cellular signalling. There is a need for reliableand precise quantification of these metabolites in biological matrices as they can providecrucial information of metabolic status and potentially be used as diagnostic biomarkersfor different pathological and physiological conditions. However, their retention andseparation in traditional reversed-phase system, without chemical derivatization, is oftenproblematic due to their volatile and hydrophilic characteristics. The aim of this studywas to implement a facile and effective derivatization method for the simultaneousquantitation of SCFAs and TCA cycle metabolites by LC-qToF-MS in negative ion mode. Inthis work, 3-nitrophenylhydrazine (3-NPH) was employed for preanalyticalderivatization to convert the compounds to their respective 3-nitrophenylhydrazones.Analytical standards and faecal samples were used to assess the linearity, matrix effect,accuracy, extraction efficiency, precision, retention-time shift and short-term stability.The compounds were successfully separated within 6 minutes on a reverse-phase C18column. All the compounds showed good linearity (R2≥ 0.97) in both solvent-only andfaecal samples. The matrix effect was minimal and did not affect the compoundsquantitation. The extraction efficiency ranged from 80% to 110% (CV≤9.7%, n = 6). Theaccuracy of quantitation was determined to be between 82.8% to 113.8% (CV≤9.0%, n =6). The intra-day (CV%) demonstrated good precision for all analytes, the inter-day (%)were more variable due to the derivatives’ chemical instability. However, most of thederivatives were chemical stable up to 5 days in the autosampler (10°C). The method wasalso applied to explore the levels of these metabolites in human faecal samples and mousebrain samples.

Capillary electrophoresis mass spectrometry (CE-MS) is a versatile instrumental method for metabolomics, which allows for comprehensive metabolite profiling of volume-limited biological specimens in order to better understand the molecular mechanisms associated with chronic diseases, including an alarming epidemic of obesity worldwide. Multiplexed CE separations enable high-throughput metabolite screening with quality assurance to prevent false discoveries when combined with rigorous method validation, robust experimental designs, complementary statistical methods, and high-resolution tandem mass spectrometry (MS/MS) for unknown metabolite identification. In this thesis, multiplexed CE-MS technology is applied for both targeted and untargeted metabolite profiling of various biological fluids, including covalently bound thiol-protein conjugates, as well as free circulating metabolites in serum and plasma, and excreted/bio-transformed compounds in urine due to complex host-gut microflora co-metabolism. This work was applied to characterize aberrant metabolic responses of obese subjects in response to dietary challenges, and measure the benefits of dietary interventions that reduce adiposity without deleterious muscle loss. Chapter 2 presents, a simple, sensitive yet robust analytical protocol to expand metabolome coverage in CE-MS for the discovery of labile protein thiols in human plasma using a rapid chemical derivatization method based on N-tert-butylmaleimide (NTBM). Chapter 3 describes targeted metabolite profiling of serum and plasma to investigate the differential metabolic responses between healthy and unhealthy obese individuals before and after consumption of a standardized high-caloric meal, respectively. Chapter 4 of this thesis describes an untargeted metabolite profiling strategy for urine using multisegment-injection (MSI)-CE-MS for elucidating the effects of protein supplementation following a short-term dietary weight-loss intervention study. This work revealed six urinary metabolites that were classified as top-ranking treatment response biomarkers useful for discriminating between subjects consuming carbohydrate (control), soy, and whey supplemented diets. In summary, this thesis demonstrated the successful implementation of multiplexed CE-MS technology for biomarker discovery in nutritional-based metabolomic studies as required for more effective treatment and prevention of obesity for innovations in public health. / Thesis / Doctor of Philosophy (PhD)