Biochemical markers (i.e., biomarkers) are of key importance to guide clinical decisions and public health policies by enabling early detection, accurate diagnosis and/or prevention of human diseases. However, major challenges remain due to the limited clinical utility of many biomarkers and the lack of robust analytical methods for their reliable measurements in a routine clinical setting that is also suitable to large-scale epidemiological studies. This thesis includes two major research themes involving the analysis of known biomarkers that lack appropriate methods, and the discovery of new biomarkers for complex disease conditions. In particular, this thesis describes the (1) development and validation of robust analytical methods based on capillary electrophoresis (CE) with photometric detection for the measurement of inorganic anions in volume-restricted biofluids; and (2) characterization of the sweat metabolome in infants and identification of sweat biomarkers in asymptomatic cystic fibrosis (CF) infants using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Chapter II introduces a simple CE assay that is remarkably selective for the analysis of sulfate, sulfite and chloride, which was applied in a pilot study to investigate the role of urinary sulfate as a biomarker for kidney stone risk assessment in children. Chapter III introduces a novel CE method for assessing iodine nutrition, which is followed by an inter-method comparison with inductively coupled plasma-mass spectrometry as described in Chapter IV. This led to the development of a fully validated CE method for monitoring the prevalence of iodine deficiency on a population level as required for global health initiatives. Chapter V demonstrates that more than 64 endogenous and exogenous compounds are present in the sweat of screen-positive CF infants, including a panel of differentiating metabolites that are associated with CF status, as well as treatment responsivity to nutritional and/or drug intervention. In summary, this thesis has contributed novel analytical methods suitable for routine biomarker analysis, in addition to the first non-targeted characterization of the sweat metabolome from infants, which require further studies to evaluate their clinical utility as biomarkers that allow for improved diagnosis, prognosis and treatment monitoring of the highly variable CF disease spectrum. / Thesis / Doctor of Philosophy (PhD)

<p> Affinity capillary electrophoresis (ACE) is a versatile technique for assessing non-covalent molecular interactions in free solution provided that there are significant changes in apparent analyte mobility as a result of specific complexation. The thermodynamics of receptor binding are vital for controlling the selectivity in molecular recognition, which are dependent on the electrolyte composition of solution. In addition, the conformational properties of the complex (e.g., size, shape) can also contribute a secondary influence on receptor selectivity that has been relatively unexplored in ACE to date. In this study, dynamic 1:1 host-guest inclusion complexation involving a anionic resorcin[4]arene with a group of neutral corticosteroids was examined by ACE, where the macrocycle serves as both an intrinsic buffer and electrokinetic host. The tetraethylsulphonate derivative of 2-methylresorcin[4]arene (TESMR) was first synthesized via an acid-catalyzed condensation reaction, which was then fully characterized in terms of its weak acidity (pKa), mobility, UV spectral and buffer capacity properties. TESMR solutions were demonstrated to have stable intrinsic buffer and ion transport properties at pH 7.5 even at low ionic strength. It was determined that over a 200 % enhancement in the apparent binding constant (KB) was realized by ACE when using TESMR as an intrinsic buffer at pH 7.5 relative to an extrinsic sodium phosphate buffer system, which was also confirmed by 1H-NMR. The coupling of thermodynamic (KB) and electrokinetic (μep, AC) factors associated with complex formation in buffered aqueous solutions that minimize the effects of extrinsic electrolytes serves to enhance enthalpy-driven molecular recognition processes by ACE.</p> / Thesis / Master of Science (MSc)

Irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) are two of the most commonly diagnosed chronic digestive disorders in Western countries with increasing prevalence among Canadians. However, the etiology of IBS and IBD remain poorly understood due to a complex interplay of genetic, psychosocial and environmental factors, which hampers efforts at early detection/screening, accurate diagnosis and effective treatments notably in children. This thesis aims to reveal new biochemical insights into the pathophysiology underlying IBS and IBD when using an untargeted metabolite profiling (i.e., metabolomics) approach on urine and stool specimens based on multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Chapter I reviews brief history and current challenges in diagnosis and treatment, as well as current metabolomics literature of IBS and IBD. Chapter II first develops a robust method for high throughput profiling of anionic metabolites in human urine samples when using MSI-CE-MS. For the first time, we demonstrate that incidental capillary fractures are caused by irreversible aminolysis of the outer polyimide coating due to the frequent use of volatile ammonia based buffers under alkaline conditions (pH > 9) in electrospray ionization-MS. Chapter III subsequently applies this validated method to investigate differentially excreted urinary metabolites between adult IBS patients and healthy controls, which indicated significantly accelerated rates of collagen degradation and cell turn-over in IBS patients. Chapter IV later develops a novel stool extraction protocol for characterization of the fecal metabolome together with meta-genomic data for elucidating complex host-gut microflora interactions from a cohort of pediatric IBD patients, including Crohn’s disease and ulcerative colitis. In this pilot study, a panel of discriminating metabolites in urine is shown to allow for differential diagnosis of major pediatric IBD sub-types as an alternative to colonoscopy and histopathology that are invasive, expensive and prone to ambiguous test results. Finally, Chapter V involves a longitudinal metabolomics study that aims to identify metabolic trajectories that predict treatment responses of a cohort of pediatric Crohn’s disease patients following initiation of exclusive enteral nutrition (EEN) therapy. In the end, Chapter VI highlights major outcomes of thesis and future direction of metabolomics in IBS and IBD with a specific focus on improved stool specimen collection and validation of biomarker specificity relative to other related gastrointestinal disorders. In summary, this thesis has demonstrated metabolic processes that are associated with exacerbation of symptoms or remission in subset of IBS and pediatric IBD patients. With follow up studies with larger cohort of patients, potential biomarkers identified in this thesis will contribute the development of more accurate and non-invasive decision making process for diagnosis and treatment, resulting in long-lasting remission and improved quality of life of patients suffering from chronic digestive disorders. / Thesis / Doctor of Science (PhD)

PhD Thesis / Metabolomics offers a systemic approach to discover clinical biomarkers for early detection of chronic diseases while also revealing underlying mechanisms relevant to human disorders of complex aetiology. Metabolomic studies in support of chronic disease prevention have focused primarily on surrogate biofluids (e.g., serum, plasma) for analysis due to their routine and less invasive sample collection in a clinical setting. However, biofluids are non-organ specific and thus are reflective of confounding biochemical processes within the body that are often difficult to interpret. As a result, it is necessary to assess metabolite changes localized within tissues since they are the direct site of pathogenic processes, in order to obtain more robust and specific biomarkers. This thesis aims to contribute to new advances in biomarker discovery and tissue metabolomic studies using multiplexed separations together with innovative data workflows based on multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Chapter II introduces a high throughput yet targeted screening method for accurate quantification of serum γ‐glutamyl dipeptides from a cohort of overweight Japanese non-alcoholic steatohepatitis (NASH) patients that may allow for better risk assessment of long-term survivorship complementary to histopathology. Chapter III introduces a non-targeted metabolite profiling strategy for fasting plasma samples from prediabetic, older adults undergoing short-term step reduction (<1000 steps/day) in order to identify adaptive metabolic responses to abrupt changes in physical inactivity for early detection of sarcopenia in high-risk older persons. Chapter IV describes the first metabolomics study to characterize the human skeletal muscle metabolome from mass-restricted tissue biopsies together with matching plasma samples, which identified novel metabolic signatures associated with strenuous interval exercise, as well as treatment effects from high-dose bicarbonate pretreatment that delays the onset of muscle fatigue. Lastly, in Chapter V, metabolite coverage was expanded to include fatty acids for comprehensive characterization of murine placental tissue metabolome, which revealed sex-specific metabolic adaptations during gestation from maternal dams fed a standardized diet. In summary, this thesis contributes to new innovations in metabolomics for the discovery of novel biomarkers from blood and/or tissue specimens as required for early detection of chronic diseases relevant to population health, which were also used to validate the efficacy of therapeutic interventions based on physical activity to support healthy ageing. / Thesis / Doctor of Philosophy (PhD)

Scanning Electrochemical Microscopy (SECM) is most commonly used to spatially resolve reaction rates, image surface topography and surface reactivity. In this research, SECM is applied to various chemical systems in order to resolve local reaction chemistry and to produce patterns with dimensions of tens of microns in n-alkanethiol passivated gold substrates. Upon completing construction of the instrumentation, SECM was applied to capillary electrophoresis to accurately and reproducibly place the electrode directly above a very small capillary opening. Feedback SECM was then used to image and pattern surfaces, effectively distinguishing between insulating and conductive domains. Finally, the size of desorbed features patterned on a passivated gold substrate were studied as a function of both applied potential and ionic strength. Electrochemical detection in capillary electrophoresis requires decoupling the voltage applied to the working electrode from the separation voltage applied across the capillary. End-capillary electrochemical detection achieves this by placing the electrode just outside the ground end of the separation capillary. Obtaining adequate signal-to-noise in this arrangement requires using small inner diameter capillaries. Decreasing the inner diameter of the separation capillary, however, increases the difficulty of aligning the microelectrode with the open end of the capillary. Using SECM, the position of the capillary opening is determined while electroactive material is continuously emerging from the end of the capillary. The SECM instrument is then used to place the electrode at the position of maximum current for subsequent separations. Subsequent measurements found that the best signal-to-noise is obtained when the detection electrode is placed directly opposite the capillary opening and just outside of the capillary opening. When the electrode is further above the opening (but still opposite the capillary opening), the signal-to-noise does not dramatically decrease until the electrode is more than 30 μm above the 10 μm inner-diameter capillary. Limits of detection for 2,3-dihydroxybenzoic acid were found to be 8.2 fmol when aligned manually, and 3.8 fmol when the SECM is used to automatically align the microelectrode. SECM was then used to image a series of multi-disk electrode arrays in order to demonstrate the ability of the instrument to discriminate between conductive and insulating domains. Upon demonstrating the capacity of the SECM to image very small domains of conductor on an insulating substrate, n-alkanethiol passivated gold surfaces were patterned using site-selective desorption. A number patterns, potentially useful for enzyme deposition, were subsequently produced in the passivated gold substrate. The feature size of the desorbed domains was monitored as a function of applied potential and the ionic strength of the solution used for desorption. Results showed that applying a more negative potential or increasing the ionic strength of the solution increased the magnitude of the electric field at the surface of the passivated gold substrate and resulted in a more complete, larger desorption. Both ionic strength and applied desorption potential prove to be parameters useful for controlling the size of patterned features in site selective desorption. / Master of Science

No / Ten different mathematical models representing the electrophoretic mobility of analytes in capillary electrophoresis in mixed solvents of different composition have been compared using 32 experimental data sets. The solvents are binary mixtures of water-methanol, water-ethanol and methanol-ethanol, respectively. Mean percentage deviation (MPD), overall MPD (OMPD) and individual percentage deviation (IPD) have been considered as comparison criteria. The results showed that a reorganized solution model, namely the combined nearly ideal binary solvent/Redlich-Kister equation, is the most accurate model among other similar models concerning both correlation ability and prediction capability

Capillary electrophoresis is a widely used separation method of analytical chemistry. Addition of a selector into the background electrolyte extends its applicability to separation of enantiomers or of compounds of similar physicochemical properties. In analytical practice, mixtures of selectors are also commonly used - either prepared intentionally to achieve better separation or because commercially available selectors may be mixtures of compounds differing in the degree of substitution and substituent positions. Mathematical description of these systems, which are highly relevant in analytical practice, can simplify search for optimal separation conditions. Also, it provides a useful insight into the separation mechanism. In this work, a model of electromigration of an analyte interacting with a mixture of two selectors is proposed and experimentally verified. This model results from a more general description of systems with an arbitrary number of selectors. The model shows that a selector mixture can be treated as a single selector if the ratio of the respective selector concentrations is kept constant. When the mixture is prepared intentionally, this description predicts, how separation potential of the mixture changes with its composition. Thus it allows the optimal composition and total...

Micellar electrokinetic chromatography (MEKC), one of the major capillary electrophoresis (CE) modes, has been interfaced to mass spectrometry (MS) to provide high sensitivity and selectivity for analysis of chiral compounds. The research in this dissertation presents the development of novel polymeric glucopyranoside based molecular micelles (MoMs) (aka. polymeric surfactants) and their application in chiral MEKC-MS. Chapter 1 is a review of chiral CE-MS - in the period 2010-2015. In this chapter, the fundamental of chiral CE and CE-MS is illustrated and the recent developments of chiral selectors and their applications in chiral EKC-MS, CEC-MS and MEKC-MS are discussed in details. Chapter 2 introduces the development of a novel polymeric α-D-glucopyranoside based surfactants, n-alkyl-α-D-glucopyranoside 4,6-hydrogen phosphate, sodium salt. In this chapter, polymeric α-D-glucopyranoside-based surfactants with different chain length and head groups have been successfully synthesized, characterized and applied as compatible chiral selector in MEKC-ESI-MS/MS. or the enantioseparation of ephedrines and β-blockers. Chapter 3 continues to describe the employment of polymeric glucopyranoside based surfactants as chiral selector in MEKC-MS/MS. The polymeric β-D-glucopyranoside based surfactants, containing charged head groups such as n-alkyl β-D-glucopyranoside 4,6-hydrogen phosphate, sodium salt and n-alkyl β-D-glucopyranoside 6-hydrogen sulfate, monosodium salt were able to enantioseparate 21 cationic drugs and 8 binaphthyl atropisomers (BAIs) in MEKC-MS/MS, which promises to open up the possibility of turning an analytical technique into high throughput screening of chiral compounds. Physicochemical properties and enantioseparation capability of polymeric β-D-glucopyranoside based surfactants with different head groups and chain lengths were compared. Moreover, the comparison of polymeric α- and β-D-glucopyranoside 4,6-hydrogen phosphate, sodium salt were further explored with regard to enantioseparations of ephedrine alkaloids and b-blockers. The concept of multiplex chiral MEKC-MS for high throughput quantitation is demonstrated for the first time in scientific literature.

This work deals with dual detection of organic and inorganic analytes after separation by capillary zone electrophoresis. In the first part, two types of hydrodynamic sampling are tested. Standard hydrodynamic sampling most often used in laboratory-made electrophoretic apparatus, based on lifting the vessel with the sample, in which the sampling end of the capillary is immersed, and a new method based on increasing the pressure in the sampling vessel without moving the capillary. This sampling procedure minimizes experimenter activity because it is controlled by software. Experimenter only changes vessel containing the sample solution for one with separation electrolyte. The experimental parameters, the sampling time and pressure, are optimized to achieve maximum separation efficiency and adequate detection sensitivity. In the second part of the work, the developed method is tested for the separation of amino-acids in a biological sample (urine).

Liquid crystals are widely used in electronics, medicine and other fields. Analytical separations are important in the development of new liquid crystals to control the purity of synthesized substances. The sample analysis is important for detection of impurities formed during synthesis and for determination of chiral purity of the substance. Liquid crystal-forming substances cannot be separated by capillary zone electrophoresis due to the absence of readily ionizable groups. Electrokinetic chromatography is a method in which a suitable surfactant is added to the background electrolyte. The uncharged substances then interact with the electrically charged surfactant to obtain an effective charge. Separation can occur if they interact differently with the added surfactant. Another problem complicating the analysis is the very low solubility of analytes in water. Separations in this work were therefore carried out under non-aqueous conditions in acetonitrile. However, under these conditions a poor repeatability of the migration times of the substances was observed. Therefore, capillaries with differently coated inner walls were used in subsequent measurements. Surface modification should improve the repeatability of migration times. Several types of capillary coating have been tested. Dynamic coating...