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Quantitative analysis of small-molecule biomarkers by capillary electrophoresis-mass spectrometry

University of British Columbia

Coupling the capillary electrophoresis (CE) to mass spectrometry (MS) provides an attractive analytical platform with minimal sample consumption, short analysis time, and high separation efficiency. Small-molecule biomarkers are metabolites associated with biological or pathogenic processes. The comprehensive study of small molecules found within the biological system is known as metabolomics. The work presented herein aimed to fulfill the advantage of CE-MS in analytical efficiency and fill the gaps of current methods by achieving the nonaqueous-CE-MS analysis of isomeric prostaglandins and enabling the peak alignment in CE-based metabolomics with inconsistent flow velocities. Urinary creatinine is commonly measured to normalize urinary metabolite concentrations or to assess renal function. In Chapter 2, multisegment injection-CE-MS was combined with a dilute-and-shoot strategy to analyze urinary creatinine. The diluted urines can be directly analyzed with a total analysis time of < 2 min/sample, providing a high-throughput method with reliable quantitative performance. A ureteral stent is widely used to treat or prevent ureteral obstruction, but little is known about the mechanisms that drive indwelling stent-associated pain and discomfort. Chapter 3 presents the first study indicating that prostaglandin E2 (PGE2) may be a biomarker for ureteral stent-associated inflammation. The stent-induced inflammation and the associated elevation of cyclooxygenase-2 (COX-2) expression were observed using porcine models. The level of PGE2, a key metabolite of COX-2 signaling, was found to be significantly higher in urines of stented animals compared to that of controls. Migration time fluctuation strongly affects the peak alignment and identification of unknown compounds, making migration time correction an essential step in large-scale metabolomics. Chapter 4 presents a correction method supporting pressure-assisted CE with varied external pressures. Additionally, the idea of using endogenous metabolites as correction markers instead of adding additional compounds was proposed. Multiple reaction monitoring (MRM)-based metabolomics has the advantages of simple data processing and high quantitative reliability. Nevertheless, the difficulty of using this approach lies in the method construction. Chapter 5 presents a straightforward workflow for developing the MRM-based metabolomics assay. Urine metabolic changes associated with stent-induced inflammation were investigated using this assay, and 13 metabolites were found to have concentrations associated with inflammatory levels.

Text

2023-04-30

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/77793

Chemistry

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/