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Permanent link (DOI): https://doi.org/10.7939/R3KD1QT7G

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Development of Liquid Chromatography Mass Spectrometry Methods for the Identification and Quantification of Acylcarnitines in Biological Samples Open Access

Descriptions

Other title
Subject/Keyword
Urine
Acylcarnitines
Dried blood spots
Plasma
Metabolomics
Liquid chromatography
Quantification
Metabolite identification
Mass spectrometry
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Zuniga, Azeret
Supervisor and department
Li, Liang (Chemistry)
Examining committee member and department
Bamforth, Fiona (Laboratory Medicine and Pathology)
Vederas, John (Chemistry)
Harynuk, James (Chemistry)
Harrison, Jed (Chemistry)
Doucette, Alan (Chemistry, Dalhousie University)
Department
Department of Chemistry
Specialization

Date accepted
2012-05-28T14:07:48Z
Graduation date
2012-11
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
The field of metabolomics follows the Greek premise where metabolic changes are believed to be indicative of disease. Acylcarnitines, for example, can be dysregulated in the presence of various diseases including genetic metabolic disorders and multiple sclerosis. Liquid chromatography mass spectrometry-based quantitative metabolomics using stable isotope-labeled internal standards has proved to be one of the most accurate and reliable approaches for biomarker discovery. The main objective of this work was to develop, validate and apply both qualitative and quantitative ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) platforms for the detection, identification and quantification of acylcarnitines in various biological samples. Comprehensive acylcarnitine profiling was performed in urine, plasma, dried blood spots and red blood cell pellets. Compounds were putatively identified based on mass, relative retention times and fragmentation pattern. Only by analyzing various sample types can a truly comprehensive acylcarnitine profile be obtained. In an effort to improve metabolite identification strategies a web-based tool called MyCompoundID was developed. It is an expansion of the Human Metabolome Database and makes use of the fragmentation tools of the software package ChemDraw. Using this tool, the identification rate of metabolites in urine and plasma were greatly increased. Another major area of this work focused on the quantification of acylcarnitines in urine and plasma. A simple and robust esterification reaction was employed to introduce a 12C2 or 13C2 labeled ethyl group to acylcarnitines in order to produce a series of reference and internal standards. Calibration curves were prepared in unesterified urine and plasma to overcome the lack of analyte-free matrices. Method validation was performed to assess accuracy, precision, limits of detection and quantification as well as linear dynamic range. The results obtained correlated well with previously published values. Future work could focus on the application of these methods to clinical samples to search for biomarkers for various diseases. Additionally, analysis of acylcarnitines in dried biofluid spots would be an interesting application. Sample preparation times could be reduced by combining analyte extraction and derivatization into a single step using microwave technology. The use of this technology could be useful for many applications.
Language
English
DOI
doi:10.7939/R3KD1QT7G
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
Zuniga, A.; Li, L. Analytica Chimica Acta 2011, 689, 77-84.

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