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

• Author / Creator

  Zuniga, Azeret

• 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.

• Subjects / Keywords

  • Urine
  • Metabolite identification
  • Metabolomics
  • Quantification
  • Mass-spectrometry
  • Liquid chromatography
  • Plasma
  • Dried blood spots
  • Acylcarnitines

• Graduation date

  Fall 2012

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3KD1QT7G

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.