Date of Award

Fall 1-1-2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy In Clinical-bioanalytical Chemistry Degree

Department

Chemistry

First Advisor

Xu, Yan

Second Advisor

Dr. Anthony Berdis

Third Advisor

Dr. Xue-Long Sun

Abstract

In this dissertation work, a semi-quantitative assay using a lipidomics approach and an absolute quantitative assay using liquid chromatography-mass spectrometry techniques were developed (LC-MS). In the lipidomics approach, ultra-high pressure liquid chromatography in tandem with quadrupole time of flight (UHPLC-QTOF) mass spectrometry was used to profile, compare and quantitate the human plasma lipids in Alzheimer’s disease subjects (AD) and normal cognitive controls (NCCs). The purpose of this study is to identify potential plasma lipid markers of AD and to explore the relationships between AD and lipid pathways in humans by using bioinformatic tools. The plasma samples of study subjects were first spiked with a mix of 12 synthetic-lipid internal standards, then total lipids were extracted using a modified Blight-Dyer method and fractionated into phospholipids (PL) and neutral lipids (NL) using the aminopropyl cartridge. The UHPLC-QTOF-MS/MS data were processed and analyzed with corrections of retention time and mass shifts. Molecular features were extracted for lipid identifications based on mass-to-charge ratios, isotopic patterns, adducts, and charge states. Venn diagrams were plotted to group the common and the unique features of lipids between AD patients and NCCs. The common significant molecular features between these two study groups were analyzed using principal component analysis (PCA), partial least vii squares-discriminant analysis (PLS-DA), and non-parametric Wilcoxon rank-sum t-test with false discovery rate calculated p-values. Quantitative lipidomics was performed on the twenty-eight identified significant common lipids. Our results indicate these significant common lipids between AD patients and NCCs were belong to glycerophospholipids, glycerolipids, and sphingolipids. Gene-lipid centric pathway analysis was performed on these significant lipids to obtain the implicated pathways in AD and to understand it’s relation to AD. In absolute quantitation work, an assay using a liquid chromatography system in tandem with triple quadrupole mass spectrometry (LC-QqQ) was developed and validated to measure the O6Benzylguanine (O6BG) and its metabolite, 8-oxo-O6Benzylguanine (8-oxoO6BG) in human plasma. O6BG and 8-oxo-O6BG along with the analog internal standard, pCl-O6BG, were extracted from alkalinized human plasma by liquid-liquid extraction (LLE) using ethyl acetate, dried under nitrogen and reconstituted in the mobile phase. Reverse-phase chromatographic separation was achieved using isocratic elution with a mobile phase containing 80% acetonitrile and 0.05% formic acid in water at a flow rate of 0.600 mL/min. Quantification was performed using multiple-reaction-monitoring (MRM) mode with positive ion-spray ionization. The linear calibration ranges of the method for O6BG and 8-oxo-O6BG were 1.25 to 250 ng/mL and 5.00 to 1.00 x 103 ng/mL respectively with acceptable assay accuracy, precision, recovery and matrix factor. The method was validated as per the Food and Drug Administration (FDA) guidelines and was applied to the measurement of O6BG and 8-oxo-O6BG in patient plasma samples from the prior phase I clinical trial.

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