Date of Award

Fall 1-1-2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy In Clinical-bioanalytical Chemistry

Department

Chemistry

First Advisor

Guo, Baochuan

Second Advisor

Dr. Xue-Long Sun

Third Advisor

Dr. John F Turner

Abstract

Liquid Chromatography-Mass Spectrometry is an advanced analytical technique that offers high sensitivity and specificity and has been increasingly used for analysis of a wide variety of compounds including clinically and pharmacologically relevant molecules. In this dissertation we describe qualitative and quantitative liquid chromatographic mass spectrometric methods to analyze both small molecules and larger macromolecules that provide useful insights into diagnosis and management of several diseases. An LCMS(/MS) analytical method includes extraction of analytes of interest from the matrix followed by liquid chromatographic separation and mass spectrometric detection. Chapter I describes pre-analytical workflows and sample pretreatment techniques and theories underlying LC-MS and instrumentation that are relevant to this work. The first chapter also describes the process of method development followed by validation guidelines for quantitative bio-analytical assays. Chapter II describes a novel, rapid, and simple quantitative mass spectrometric method for endogenous molecules in human bile that are associated with Cholangiocarcinoma and Cholelithiasis. The method was designed and validated to overcome problems suffered by conventional methods such as time-consuming extraction steps, carryover and unavailability of blank bile by employing simple dilution, flow injection and standard addition to matrix effects respectively. In Chapter III, a quantitative LC-MS/MS method was developed and validated for the determination of an antitumor drug in mouse brain to support an investigation to study the effectiveness of v intracerebral microdialysis as an alternative route of administration. This method describes a two-step extraction process using Proteinase K and ethanol protein precipitation to overcome the low recovery and high matrix effects faced by previously reported methods. Chapter IV describes investigation of feasibility of employing a less commonly used proteolytic enzyme, aspartic acid N endopeptidase, in the digestion of prothrombin for qualitative LC-MS analysis. This study could be employed to study distribution of variants of des-gamma-carboxy-prothrombin, a biomarker which is elevated in hepatocellular carcinoma and vitamin K deficiency to further identify a more specific variant(s) as a biomarker. Finally, this dissertation is concluded with recommendations for qualitative and quantitative LC-MS research methodology based on the findings herein and future directions implicated by the impact of this work.

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