Development of novel analytical methods to elucidate the roles of active xenobiotics and endogenous molecules in toxicity, metabolism and posttranslational modification

Abstract

Bioanalysis of active xenobiotics, including drugs, chemicals and toxicants, as well as endogenous compounds, is important not only for pharmacokinetics/toxicokinetic studies, but also for understanding their mechanisms of actions including toxicity, metabolism and protein post-translational modifications. However, these molecules are challenging to analyze, mainly due to their chemical instability and/or biological activity, and lack of blank matrices for endogenous molecules. In this dissertation, a series of novel, sensitive and specific analytical methods were developed, validated and applied to quantitate these molecules and their metabolites in complex biological matrices to investigate their mechanisms of actions. Chapter 1 is the introduction and describes the layout of the dissertation. Chapter 2 is a literature review of xenobiotic toxicity studies by liquid chromatography tandem mass spectrometry (LC-MS/MS), particularly from the perspective of protein adducts. Chapter 3 describes method development and validation for the determination of chlorpyrifos and its metabolites in cells and culture media by LC-MS/MS. Chapter 4 describes the determination of Lys-40 acetylated α-tubulin in rat brain tissue by immunoprecipitation–mass spectrometry, a key post-translational modification hypothesized to be disrupted by exposure to organophosphates. Chapter 5 describes the development of a method for the quantitative profiling of cellular acyl-Coenzyme As (acyl-CoAs) compounds by LC-MS/MS to probe the metabolism of fatty acids. In Chapter 6, a method for the simultaneous quantitation of quetiapine and its active metabolite norquetiapine in rat plasma and brain tissue by LC-MS/MS is presented.