Date of Award

2011

Degree Type

Dissertation

Department

Chemistry

First Advisor

Guo, Baochuan

Subject Headings

Drug development -- Methodology, Drugs -- Analysis, Liquid chromatography, Mass spectrometry, Proteomics, DNA -- Methylation, LC-MS(/MS), method development, proteomics, gene methylation

Abstract

In the past decade, bioanalytical method development has become an integral part of clinical diagnosis, biomarker discovery, and drug discovery and development. The new and emerged bioanalytical technologies allow the quantitative and qualitative analysis of small molecules and biomolecules with high sensitivity and specificity. Specifically, the bioanalytical methods based on LC-MS and methylation-specific PCR are well suited for detecting low-abundance metabolites, proteins, and DNA in biofluids and tissues for biomarker investigation. They offer great clinical promises for early diseases diagnosis and therapeutic interventions. Besides, the LC-MS/MS quantitative method is essential for the estimation of pharmacokinetic and toxicological properties in drug screening.In this work, modern bioanalytical technologies, together with their applications from biomarker discovery and validation in metabonomics, genomics and proteomics to drug discovery, were reviewed. Dependent on the type of molecules analyzed, different methods were established to achieve accurate and reliable detection. LC-MS/(MS) methods were developed and validated for quantitative analysis of bile acids and anti-cancer agent JCC76. The former has been successfully applied in a clinical study for the diagnosis of inflammatory bowel diseases and the latter has been utilized in a pharmacokinetics study for drug screening and optimization. In terms of proteomics profiling, a LC-MS/MS method was demonstrated for comparative analysis of serum peptides with the successful identification of a potential biomarker for ovarian cancer. Lastly, a comprehensive DNA methylation profiling for hepatocellular carcinoma was conducted through methylation-specific PCR methods. These methods enabled sensitive and specific detection of DNA hypermethylation on several tumor-associated genes. In addition, this work discussed a major challenge of matrix effect in quantitative method development. Possible solutions were proposed for matrix effect prevention and troublesho

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