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Advanced Separation Methods for the Analysis of Drugs and Other Targets by High-Performance Liquid Chromatography

Ashley G Woolfork, University of Nebraska - Lincoln


Human serum albumin (HSA) is a transport protein known to bind to many drugs. The modification of HSA is linked to numerous diseases such as diabetes. High serum levels of glucose can result in HSA modification, affecting the structure and function of this protein and its binding to solutes in the circulation. Modifications that can occur during diabetes are glycation and the formation of advanced glycation end-products (AGEs). The work presented in this dissertation used high-performance affinity chromatography (HPAC) to examine solute-protein interactions. The first portion of this research employed zonal elution to examine the interactions between serum proteins and imidazoline drugs. The trends observed for these drugs varied by their structure and extent of binding to the proteins. Part of this research involved rapidly screening sulfonylurea drugs (as used to treat type II diabetes) by utilizing zonal elution to characterize the interactions between sulfonylureas and normal or AGE-modified HSA. Competition studies were also conducted to examine binding by first-generation sulfonylureas to the two major drug binding sites of normal and modified HSA. The affinity of the drugs was found to vary with the levels of protein modification. The next portion of this research involved examination of the effects of a drug’s stability on its measured binding by HPAC. This study was conducted using HSA and biguanide or glitazone anti-diabetic drugs. Zonal elution was used to identify changes in binding of the drugs to HSA under various experimental conditions. The last portion of this research involved using the known properties of HSA to develop a fluorometric sandwich assay for the detection of COVID-19 antibodies. The assay employed silica magnetic particles and fluorescent labels to selectively detect antibodies for COVID-19. The assay was found to be selective against other antibodies to known common infectious agents. The assay was also rapid and sensitive and can be employed in a microchip device for affordable and self-COVID-19 or similar antibody test.

Subject Area


Recommended Citation

Woolfork, Ashley G, "Advanced Separation Methods for the Analysis of Drugs and Other Targets by High-Performance Liquid Chromatography" (2021). ETD collection for University of Nebraska - Lincoln. AAI28489841.