Affinity Chromatographic Studies of Drug Interactions with Alpha1-Acid Glycoprotein and Use of New Nanomaterials in Ultrathin-Layer Chromatography

AFFINITY CHROMATOGRAPHIC STUDIES OF DRUG INTERACTIONS WITH ALPHA1-ACID GLYCOPROTEIN AND USE OF NEW NANOMATERIALS IN ULTRATHIN-LAYER CHROMATOGRAPHY Sandya Rani Beeram, Ph.D. University of Nebraska, 2017 Advisor: David S. Hage The overall extent of drug binding to serum proteins and the rate of drug-protein dissociation can have a significant effect on the pharmacokinetics and pharmacodynamics of a drug. Alpha1-acid glycoprotein (AGP) is an important acute phase protein and is the principal binding protein for many basic and neutral drugs in blood. This dissertation describes the use of high-performance affinity chromatography (HPAC) and AGP microcolumns to examine the binding and kinetics of various drugs to AGP to better understand the delivery of these drugs in the circulation. The first part of this research examined the use of ultrafast affinity extraction for the analysis of free drug fractions and for determination of equilibrium constants and rate constants for various model drugs with soluble AGP in the samples. The effect of varying the concentrations of the drugs and soluble AGP was also investigated and the results were examined to see if they indicate the presence of multiple types of interactions for these drugs with soluble AGP. The second part of this research examined the use of ultrafast affinity extraction with a two-dimensional affinity system to measure the free fractions of drugs with AGP in systemic lupus erythematosus (SLE) clinical samples. The binding constants for the drugs with AGP were found to be significantly different in clinical samples when compared to normal serum samples. The increase in affinity for the SLE samples was determined to be due, in part, to the elevated levels of AGP in these samples; however, changes in the glycoforms of AGP were also noted that may have contributed to these variations. The third part of this dissertation examined the use of new nanomaterial-based supports/stationary phases and a label-free detection technique for ultra-thin layer chromatography (UTLC). This work involved the detection of spatial- and time-resolved images of linear birefringence variations in the nanomaterials-based support/stationary phases upon their interaction with analytes. This support/stationary phase was also modified to immobilize human serum albumin (HSA) for possible use in screening solute-protein interactions or chiral separations