Ultrafast Affinity Extraction and High-Performance Affinity Chromatography Applications for Measuring Free Drug Fractions: Interactions of Sulfonylurea Drugs with Normal and Glycated Human Serum Albumin
Dr. David. S. Hage
Date of this Version
Ultrafast affinity extraction was used with microcolumns containing human serum albumin (HSA) to measure the free fractions of several sulfonylurea drugs in the presence of solution-phase HSA and for the analysis of these drugs at therapeutic levels or determining their binding strengths with this protein. This approach was first used with normal HSA and the drugs glibenclamide, glipizide and glimepiride. This work used both one- and two-dimensional affinity systems based on HSA microcolumns. The first microcolumn was used to extract the free drug fractions in injected drug/protein mixtures. As the retained drug eluted from the first microcolumn, it was passed through a second HSA column for further separation and measurement. Items that were considered during the optimization of this approach included the column sizes and flow rates that were used and the time at which the second column was placed on-line with the first HSA microcolumn. Global affinity constants that were obtained from the measured free fractions gave good agreement with those predicted from previous binding studies.
The second part of this study examined the binding of sulfonylurea drugs with HSA at various stages of glycation. Affinity microcolumns containing immobilized HSA were used to extract the free drug fractions of drug/glycated HSA mixtures. Conditions that were considered during method optimization again included the column sizes and flow rates that were used for ultrafast affinity extraction. An apparent change in the affinity of the given drugs for HSA was seen at moderate to high levels of glycation. The results throughout this work indicated that affinity microcolumns and ultrafast affinity extraction could be used as tools for measuring free drug fractions and for examining the interactions of sulfonylurea drugs with normal or modified forms of HSA. The same approach could be utilized with other drugs and proteins or modified binding agents of clinical or pharmaceutical interest.
Advisor: David S. Hage