The rate at which a drug or other small solute interacts with a protein is important in understanding the biological and pharmacokinetic behavior of these agents. One approach that has been developed for examining these rates involved the use of high-performance affinity chromatography (HPAC) and estimates of band-broadening through peak profiling. This dissertation describes an extension of peak profiling that allows a broad range of flow rates for examining solute-protein dissociation rates. Chromatographic theory was employed to derive equations for use with this approach on a single column, as well as with multiple columns to evaluate and correct for the impact of stagnant mobile phase mass transfer. The interaction of L-tryptophan with human serum albumin (HSA) demonstrated peak profiling's capabilities for a model system. The methodology was then extended to more complicated systems that display binding to both immobilized HSA and support particles during passage through an HPAC column. The techniques described herein can be applied to other biomolecular systems and will be valuable for the determination of drug-protein dissociation rates.

This dissertation also describes a novel high-performance immunoaffinity chromatographic method for measuring free drug fractions using a reversed displacement immunoassay (RDIA) format with near-infrared (NIR) fluorescent labels. This approach gave accurate quantitation of free phenytoin concentration in samples containing the binding protein HSA. Items considered in the design of the phenytoin RDIA included the dissociation rate of phenytoin's bound fraction from HSA, the development of an immobilized phenytoin affinity microcolumn, and the preparation and behavior of labeled monoclonal antibodies (mAb) in a displacement format. In the final method, the free fraction of phenytoin displaced a proportionate quantity of labeled mAb in approximately 140 ms during sample passage through the microcolumn. RDIA using affinity microcolumns and NIR fluorescent labels offers a high throughput alternative to current free drug assays that is less prone to biological interferences. This approach is not limited to phenytoin, but can be adapted for other analytes through the use of appropriate columns and labeled antibodies.