Development of microaffinity columns for drug analysis

This dissertation describes the use of high-performance affinity chromatography (HPAC) and monolithic columns to study drug-protein interactions. The first project studied the binding of imipramine to human serum albumin (HSA) by using HPAC. Frontal analysis and zonal elution competition studies determined that imipramine had one major binding site and a second group of weaker, non-specific binding regions on HSA.;The overall objective of the remainder of this dissertation was to develop microaffinity columns for use in HPAC and drug-protein binding studies. The second project used silica monoliths in affinity microcolumns for the high-throughput analysis of drug-protein interactions. It was found that HSA silica monoliths in affinity microcolumns as short as 1 to 3 mm could be used to provide reliable estimates of retention factor and plate height. A comparison between silica monoliths and silica particles in microcolumns showed that silica monoliths allowed for the use of faster flow rates because of their better mass transfer properties, which decreased analysis times.;The third project used silica particles in columns with lengths from 1 mm to 2 cm to compare affinity microcolumns to traditional longer affinity columns in HPAC. Zonal elution studies showed that retention factor, plate height, and peak asymmetry measurements were fairly consistent across all of the tested flow rates. However, there was a decrease in precision as shorter column lengths were used. Frontal analysis studies determined that association equilibrium constant measurements for all column lengths were consistent. In these studies, using affinity microcolumns for frontal analysis studies to decrease analysis times did not compromise the accuracy of these measurements.;The last project used the noncompetitive peak decay method with HSA silica monolith affinity microcolumns to measure the dissociation rate constants of several drugs from HSA. Work with flow rates as high as 10 mL/min made it possible to provide dissociation rate constants for warfarin in less than 40 s. This method was then extended to other drugs known to bind to HSA, including diazepam, imipramine, acetohexamide, and tolbutamide.