Analysis of drug-protein binding by ultrafast affinity chromatography using immobilized human serum albumin

Abstract

Human serum albumin (HSA) was explored for use as a stationary phase and ligand in affinity microcolumns for the ultrafast extraction of free drug fractions and the use of this information for the analysis of drug-protein binding. Warfarin, imipramine, and ibuprofen were used as model analytes in this study. It was found that greater than 95% extraction of all these drugs could be achieved in as little as 250 ms on HSA microcolumns. The retained drug fraction was then eluted from the same column under isocratic conditions, giving elution in less than 40 s when working at 4.5 mL/min. The chromatographic behavior of this system gave a good fit with that predicted by computer simulations based on a reversible, saturable model for the binding of an injected drug with immobilized HSA. The free fractions measured by this method were found to be comparable to those determined by ultrafiltration, and equilibrium constants estimated by this approach gave good agreement with literature values. Advantages of this method include its speed and the relatively low cost of microcolumns that contain HSA. The ability of HSA to bind many types of drugs also creates the possibility of using the same affinity microcolumn to study and measure the free fractions for a variety of pharmaceutical agents. These properties make this technique appealing for use in drug-binding studies and in the high-throughput screening of new drug candidates.

Figure 1

General scheme for the separation of the free and protein-bound fractions of a drug in a sample through the use of an affinity microcolumn that contains immobilized human serum albumin (HSA).

Figure 2

Typical chromatograms obtained at 4.5 mL/min for 1.1 μL injections of (a) 10 μM R-warfarin onto an HSA microcolumn, (b) 20.0 μM HSA plus 10.0 μM R-warfarin onto an HSA microcolumn, or (c) 20.0 μM HSA plus 10.0 μM R-warfarin onto a control column. The HSA microcolumn was prepared by the Schiff base method. The size of both this column and the control column was 4.5 mm × 2.1 mm i.d. Other conditions are given in the text.

Figure 3

Simulated chromatograms for the binding and elution of samples containing R-warfarin on an HSA microcolumn. The results in (a) show the predicted response for the injection of R-warfarin or a mixture of R-warfarin and HSA onto an HSA microcolumn at 25 °C and pH 7.4 under the same sample, column, and flow rate conditions as were present for the experimental results shown in Figure 2. The R-warfarin/HSA sample used in these simulations had 30% R-warfarin in the free form, in line with experimental results presented later in this report. Estimates of the activity for the original immobilized HSA (i.e., around 70–75%) were based on previous data from the literature for similar columns [8,13]. The plots in (b) show how the peak for the retained fraction of R-warfarin would be expected to change under these same conditions as the relative amount of active protein is decreased by 80%, 60% or 40% of its initial value. Further details on the simulation model can be found in the Experimental section.

Figure 4

Effect of flow rate on the apparent free fractions measured for S-warfarin in samples containing soluble HSA and injected onto an HSA microcolumn. These results were obtained for 1.1 μL injections of a sample containing 20.0 μM HSA plus 8.5 μM S-warfarin. The HSA microcolumn was prepared by the Schiff base method and had a size of 4.5 mm × 2.1 mm i.d. Other conditions are given in the text.

Figure 5

Chromatograms obtained on 2.5 mm × 2.1 mm i.d. HSA microcolumns that had been prepared by the SMCC method. The results shown in (a) were obtained at 2.0 mL/min for 20 μL samples of 20.6 μM S-warfarin or 20.6 μM S-warfarin plus 25.0 μM HSA. The results shown in (b) were obtained at 3.0 mL/min for 40.0 μM imipramine or 40.0 μM imipramine plus 45.0 μM HSA. Other conditions are given in the text.