Evaluation of affinity microcolumns containing human serum albumin for rapid analysis of drug-protein binding

Abstract

This study examined the use of affinity microcolumns as tools for the rapid analysis and high-throughput screening of drug-protein binding. The protein used was immobilized human serum albumin (HSA) and the model analytes were warfarin and L-tryptophan, two solutes often used as site-specific probes for drug binding to Sudlow sites I and II of HSA, respectively. The use of HSA microcolumns in binding studies was examined by using both zonal elution and frontal analysis formats. The zonal elution studies were conducted by injecting the probe compounds onto HSA microcolumns of varying lengths while measuring the resulting retention factors, plate heights and peak asymmetries. A decrease in the retention factor was noted when moving from longer to shorter column lengths while using a constant amount of injected solute. However, this change could be corrected, in part, by determining the relative retention factor of a solute versus a reference compound injected onto the same microcolumn. The plate height values were relatively consistent for all column lengths and gave an expected increase at higher linear velocities. The peak asymmetries were similar for all columns up to 1 mL/min but shifted to larger values at higher flow rates and when using short microcolumns (e.g., 1 mm length). The association equilibrium constants and number of binding sites estimated by frontal analysis for warfarin with HSA were consistent at the various column sizes that were tested and gave good agreement with previous literature values. These results confirmed affinity microcolumns provide comparable results to those obtained with longer columns and can be used in the rapid analysis of drug-protein binding and in the high-throughput screening of such interactions.

Figure 1

Retention factors measured for R-warfarin at various flow rates for 5 μL injections of 20 μM R-warfarin onto HSA microcolumns with lengths of (a) 2 cm, (b) 5 mm, or (c) 1 mm and an inner diameter of 2.1 mm. Other experimental conditions are given in the text. The error bars represent a range of ± 1 S.D. of the mean.

Figure 2

Retention factors measured for L-tryptophan at various flow rates for 5 μL injections of 20 μM L-tryptophan onto HSA microcolumns with lengths of (a) 2 cm, (b) 5 mm, or (c) 1 mm and an inner diameter of 2.1 mm. Other experimental conditions are given in the text. The error bars represent a range of ± 1 S.D. of the mean.

Figure 3

(a) Typical chromatograms obtained for R-warfarin and L-tryptophan and (b) the resulting selectivity factors determined R-warfarin versus L-tryptophan for HSA microcolumns. The results in (a) were generated using HSA microcolumns with lengths of 20, 5 or 1 mm and an inner diameter of 2.1 mm. Other experimental conditions are given in the text. The error bars represent a range of ± 1 S.D. of the mean.

Figure 4

Plate height plots obtained for 5 μL injections of 20 μM R-warfarin onto HSA microcolumns with lengths of (a) 2 cm, (b) 5 mm, or (c) 1 mm and an inner diameter of 2.1 mm. Other experimental conditions are given in the text. The error bars represent a range of ± 1 S.D. of the mean.

Figure 5

Measured peak asymmetry at tenth height width for 5 μL injections of 20 μM R-warfarin onto HSA microcolumns with lengths of (a) 2 cm or (b) 1 mm and an inner diameter of 2.1 mm. Other experimental conditions are given in the text. The error bars represent a range of ± 1 S.D. of the mean.

Figure 6

Typical breakthrough curves obtained for racemic warfarin on HSA microcolumns. The results in (a) and (b) were obtained at 0.5 mL/min on 2 cm and 1 mm long HSA microcolumns with inner diameters of 2.1 mm and using warfarin concentrations (from left-to-right and top-to-bottom) of 10, 5, 2.5, and 1 μM. The results in (c) were obtained for a 5 μM sample of warfarin applied to a 1 mm × 2.1 mm i.d. HSA microcolumn at flow rates of 2.0, 1.0, 0.5, or 0.2 mL/min. All of these experiments were conducted at pH 7.4 and 37 °C. Other experimental conditions are given in the text.

Figure 7

Double-reciprocal frontal analysis plots prepared according to Eqn. (3) for the application of warfarin at 0.5 mL/min to (a) a 2 cm or (b) a 1 mm long HSA microcolumn and an inner diameter of 2.1 mm for samples with warfarin concentrations that ranged from 1 to 10 μM. The equation for the best-fit lines were (a) y = 189 (± 3) x + 3.6 (± 0.2) × 10⁷ (r = 0.9998) and (b) y = 3895 (± 230) x + 8.8 (± 1.1) × 10⁸ (r = 0.9965). The error bars represent a range of ± 1 S.D. of the mean.