Analysis of free drug fractions in serum by ultrafast affinity extraction and two-dimensional affinity chromatography using α1-acid glycoprotein microcolumns

Abstract

In the circulatory system, many drugs are reversibly bound to serum proteins such as human serum albumin (HSA) and alpha1-acid glycoprotein (AGP), resulting in both free and protein-bound fractions for these drugs. This report examined the use of microcolumns containing immobilized AGP for the measurement of free drug fractions by ultrafast affinity extraction and a two-dimensional affinity system. Several drugs known to bind AGP were used as models to develop and evaluate this approach. Factors considered during the creation of this method included the retention of the drugs on the microcolumns, the injection flow rate, the microcolumn size, and the times at which a second AGP column was placed on-line with the microcolumn. The final system had residence times of only 110-830ms during sample passage through the AGP microcolumns and allowed free drug fractions to be determined within 10-20min when using only 3-10μL of sample per injection. This method was used to measure the free fractions of the model drugs at typical therapeutic levels in serum, giving good agreement with the results obtained by ultrafiltration. This approach was also used to estimate the binding constants for each drug with AGP in serum, even for drugs that had significant interactions with both AGP and HSA in such samples. These results indicated that AGP microcolumns could be used with ultrafast affinity extraction to measure free drug fractions in a label-free manner and to study the binding of drugs with AGP in complex samples such as serum.

Keywords: Drug-protein binding; Free drug fraction; Human serum albumin; Ultrafast affinity extraction; α(1)-Acid glycoprotein.

Figure 1

General scheme for the separation of the free and protein-bound fractions of a drug and measurement of the free drug fraction by using immobilized AGP in an affinity microcolumn for ultrafast affinity extraction, followed by an AGP analytical column as part of a two-dimensional affinity system.

Figure 2

Effect of the injection flow rate on the apparent free fractions that were measured for samples containing 20 µM AGP and 10 µM of carbamazepine (□), disopyramide (Δ), lidocaine (◊), propafenone (○) or warfarin (×) when injected at pH 7.4 and 37 °C onto AGP microcolumns. The AGP microcolumns were 5 mm × 2.1 mm i.d. for carbamazepine or lidocaine and 2 mm × 2.1 mm i.d. for disopyramide, propafenone or warfarin. The injection volume was 3 µL for the carbamazepine or lidocaine samples and 10 µL for the samples containing disopyramide, propafenone or warfarin. The error bars represent a range of ± 1 S.D. (n = 3).

Figure 3

Typical chromatograms obtained at 3.0 mL/min during the injection of (a) 3 µL of 15 µM carbamazepine/20 µM AGP/481 µM HSA (top) or 15 µM of carbamazepine alone (bottom) onto a 5 mm × 2.1 mm i.d. AGP microcolumn, and (b) 10 µL of 7.5 µM warfarin/20 µM AGP/481 µM HSA (top) or 7.5 µM of warfarin alone (bottom) onto a 2 mm × 2.1 mm i.d. AGP microcolumn. These chromatograms were processed by using the locally weighted scatterplot smoothing function of PeakFit 4.12.

Figure 4

Chromatograms obtained on the second AGP column in a two-dimensional affinity system for the retained peaks from an AGP microcolumn, as observed when using various times to place the second column on-line in this system. These results are for 3 µL injections that were made onto the AGP microcolumn of a mixture containing 15 µM carbamazepine/20 µM AGP/481 µM HSA. The chromatograms shown in this figure were acquired at 0.50 mL/min on the second AGP column, which had dimensions of 10 mm × 2.1 mm i.d. The x-axis shows the total time that had elapsed following sample injection onto the system. The times shown by the peaks are the times at which the second column was placed on-line with the AGP microcolumn.

Figure 5

Effect of changing the time at which the second AGP column was placed on-line (i.e., the valve switching time) on the apparent free fractions that were measured for samples containing (a) 15 µM carbamazepine/20 µM AGP/481 µM HSA or (b) 7.5 µM warfarin/20 µM AGP/481 µM HSA. The times on the x-axis represent the interval that had elapsed from sample injection to placement of the second affinity column on-line with the AGP microcolumn. Other experimental conditions were the same as described in the text. The error bars represent a range of ± 1 standard error of the mean (n = 3).