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.

Analysis of free drug fractions by ultrafast affinity extraction: Interactions of sulfonylurea drugs with normal or glycated human serum albumin

“…The mixtures of the drugs and normal HSA or glycated HSA were prepared by dissolving each protein in the corresponding drug solutions. These drug/protein mixtures were incubated for at least 30 min at 37 °C before injection to allow equilibrium to be established between the free and protein-bound fractions of the drug in the sample [37]. Replicate injections ( n = 4) were made for all samples and standards onto the system.…”

“…However, these techniques often require long separation or analysis times (ranging from 15–30 min to hours) and relatively large sample volumes (i.e., typically in the milliliter range). In addition, the non-specific adsorption of drugs to the membranes or components of these methods can introduce errors in the free fraction measurements [35,37–41]. Ultrafast affinity extraction is an alternative technique that has been used to measure the free and protein-bound forms of drugs and hormones in clinical and pharmaceutical samples [37–40,45–47].…”

“…In addition, the non-specific adsorption of drugs to the membranes or components of these methods can introduce errors in the free fraction measurements [35,37–41]. Ultrafast affinity extraction is an alternative technique that has been used to measure the free and protein-bound forms of drugs and hormones in clinical and pharmaceutical samples [37–40,45–47]. In this method, an affinity microcolumn that contains an immobilized binding agent with relatively fast and strong binding for the drug or hormone of interest is used to extract the free form of this analyte on a time scale that minimizes dissociation of the same analyte from its protein-bound form in the sample.…”

“…This method has been used to measure the free fractions of thyroxine, warfarin and phenytoin in clinical samples [38–40,45,46]. This technique has also been used to estimate the association equilibrium constants for various drugs with normal HSA [37,47]. In addition, a multidimensional system combining ultrafast affinity extraction with a chiral stationary phase has recently been used to simultaneously measure the free fractions of warfarin enantiomers in complex samples [38].…”

Pretreatment of plasma samples by a novel hollow fiber centrifugal ultrafiltration technique for the determination of plasma protein binding of three coumarins using acetone as protein binding releasing agent

“…Usually, adding water miscible organic solvent can make intramolecular and intermolecular hydrogen bonding of protein change so that the drug with protein-bound fraction can be released [43][44][45][46]. The principle is based on the following aspects: (1) the hydrophilic organic solvent introduced as protein binding releasing agent can reduce the dielectric constant of the sample solution, resulting in enhanced electrostatic attraction between solute molecules; (2) the competitive binding for water between hydrosoluble organic solvent and protein can directly damage the protein surface hydration layer, make the protein spatial structure expand and release drug from drug-bound fraction [47].…”