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

Rangan Mallik, Michelle J. Yoo, Chad J. Briscoe, David S. Hage

Research output: Contribution to journalArticlepeer-review

65 Scopus citations


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 250ms 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.

Original languageEnglish (US)
Pages (from-to)2796-2803
Number of pages8
JournalJournal of Chromatography A
Issue number17
StatePublished - Apr 2010
Externally publishedYes


  • Affinity chromatography
  • Affinity microcolumns
  • Drug-protein binding
  • High-throughput screening
  • Human serum albumin
  • Ibuprofen
  • Imipramine
  • Warfarin

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry


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