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Title: Determination of binding constants by ultrafast affinity extraction: Theoretical and experimental studies of optimum conditions for analysis
Ultrafast affinity extraction (UAE) is a form of microscale affinity HPLC that can be employed to quickly measure equilibrium constants for solute-binding agent interactions in solution. This study used chromatographic and equilibrium theory with universal plots to examine the general conditions that are needed in UAE to obtain accurate, precise, and robust measurements of equilibrium constants for such interactions. The predicted results were compared to those obtained by UAE in studies that examined the binding of various drugs with two transport proteins: human serum albumin and α1-acid glycoprotein. The most precise and robust conditions for these binding studies occurred for systems with intermediate values for their equilibrium free fraction for the solute (F0 ≈ 0.20-0.80). These trends showed good agreement with those seen in prior studies using UAE. It was further determined how the apparent free fraction of a solute was related to the dissociation rate of this solute, the time allowed for solute dissociation during UAE, and the equilibrium free fraction for the solute. These results also agreed with experimental results, as obtained for the binding of warfarin and gliclazide with human serum albumin. The final section examined how a change in the apparent free fraction, as caused by solute dissociation, affected the accuracy of an equilibrium constant that was measured by UAE. In addition, theoretical plots were generated to allow the selection of conditions for UAE that provided a given level of accuracy during the measurement of an equilibrium constant. The equations created and trends identified for UAE were general ones that can be extended in future work to other solutes and binding agents.  more » « less
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Author(s) / Creator(s):
; ; ;
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Date Published:
Journal Name:
Journal of Chromatography A
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Subject(s) / Keyword(s):
["Ultrafast affinity extraction","Drug-protein binding","Free drug fraction","Association equilibrium constant","Affinity microcolumn"]
Medium: X
Sponsoring Org:
National Science Foundation
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