Characterization of the protein binding of chiral drugs by high-performance affinity chromatography interactions of R- and S-ibuprofen with human serum albumin

Zonal elution and high-performance affinity chromatography were used to study the different binding characteristics of R- and S-ibuprofen with the protein human serum albumin (HSA). This was done by injecting small amounts of R- and S-ibuprofen onto an immobilized HSA column in the presence of a mobile phase that contained a known concentration of R- or S-ibuprofen as a competing agent. These studies indicated that R- and S-ibuprofen had one common binding site on the immobilized HSA column. In addition, S-ibuprofen had at least one other major binding region. The association equilibrium constant for R-ibuprofen with HSA was found to be 5.3·10⁵ M^-1 at pH 6.9 and 25°C. Under the same conditions, the association constants for S-ibuprofen at its two sites were 1.1·10⁵ M^-1 and 1.2·10⁵ M^-1. The S-ibuprofen sites were present in about a 1:1 ratio and appeared to exhibit some allosteric interactions at high S-ibuprofen concentrations. Thee chromatographic technique used in this work is a general one which can be adapted for use in studying the interactions of other chiral compounds with either HSA or additional proteins.