Hummel-Dreyer method in high-performance liquid chromatography for the determination of drug-protein binding parameters

Two types of Hummel-Dreyer elution pattern were developed, one with a positive peak followed by a negative peak and the other with two positive peaks. The evaluation of the area of the second peak, whether positive or negative, makes it possible to determine the value of L(b) (the number of moles of the ligand bound to macromolecule) or [L]b (the concentration of the ligand bound to macromolecule). Three techniques were employed for the evaluation of the area: planimeter, geometry and integrator. Planimeter and geometry can be used for both types of elution profiles and for both external calibration and internal calibration. The integrator can only be used for the second type of elution profile, namely two positive peaks and hence, can only be used in conjunction with internal calibration. The results in terms of the two binding parameters, n (the number of binding sites) and k (the affinity constant) in binding equilibrium for L-tryptophan-bovine serum albumin system were compared. Realizing that uncertainty involved is large for the binding studies in any experimental method for the binding studies, we believe that any of the five combinations (among external calibration, internal calibration, planimeter reading. geometry reading and integrator reading) would lead to a reasonably accurate result.