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 posit ive peaks. The evaluation of the area of the second peak, whether posi tive or negative, makes it possible to determine the value of L(b) (th e number of moles of the ligand bound to macromolecule) or [L]b (the c oncentration of the ligand bound to macromolecule). Three techniques w ere employed for the evaluation of the area: planimeter, geometry and integrator. Planimeter and geometry can be used for both types of elut ion profiles and for both external calibration and internal calibratio n. The integrator can only be used for the second type of elution prof ile, namely two positive peaks and hence, can only be used in conjunct ion with internal calibration. The results in terms of the two binding parameters, n (the number of binding sites) and k (the affinity const ant) in binding equilibrium for L-tryptophan-bovine serum albumin syst em were compared. Realizing that uncertainty involved is large for the binding studies in any experimental method for the binding studies, w e believe that any of the five combinations (among external calibratio n, internal calibration, planimeter reading. geometry reading and inte grator reading) would lead to a reasonably accurate result.