In this study affinity chromatographic technique has been used to derive binding affinities. With the application of affinity chromatography to direct determination of functional activities, there is problem with determination of functional activities, indicating the fact that affinity in binding may not be directly correlated with the pharmacological action of the drug on the pharmacological target. In case of competitive agonists and antagonists, these properties are related using Cheng-Prusoff relationship,
where Ki is the binding affinity of the inhibitor, IC50 is the functional strength of the inhibitor, S is substrate concentration, and Km is the affinity of the substrate for the enzyme. The Cheng-Prusoff relationship cannot be used with NCAs due to occurrence of allosteric interactions. The relative antagonistic activities of a series of noncompetitive antagonists, namely, imipramine, ethidiuum, phencyclidine, dextromethorphan, and mecamylamine expressed as IC50 values towards nAChRs can be measured using data from affinity chromatography on an immobilized nAChR stationary phase using nondirect method of multivariate analysis for assessment of IC50 values.