New approach for the potentiometric-enzymatic assay of reversible-competitive enzyme inhibitors. Application to acetylcholinesterase inhibitor galantamine and its determination in pharmaceuticals and human urine

Abstract

A new kinetic-potentiometric method for the characterization and analytical determination of competitive reversible enzyme inhibitors was developed. The method is based on a mathematical approach, assuming that the reaction proceeds at the steady state, which permits calculation of a tentative substrate concentration to be used to determine low inhibitor concentrations and to obtain the value of inhibition constant corresponding to the inhibitor. The mathematical approach predicts a linear relationship between the inverse of the relative inhibition and the inverse of the inhibitor concentration. The method developed is applied to the acetylcholinesterase inhibitor galantamine, using an acetylcholine-selective electrode. A linear relationship for galantamine concentration from 2×10−8 to 1×10−6M and a limit of detection of 5.4×10−9M was found. A value for KIGal of 2.0×10−7±0.1×10−7M was obtained. The effect of several other drugs and of the main galantamine metabolite excreted in urine was studied. The method was satisfactorily applied to the determination of galantamine in pharmaceuticals and human urine.