Development of an Enzymatic Method for the De- termination of Decamethoxine in Liquid Dosage Forms

Abstract

The Covid-19 pandemic has triggered scientists around the world to focus on the development of antiviral agents and the impovement of methods for their analysis. Decamethoxine (DM) is a quaternary ammonium compound. Its mechanism of action is to disrupt the permeability of the cytoplasmic membrane of bacteria and fungi by binding to the phosphatide groups of membrane lipids. In addition, DM is capable of inhibiting the enzyme acetylcholinesterase (AChE). Based on this property, we have developed, а new simple and sensitive enzymatic kinetic-spectrophotometric method for quantitative determination of DM in ear and eye drops, as well as in antiseptic formulations,. Changes in the activity of AChE alters the amount of unreacted acetylcholine (АСh) in the enzymatic hydrolysis reaction. Addition of hydrogen peroxide to the unreacted ACh leads to the formation of an equivalent amount of peracetic acid. Рeracetic acid can react with р-phenetidine giving a colored product, allowing the quantitative estimation of peracetic acid and subsequently the amount of АСh. The method involves determination of the absorption of the product at λmax = 358 nm (logε = 4.2) over time. The rates of biochemical reactions are calulated from the the slopes of the linear portions of the kinetic curves in experiments with the inhibitor, as well as in two additional control experiments in the absence of the inhibitor, (control 1), and in the absence of the AChE (control 2), The degree of inhibition (U, %) is calculated from these reaction rates. The dependence of the U, % on the concentration of the inhibitor is used as a calibration curve. Linearity (r=0.996) is maintained in the range 1 to 6 ng/ml. The LOQ, evaluated as the concentration corresponding to 20% inhibition, is 1,0 ng/mL. The potential of quantitative determination of DM in factory-produced pharmaceuticals in the presence of various excipients has been demonstrated.