Application of Enzymatic Photometric Kinetic Method for Determination of Benzalkonium Chloride in Various Dosage Forms

Abstract

A kinetic-photometric method for accurate and sensitive determination of benzalconium chloride has been described. The method is based on inhibition of enzymatic hydrolysis of acetylcholine by enzyme acethylcholinestherase reaction. The amount of benzalconium chloride was determined by the degree of inhibition of the enzymatic reaction, which was evaluated by the residual unreacted substrate - acetylcholine. Determination of the residual amount of acetylcholine in the reaction mixture was performed by a kineticphotometric method using an indicator oxidation reaction of p-phenetidine with peracetic acid, which is formed during the auxiliration reaction of perhydrolysis with addition of excess hydrogen peroxide in the reaction mixture over a period of time. The inhibition degree –concentration plot is linear over the range of 1.4 ∙10-6 – 7.0∙10-6 mol/ L with correlation coefficient of 0.998. The LOQ was 1.9∙10-6 mol/L. The different experimental parameters pH, buffer solution was carefully studied and optimized. The proposed method has been successfully applied to pharmaceutical formulations. Statistical comparison of the results with a good established reported method showed excellent agreement and proved that there is no significant difference in the accuracy and precision. For “Virotec-intim” preparation RSD was 3.2 % (δ*=- 0.3 %).