The enzymatic method for the quantitative determination of benzalkonium chloride in the antiseptic solution “CUTASEPT® F”




benzalkonium chloride, CUTASEPT® F, cholinesterase, acetylcholine


Aim. To develop an alternative method for the quantitative determination of the benzalkonium chloride content as an active pharmaceutical ingredient in the disinfectant solution “CUTASEPT® F”.
Materials and methods. The method is based on the ability of benzalkonium chloride to inhibit the enzymatic hydrolysis of acetylcholine by acetylcholinesterase. The reaction rate is assessed by the non-hydrolyzed acetylcholine residue, which is determined by the amount of peracetic acid produced during the interaction with the excess of the hydrogen peroxide solution. The indicator reaction is the interaction of p-phenetidine with peracetic acid that leads to the formation of 4,4’-azoxyphenetole with λmax = 358 nm (log10 ε = 4.2).
Results and discussion. As a result of the research conducted the linear dependence of the degree of inhibition of the enzymatic hydrolysis of acetylcholine (U, %) on the concentration of benzalkonium chloride was determined in the concentration range of (0.5 – 7.0) × 10–6 mol L-1 with the correlation coefficient of 0.999. The limit of quantitation was 1.9 × 10–6 mol L-1.
Conclusions. As a result of the research conducted the kinetic enzymatic method for the quantitative determination of benzalkonium chloride has been developed by its inhibitory effect in the biochemical reaction of acetylcholine hydrolysis. This method is fast, cheap and easy to perform, does not require expensive equipment, and available for use in the field.

Supporting Agency

  • The work is a part of researches of the National University of Pharmacy on the topic «Organic synthesis and analysis of biologically active compounds, drugs development on the basis of synthetic substances» (the state registration No. 01144000943; the research period 2019 – 2024).


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How to Cite

Koval’ska, O. V.; Blazheyevskіy M. Y. The Enzymatic Method for the Quantitative Determination of Benzalkonium Chloride in the Antiseptic Solution “CUTASEPT® F.” J. Org. Pharm. Chem. 2022, 19, 33-39.



Original Researches