A New Colorimetric kinetic method for determination of Scopolamine butylbromide based on the cholinesterase activity inhibition

Abstract

A new method for enzyme-kinetic determination of scopolamine butylbromide (SBB), based on the SBB ability to inhibit the reaction of hydrolytic acetylcholine decay over cholinesterase enzyme is elaborated. Scopolamine butylbromide content is determined by the degree of enzyme reaction inhibition, which is measured using unreacted acetylcholine in enzyme reaction: determination of acetylcholine is carried out by kinetic tangent method with p-phenetidine oxidizing reaction, formed in the previous perhydrolysis reaction (with excess hydrogen peroxide) by peracetic acid. Indicator reaction rate is determined by the photometric method by the increase of absorbance formed azoxyphenetole (λmax=350 nm). The proposed method was applied successfully to analyze scopolamine butylbromide in tablets dosage form. No interference was observed from common pharmaceutical excipients. In the optimum conditions, the calibration curve is linier over the range 1 ... 6 μmol / L (r=0,996). The relative standard deviation was ± 7.4% (n=5) for 1.15 μmol / L and ± 1.95% (n=5) for 5.75 μmol / L (δ =-1.22 % and 0.03% respectively). The limit of quantitation (for the reaction inhibition grade 20%) 2 ∙ 10-6 mol / L (n=5). For seven determinations of active substance content in scopolamine buthylbromide substance drug was found to be 99.62%, RSD=1.72% (δ = + 0.41%; compared with HPLC method). A recovery of scopolamine buthylbromide in “Spasmobru” tablets 10 mg is 100.51%, RSD=1.65% (δ = + 0.5%).