Potentiometric flow injection determination of amylase activity by using hexacyanoferrate(III)-hexacyanoferrate(II) potential buffer

A highly sensitive potentiometric flow injection determination of amylase activity was carried out, utilizing a redox reaction of hexacyanoferrate(III) in alkaline media with reducing sugar as product of the enzymatic hydrolysis reaction of starch with amylase. The analytical method is based on the potential change detection of a flow-through type redox electrode detector due to the composition change of a [Fe(CN)₆]^3--[Fe(CN)₆]^4- potential buffer solution, which is caused by the redox reaction with the product of the enzymatic reaction. A linear relationship exists between the potential change (peak height) and the activity of amylase. Amylase of a wide activity range from 2.5 x 10^-2 to 1.2 x 10^-4 U ml^-1 can be determined by the changing the concentrations of the [Fe(CN)₆]^3--[Fe(CN)₆]^4- potential buffer from 10^-3 to 10^-5 M. The lower detection limit of amylase activity is 6.0 x 10^-5 U ml^-1. The sampling rate and relative standard deviation are 15 h^-1 and 0.9% (n = 5) for 3.8 x 10^-3 U ml^-1 of amylase. The present method was successfully applied to determine amylase activity in real samples (commercial digestive medicines) with an accuracy of 4% compared with analytical results obtained using the present method with those achieved using the conventional titration method.