Quantification of acetaminophen by oxidation with tyrosinase in the presence of Besthorn's hydrazone.

A method for the quantitative determination of acetaminophen by measuring the kinetics of its oxidation by the enzyme tyrosinase in the presence of the nucleophilic agent 3-methyl-2-benzothiazolone hydrazone (Besthorn's hydrazone) is described. The hydrazone traps the o-quinone product of the catalytic activity, 4-acetamido-o-benzoquinone, thus increasing the level of the catecholic intermediate, 3'-hydroxyacetaminophen, in the pathway, as was shown by liquid chromatography. This reduces the time necessary for the steady state to be reached and increases the steady state oxidation rate, reaching activations of 124 times at an initial concentration of 3mM hydrazone. Despite being rapid and simple the method is thorough and provides a linear range of quantification of the drug from 10 nmol/ml to 1.4 micromol/ml. The assay can be performed in continuous or discontinuous form, meaning that it can be used with the immobilized enzyme for the design of specific biosensors.