Visual discrimination of phenolic group β₂-agonists and the ultrasensitive identification of their oxidation products by use of a tyrosinase-based catalytic reaction.

Abstract

The fast, visual discrimination of β2-agonist drugs is needed for the on-site screening of various types of β2-agonists in blood and urine samples. We developed a simple, rapid, one-step colorimetric method to detect phenolic β2-agonists by use of a tyrosinase catalytic reaction, which involved the oxidation of the phenol group on the benzene rings of β2-agonists. The enzymatic oxidation products of β2-agonists with phenolic groups exhibited different color transitions based on the different substituent groups on the aromatic ring, whereas β2-agonists with the aniline group or the resorcinol group remained colorless. This visual color discrepancy has been used to intuitively and conveniently differentiate the phenolic group β2-agonists, such as ractopamine, isoxsuprine, ritodrine, and fenoterol. The oxidation products of these compounds have been identified using mass spectrometry, and the possible reaction mechanisms between β2-agonists and tyrosinase have been deduced. The parameters that govern the analytical performance of the reaction product, including the pH of the buffer solution, the concentration of tyrosinase, and the incubation time, have been studied and optimized using ultraviolet-visible (UV-vis) spectroscopy and electrochemical methods. Under the optimal experimental conditions, the absorbance intensity and electrochemical signal were found to increase proportionally to the concentrations of the phenolic group β2-agonists, which gave a quantitative description of the β2-agonists in solution.