Spectrofluorimetric determination of artemisinin with pyronine B as the substrate for horseradish peroxidase

Abstract

Enzyme-catalytic fluorescence determination of artemisinin (qinghaosu, QHS) was developed using pyronine B (PB) as substrate of horseradish peroxidase (HRP). The interaction between HRP and QHS was an enzyme-substrate model. The catalytic characteristic of HRP in the oxidation reaction, in which the fluorescence of PB was decreased in the presence of QHS, was studied. The steady-state catalytic rate depended upon enzyme and substrate concentrations, and the Michaelis-Menten parameters K m Vmax and K cat were 8.4×10−5 mol · L−1, 7.4×10−6 mol · L−1v s−1 and 50.23 s−1. The catalytic activity of enzyme was inhibited in the presence of deactivated agents and at high temperature, respectively. Under optimum conditions, linear relationship between fluorescence intensity change (F 0- F) of pyronine B and concentration of QHS was in the range of 1.41×10−7-1.27×10−6 mol · L−1. The detection limit (3σ) was determined to be 2.7×10−8mol· L−1. The proposed method was applied to the concentration determination of QHS in the media of plasma or urine samples.