Fluorescence Determination of Artemisinin Using Cytochrome c as Catalyst and Pyronine B as Indicator

The cytochrome c-catalyzed fluorescence determination of artemisinin (qinghaosu, QHS) was developed using pyronine B (PB) as substrate. The interaction between cytochrome c and QHS was an enzyme-substrate model. The kinetic studies demonstrated that the steady-state catalytic rate depended upon enzyme and substrate concentrations, and the Michaelis-Menten parameters, Km, Vmax, and Kcat were 3.3×10−5 M, 5.4×10−6 M s−1 and 13.5 s−1, respectively. Catalytic activity of cytochrome c was inhibited in the presence of deactivated agents CN− and ethanol. Under optimal conditions (pH 5.3, 25°C and 7.6×10−8 to 1.1×10−6 M, the detection limit (3σ) was as low as 7.2×10−9 M and the percent recovery range was 96.3%–106.8%. The proposed method was applied to detect the concentration of QHS in the media of plasma and urine.