High Performance Liquid Chromatography Assay for p-Nitroanisole o-Demethylation by Cytochrome P450 Enzymes in Musca domestica L.

Abstract

Abstract A novel sensitive method was developed for measuring p -nitroanisole o -demethylation (PNOD) catalyzed by cytochrome P450 enzymes in crude homogenates of housefly, Musca domestica L., using high performance liquid chromatography (HPLC) with diode array detector. This method is based on the use of liquid-liquid extraction and HPLC with reversed-phase C 18 column and gradient ultraviolet detection at 316 nm. The detection limit ( S/N of 3/1) of p -nitrophenol ( p NP) was 0.1 ng. The relative standard deviation (RSD) of peak area response of the HPLC analysis system was 1.0%. The average recoveries of 0.351, 1.755 and 8.755 μg of p NP added to incubation mixtures ranged from 92.3% to 87.6%, and the RSD of recoveries of three added levels ranged 2.64%–5.90%. The relationships between peak areas and concentration were linear in the range of 9.72–486 ng with good coefficient of determination ( r 2 = 0.9995). This method is sensitive, precise, and reproducible and is also applicable to the assay of p -nitroanisole o -demethylases, which have low catalytic activities, present in other insects or organisms. With this method, the comparison of P450 o -demethylation activity of an imidacloprid-resistant strain with a susceptible strain of M. domestica (L.) was conducted to identify the roles of P450 enzymes involved in housefly resistance to imidacloprid. The results indicated that the resistance mechanism of housefly to imidacloprid was intensely assisted with elevated P450 activities.