Characterization of Activation Metabolism Activity of Indoxacarb in Insects by Liquid Chromatography-Triple Quadrupole Mass Spectrometry

A triple-quadrupole LC-MS/MS method was developed for the investigation of indoxacarb N-decarbomethoxyllation activity catalyzed by insect crude enzymes. The metabolite, N-decarbomethoxyllated indoxacarb (DCJW), was determined using electrospray ionization in positive ion mode. The quantitative and qualitative analyses were performed in multiple reaction monitoring (MRM) mode. The relative standard deviation (RSD, n = 5) of DCJW was 2.1%, indicating the metabolite had a good chemical stability in 30 h after the sample preparation. Five injections of DCJW standard solution in acetonitrile, five injections of the same sample in one incubation (substrates plus enzyme samples), and five injections of 5 separate metabolic reactions with the whole body of a susceptible strain of Plutella xylostella (L.) were individually injected into LC-MS/MS system. The RSD was 0.7%, 1.1% and 2.7%, respectively. The average recoveries for 440, 880 and 2200 pg of DCJW added to incubation mixtures were 96.1%–102.9%, and the RSD of recoveries of three added levels ranged from 4.8% to 9.4%. The limits of quantification and detection were 0.1 and 0.01 pg, respectively. A good linearity was achieved in the range of 46–2310 pg (R2 = 0.9996). This method with high sensitivity and simplicity was applicable to the assay of indoxacarb N-decarbomethoxyllation activity of insect metabolic enzymes. A comparison of indoxacarb N-decarbomethoxyllation activity of an avermectin-resistant strain and a susceptible strain of Plutella xylostella (L.) was carried out using this novel LC-MS/MS method. The results showed that the indoxacarb N-decarbomethoxyllation activity in the resistant strain was 3.4-fold of that in the susceptible strain, implicating that negative cross-resistance might exist between indoxacarb and avermectin in Plutella xylostella (L.).

As can be seen from the LC-MS chromatograms of blank incubations (without substrates) (a), control incubations (without enzyme samples) (b), and incubations (substrates plus enzyme samples) (c) under gradient elution program with an injection of 15 μL, a metabolic peak eluted at about 16.5 min was found, but not found in either blank incubations or control incubations, which shows that the product was the metabolite from indoxacarb mediated by H. armigera midgut.Figure optionsDownload as PowerPoint slide