Recombinant P450s Neonicotinoid Metabolism

Recombinant P450s (160 μg/well) in 0.1 M potassium phosphate buffer were incubated for 1 h (shaking at 225 rpm) with neonicotinoid insecticides, at a concentration of 10 μM, in a total assay volume of 200 μL, at 30 ± 1°C, in the presence or absence of a NADPH regeneration system. Three replicates were performed for each data point. Samples incubated without NADPH served as controls. The reactions were terminated by the addition of ice‐cold acetonitrile (to 80% final concentration). Samples were incubated at 4°C overnight and then centrifuged at 3000g for 10 min to pellet any precipitation of protein. LC–MS/MS was performed as previously described (Haas et al., 2022 (link)), using an Acquity UPLC (Waters) coupled to an API 4000 mass spectrometer (Sciex) and an Infinity II UHPLC (Agilent Technologies; reverse phase mode) coupled to a QTRAP 6500 mass spectrometer (Sciex) employing electrospray ionization. The recovery rates of parent compounds incubated without NADPH were typically close to 100%.