Total determination of residual flutolanil and its metabolites in livestock products and seafood using liquid chromatography-tandem mass spectrometry

We have developed a simple and sensitive LC-MS/MS analytical method for the determination of residual flutolanil and its principal metabolites, including ,,-trifluoro-3-hydroxy-o-toluanilide (M-4) and its conjugates, in livestock and seafood products. Both flutolanil and its metabolites contain the 2-(trifluoromethyl)benzoic acid (2-TFMBA) moiety. In this method, flutolanil and its metabolites are converted to 2-TFMBA by hydrolysis. The method involves direct hydrolysis with sodium hydroxide at 200 degrees C, acidification, partitioning into a mixture of ethyl acetate-n-hexane (1:9, v/v), clean-up using a strong anion exchange cartridge (InertSep SAX), and then quantification using LC-MS/MS. The optimal conditions for the complete hydrolysis of flutolanil to 2-TFMBA are an incubation time of 6h and a temperature of 200 degrees C. The developed method was evaluated using seven types of food: bovine samples of muscle, fat, liver and milk, as well as egg, eel, and freshwater clam. Samples were spiked both at 0.01mg/kg and at the Japanese maximum residue limit (MRL) established for each food type. The validation results show excellent recoveries (88-107%) and precision (<10%) for flutolanil and M-4. The limit of quantification (S/N10) of the developed method is 0.01mg/kg. The developed method is applicable to the definition of residual flutolanil for animal-based food commodities and MRLs established by the Codex Alimentarius, and will be useful for the regulatory monitoring of residual flutolanil and its metabolites in food products.