Trace multiresidue analysis of fenbendazole and its sulfoxide, sulfone, and p-hydroxylated metabolites in milk by liquid chromatography

A liquid chromatographic method for trace analysis of fenbendazole and its sulfoxide, sulfone, and p-hydroxylated metabolites in milk has been developed. Samples are deproteinized with acetonitrile, and the supernatants are extracted with dichloromethane. The separated bottom organic layers are washed with alkaline buffer and evaporated to dryness, and the residues are partitioned between water and ethyl acetate. The separated top organic layers are evaporated to dryness, reconstituted in mobile phase, and submitted to reversed-phase ion-pair chromatography. Overall recoveries ranged from 82% for the sulfoxide and p-hydroxylated metabolites to 91% for fenbendazole and its sulfone metabolite. The linearity of the assay was quite satisfactory, whereas the limit of detection was in the range 2-5 ng/mL. Precision studies based on within- and between-days variation suggested overall precision values better than 4.3% for all analytes. The method was successfully applied to quantitate incurred fenbendazole residues in milk from a lactating dairy cow treated with a fenbendazole formulation.