Residue Analysis of Clenbuterol in Bovine and Horse Tissues and Cow's Milk by Enzyme-Linked Immunosorbent Assay

Residue analysis of clenbuterol (CBL) in bovine and horse tissues and cow's milk using enzyme-linked immunosorbent assay (ELISA) with alkaline phosphatase as the label is described. The detection limit of the ELISA established here was 0.1ng/g (0.1ppb). Tissue samples were homogenized with 3% metaphosphoric acid and cleaned up by liquid-liquid extraction with ethyl acetate. After evaporation of the ethyl acetate extract, CBL in the residue was measured by ELISA. Recoveries of CBL were 75-96% for bovine tissues, 74-98% for horse tissues and 99% for cow's milk. CBL was not detectable in bovine tissues after the 6th day of withdrawal (usual dose: 0.3mg/head, double dose: 0.6mg/head), or in horse tissues after the 25th day of withdrawal (1.6μg/kg, twice a day, for 11 days). CBL concentration in horse plasma fell below the detection limit within 24 hours after the final administration. CBL was not detectable after 2.5 days of withdrawal in cow's milk or after 6 hours (usual dose group) or 24 hours (double dose group) in cow's plasma.