Detection of clenbuterol (Ventipulmin) in the horse

An enzyme linked immunosorbent assay (ELISA) was developed to detect the beta 2-agonist clenbuterol in equine blood and urine. The antiserum was raised in rabbits, employing clenbuterol-diazo-BSA as antigen. Clenbuterol-diazo-horseradish peroxidase served as enzyme conjugate. The concentration of clenbuterol to decrease tracer binding by 50% (IC50 value) was found to be 27.50 +/- 4.20 pg/well (1.37 ng/ml). The antibody cross-reacted with salbutamol (30%), terbutaline (14%) and cimaterol (1%). Horse serum was used directly to screen for clenbuterol, while urine was employed diluted. Positive screening results were confirmed by means of two independent HPLC systems combined with off-line detection by the clenbuterol-ELISA. Salbutamol served as internal standard to ascertain relative retention of the drug. The detection limit of clenbuterol in serum and urine amounted to 0.04 ng/ml. In addition, GC/MS technique was applied to detect clenbuterol in urine samples by a newly developed derivatization method. Confirmation of intravenously given clenbuterol in serum of horses treated with Ventipulmin (0.8 microgram clenbuterol.HCl/kg) was achieved by HPLC/ELISA up to 24 h, in urine up to 96 h. After oral administration, the beta 2-agonist was detected in serum for 48 h and in urine for 75 h.