Evaluation of commercial immunoassays for cross-reactivity to clenbuterol stereoisomers and bovine metabolites

Several commercially available immunoassay kits have been developed to dete ct the beta-adrenergic agonist clenbuterol HCl. Technical materials supplie d with the kits do not generally report cross-reactivity with clenbuterol m etabolites. Use of such kits to quantitate clenbuterol might lead to an ove restimation of parent drug if metabolites were present. The objective of th is study was to measure the cross-reactivity of clenbuterol metabolites wit h several commercially available clenbuterol immunoassays. Three clenbutero l-glucuronide conjugates, clenbuterol-sulphamate, 4-amino-3,5-dichloro-hipp uric acid (clenbuterol-hippurate), and purified clenbuterol-stereoisomers w ere tested for cross-reactivity. The clenbuterol-sulphamate metabolite show ed significant cross-reactivity (42-77%), but clenbuterol-hippurate showed very little competition (<0.2%) towards clenbuterol. Clenbuterol-glucuronid es had little (0.1-1.6%) cross-reactivity. In addition, (R)-, (S)-, and rac emic clenbuterol were used to determine the stereospecificity of the kits. Both (R) and (S)-clenbuterol competed for binding in two of the kits, howev er, in one kit the (S)- clenbuterol stereoisomer had an affinity 100 times greater than the (R) stereoisomer. The presence of significant quantities o f the sulphamate metabolite of clenbuterol in a biological matrix would cau se an overestimation of the amount of parent clenbuterol. This study illust rates the inherent problems of using unvalidated immunoassays for quantitat ion purposes.