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

Several commercially available immunoassay kits have been developed to detect the beta-adrenergic agonist clenbuterol HCl. Technical materials supplied with the kits do not generally report cross-reactivity with clenbuterol metabolites. Use of such kits to quantitate clenbuterol might lead to an overestimation of parent drug if metabolites were present. The objective of this study was to measure the cross-reactivity of clenbuterol metabolites with several commercially available clenbuterol immunoassays. Three clenbuterol-glucuronide conjugates, clenbuterol-sulphamate, 4-amino-3,5-dichloro-hippuric acid (clenbuterol-hippurate), and purified clenbuterol-stereoisomers were tested for cross-reactivity. The clenbuterol-sulphamate metabolite showed significant cross-reactivity (42-77%), but clenbuterol-hippurate showed very little competition (< 0.2%) towards clenbuterol. Clenbuterol-glucuronides had little (0.1-1.6%) cross-reactivity. In addition, (R)-, (S)-, and racemic clenbuterol were used to determine the stereospecificity of the kits. Both (R)- and (S)-clenbuterol competed for binding in two of the kits, however, in one kit the (S)-clenbuterol stereoisomer had an affinity 100 times greater than the (R)-stereoisomer. The presence of significant quantities of the sulphamate metabolite of clenbuterol in a biological matrix would cause an overestimation of the amount of parent clenbuterol. This study illustrates the inherent problems of using unvalidated immunoassays for quantitation purposes.