DETERMINATION OF CLENBUTEROL IN BOVINE URINE BY ENZYME-IMMUNOASSAY FOLLOWING CONCENTRATION AND CLEANUP BY IMMUNOAFFINITY CHROMATOGRAPHY

A method for the determination of clenbuterol in bovine urine is described, which involves clean-up and concentration of the sample by immunoaffinity chromatography, followed by microtitre plate enzyme immunoassay of the extract. Both the chromatographic procedure and the assay use ovine antiserum raised against clenbuterol. Enzyme-labelled clenbuterol was prepared by conjugation of a clenbuterol metabolite [4-amino-3,5-dichloro-alpha-(2-hy-droxy-1,1-dimethyl)ethylamino methylbenzyl alcohol) with alkaline phosphatase using a diazotization procedure. The procedure provided good recovery and linearity of response, and an acceptable limit of determination of 10 pg ml-1. The coefficient of variation of the assay ranged from 28% at 20 pg ml-1, to 4.5% at 0.5 ng ml-1. The procedure proved sufficiently robust to enable heavily pigmented samples to be analysed without undue non-specific effects.