A multi‐residue enzyme immunoassay for screening illegally usedβ‐agonists

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Abstract
A multi‐residue screening test was developed which provides reliable detection of eight ß‐agonists in urine, based on an enzyme immunoassay with peroxidase and an antibody raised against clenbuterol‐diazo‐BSA. Antibodies were compared after continuous immunization. The antibody obtained after the 12th immunization showed the best sensitivity, procedural blanks of urine samples and coefficients of variation, using analysis of spiked urine samples. The simultaneous clean‐up was performed with silicagel RP select‐B. In 125 samples from untreated female or male cattle or pigs, sample blanks gave results of <0·07 ng clenbuterol equivalents/ml urine and in two further samples, results of 0·13 and 0·15 ng/ml, respectively. The method showed coefficients of variation of 20–30% for the ß‐agonists tested, except in the case of orciprenaline, at levels ranging from 0·1 ng/ml urine (clenbuterol, mabuterol) to 10 ng/ml urine (pirbuterol, orciprenaline). These ß‐agonists were also separated by a high performance liquid chromatography (HPLC) system, which provided complete separation of orciprenaline, 2‐amino‐3‐chloro‐5‐(1'‐hydroxy‐2'amino‐tert‐butyl‐ethyl) pyridine (ACP), isoetharine, clenbuterol and mabuterol as well as good separation of carbuterol, terbutaline, salbutamol and pirbuterol.