Multi-residue analysis for beta-agonists in urine and liver samples using mixed phase columns with determination by radioimmunoassay

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Abstract
A method is described for the extraction of four β-agonists, clenbuterol, salbutamol, mabuterol and terbutaline from bovine urine and liver samples using radioimmunoassay (RIA) as the method of determination. Following enzymic digestion of the liver samples using protease enzyme, the digest is centrifuged and the harvested supernatant is saturated with sodium chloride and adjusted to pH 11.0. An ethyl acetate–propan-2-ol mixture is used to extract the β-agonists from the liver digest. The samples of urine and liver extracts are adjusted to pH 6.0 and applied to mixed phase (XtrackT) columns. The column is washed with water and methanol and the β-agonists are eluted with methanol containing 2% ammonia. After evaporation of the eluting solvent and reconstitution in ethanol the β-agonist residues are determined by RIA, with standard graphs prepared in residue-free sample extract. The procedure has been validated for clenbuterol, salbutamol, mabuterol and terbutaline. The mean recovery of the β-agonists from urine and liver is >75% and >85%, respectively. The detection limit is 0.13 ng ml–1 and 0.46 ng g–1 of clenbuterol in urine and liver, respectively. The high recoveries attained for both types of β-agonists are a result of an efficient liquid–liquid extraction step coupled with a selective mixed solid-phase extraction procedure.

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