Screening and confirmatory determination of ractopamine residues in calves treated with growth promoting doses of the beta-agonist.

Abstract

Ractopamine (RCT) is a phenethanolamine member of the family of beta-adrenergic agonists (beta-agonists). This class of compounds have become notable for their properties of enhancing the growth rates of farm animal species but are not licensed for use in Europe. An ELISA procedure employing a polyclonal antibody raised in a goat was developed to detect RCT residues in bovine urine samples. The assay had a high sensitivity (calibration curve mid-point of 22 pg per well), allowing the analysis of urine samples without the need for sample clean-up. In addition, an LC-MS-MS confirmatory procedure was developed which was able to act as a confirmatory procedure for the ELSA results. Four calves were orally treated with RCT (0.1 mg kg-1 body mass for 17 d) and urine samples collected were assayed by both analytical procedures. It was observed that RCT residues were excreted mainly in the form of glucuronides and deconjugation could be achieved using two different sources of the enzyme beta-glucuronidase (Helix pomatia and Escherichia coli). High concentrations of RCT residues were found throughout the medication period (44-473 ng ml-1; LC-MS-MS data) and remained present for several days following removal of the drug from the diet. RCT residues were no longer detectable 2 weeks after withdrawal. Good agreement (r2 = 0.73) was achieved between the ELISA and LC-MS-MS results, especially when sample deconjugation was applied to the urine samples for sets of analyses. The results show that an effective screening and confirmatory system was devised to detect RCT residues in urine samples taken during treatment and close to withdrawal. However, alternative matrices may have to be selected to allow the illegal use of the substance to be detected following prolonged withdrawal times.