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

Determination of Ractopamine Residues in Pigs by Ultra Performance Liquid Chromatography Tandem Mass Spectrometry

“…In calf, the ractopamine residue concentrations found after drug withdrawal were also substantially lower than during the medication period, and were only detectable in one animal 2 weeks after removal of medication from the diet (Elliot et al, 1998). In their study on cattle treated with 20 mg/kg dietary ractopamine for seven days, Smith and Shelver (2002) obtained similar results concerning rapid urinary excretion of ractopamine.…”

“…Control and monitoring programs mandated by government have also necessitated implementation of assays for determination of ractopamine accumulation and excretion from tissues and body fluids in farm animals. Studies of ractopamine residue detection after different treatment schedules in different animal species have been reported by several authors (Elliot et al, 1998;Qiang et al, 2007;Smith & Shelver, 2002;Thompson et al, 2008). Published studies report on tissue residues of ractopamine and its urinary excretion (Antignac et al, 2002;Blanca et al, 2005;Dickson et al, 2005;Moragues & Igualada, 2009;Nielen et al, 2008;Thompson et al, 2008;Van Hoof et al, 2005) and ractopamine residues in hair (Nielen et al, 2008).…”

Substitution of Antibody with Molecularly Imprinted Film in Enzyme-Linked Immunosorbent Assay for Determination of Trace Ractopamine in Urine and Pork Samples

“…The enzyme conjugates were synthesized according to the literature reported by Christopher et al (Elliott et al 1998). Five milligrams of HRP was dissolved in 0.5 mL of DDW with pH controlled at 10.8 using NaOH (0.5 mol L −1 ), and the whole system was degassed with nitrogen for 5 min.…”

Safety evaluation of ractopamine

“…An ELISA method for ractopamine in liver and urine was developed (Elliot et al, 1998). The cross-reactivity for ractopamine was estimated as 100 %, whereas the cross-reactivities for some other ß-agonists were below < 0.01 %.…”