Evaluation of ractopamine cross-reactivity in several commercially available β-agonist enzyme immunoassay kits

Wicker, Alan L; Turberg, Michael P; Coleman, Mark R

doi:10.1039/an9952002879

Cited by 8 publications

(5 citation statements)

References 5 publications

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“…The concentrations of RCT given to the experimental animals (0.1 mg kg 21 d 21 ) were in the region of dose required to cause growth promotion. 8,11 The concentrations of RCT residues detected as a result of this treatment were substantial, at least during the medication phase. There was considerable animal to animal variation in the residue levels measured during this phase and surprisingly the concentrations of RCT present in the urine samples appeared to decline during the first week of treatment despite a steady intake of drug by the animals.…”

Section: Discussionmentioning

confidence: 99%

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

Elliott¹,

Thompson²,

Arts³

et al. 1998

Analyst

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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.

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Section: Discussionmentioning

confidence: 99%

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

Elliott¹,

Thompson²,

Arts³

et al. 1998

Analyst

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“…The correlation between the two methods was moderate, at r 2 ) 0.73. A report using commercially available β-adrenergic agonist immunoassay kits demonstrated no crossreactivity with ractopamine (15) indicating that they would not be useful for ractopamine analysis.…”

Section: Introductionmentioning

confidence: 99%

Application of a Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay for the Determination of Ractopamine in Incurred Samples from Food Animals

Shelver

Smith

2002

J. Agric. Food Chem.

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A monoclonal antibody-based ractopamine immunoassay has been applied to incurred samples from sheep and cattle. Results obtained by immunoassay were compared with those from high-performance liquid chromatography (HPLC). Three sets of sample extracts containing primarily unmetabolized ractopamine were analyzed. Correlation of HPLC with enzyme-linked immunosorbent assay (ELISA) for beef liver samples gave an r(2) = 0.98 despite rather low ractopamine concentrations (range 1.1-13.4 ng/mL, n = 6). Ractopamine concentrations in cow urine samples treated by solid phase extraction, to remove ractopamine metabolites, also showed a high correlation between the HPLC and the ELISA results (r(2) = 0.95, range 1.0-275 ng/mL, n = 61). In contrast, HPLC and ELISA analyses of ractopamine in sheep urine were not well-correlated (r(2) = 0.58, range 0.85-51 ng/mL, n = 34). When ractopamine conjugates in urine samples were hydrolyzed with hydrolytic enzymes, ELISA and HPLC methods were highly correlated [r(2) = 0.94 for sheep (range 123-10 554 ppb, n = 60) and an r(2) = 0.98 for cattle (range 14-8159 ppb, n = 62)]. Tissues contained only minute amounts of ractopamine, and after 7-day withdrawal periods, less than 1 ppb of free ractopamine was detected. Ractopamine was rapidly metabolized in both cattle and sheep. The difference in ractopamine concentration of urine samples before and after hydrolysis indicated that only 1-5% of ractopamine was excreted unmetabolized. Results from this study indicate that the monoclonal antibody-based ELISA could be useful for a sensitive, quantitative, or qualitative ractopamine screening assay.

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“…All these compounds showed no cross-reactivity with the RAC antibody except for dobutamine. Wicker et al [18] reported that if the antibody is developed against a compound with a very similar structure, crossreactivity will likely occur. In conclusion, it was reasonable that dobutamine which has a very similar structure to RAC showed 7.5% crossreactivity with RAC.…”

Section: Resultsmentioning

confidence: 99%

Rapid Determination of Ractopamine Residues in Edible Animal Products by Enzyme‐Linked Immunosorbent Assay: Development and Investigation of Matrix Effects

Zhang

Wang

et al. 2009

BioMed Research International

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To determine ractopamine residues in animal food products (chicken muscle, pettitoes, pig muscle, and pig liver), we established a rapid direct competitive enzyme-linked immunosorbent assay (ELISA) using a polyclonal antibody generated from ractopamine-linker-BSA. The antibody showed high sensitivity and specificity in phosphate buffer, with an IC50 of 0.6 ng/mL, and the limit of detection was 0.04 ng/mL. The matrix effect of the samples was easily eliminated by one-step extraction with PBS, without any organic solution or clean-up procedure such as SPE or liquid-liquid extraction, making it a much more simple and rapid method than previously reported ones. The detection limit in blank samples was 0.2 μg/kg. To validate this new RAC (ractopamine hydrochloride) ELISA, a RAC-free pig liver sample spiked at three different concentrations was prepared and analyzed by HPLC and ELISA. The results showed a good correlation between the data of ELISA and HPLC (R2 > 0.95), which proves that the established ELISA is accurate enough to quantify the residue of RAC in the animal derived foods.

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Evaluation of ractopamine cross-reactivity in several commercially available β-agonist enzyme immunoassay kits

Cited by 8 publications

References 5 publications

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

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

Application of a Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay for the Determination of Ractopamine in Incurred Samples from Food Animals

Rapid Determination of Ractopamine Residues in Edible Animal Products by Enzyme‐Linked Immunosorbent Assay: Development and Investigation of Matrix Effects

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