Submitted to: Journal of Association of Official Analytical Chemists International
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 12, 2002
Publication Date: November 1, 2002
Citation: Shelver, W.L., Smith, D.J. 2002. Immunoaffinity column as sample cleanup method for determination of the beta-adrenergic agonist ractopamine and its metabolites. Journal of Association of Official Analytical Chemists International. 85(6):1302-1307. Interpretive Summary: Ractopamine is a growth enhancing agent approved for use in the U. S. but not in the European communities. Because of the trade issues and food safety reasons, a rapid analysis is needed to provide a quick report for food samples. We have developed a column system that can isolate ractopamine from urine, muscle, kidney, and liver in the non-approved species such as cattle and sheep. The column saves time and solvent neede for ractopamine isolation from different tissues and urine. In addition, the column can be re-used for more than 20 times and is stable for more than 3 months if stored properly.
Technical Abstract: A monoclonal antibody-based immunoaffinity column (RAC-IAC) was developed as a potential cleanup method for the analysis of ractopamine and ractopamine-glucuronides. [14C]Ractopamine (5 mg) and [14C]ractopamine glucuronides (5 mg) were fortified into 10 mL of ractopamine free cattle urine, loaded onto a column (5 mg IgG/mL), and eluted with 10 mL of 50% aqueous methanol followed by 10 mL of 100% methanol. In the initial loading and washing 22% of the radioactivity was not retained whereas 78% was recovered in the two-elution steps. By comparison, 92% of the radioactivity loaded onto a non-specific IAC was removed by the initial washing steps. The IAC columns were damaged by high methanol concentrations but could be reproducibly reused when 50 mM glycine, pH 2.8, was used as an elution buffer. The columns were stable for over 3 months and elution was reproducible over 20 runs. In addition, elution with a glycine buffer in place of methanol allowed for separation of the ractopamine/ractopamine glucuronides from fluorescence interferences. The procedure was also tested using meat, liver, and kidney samples. Liver and kidney samples had to be diluted to prevent column plugging, but the RAC- IAC eluants were suitable for HPLC analysis. A RAC-IAC can be a selective, efficient, and economical cleanup method for use in ractopamine analysis.