Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 26, 2003
Publication Date: May 1, 2003
Citation: Shelver, W.L., Smith, D.J. 2003. Determination of ractopamine in cattle and sheep urine samples using an optical biosensor analysis:comparative study with HPLC and ELISA. Journal of Agricultural and Food Chemistry 51(13):3715-3721. Interpretive Summary: This is a manuscript that purports the suitability of using an immunobiosensor for measuring ractopamine levels in food animals. It is proposed that the assay is suitable for measuring ractopamine levels in urine samples in animals, such as sheep and cattle, where ractopamine is not approved as a growth aid.
Technical Abstract: A biosensor method, using the surface plasmon resonance (SPR) principle, was developed for the determination of ractopamine in cattle and sheep urine. A monoclonal antibody was used to compete with ractopamine in the sample and ractopamine immobilized on the sensor chip. Addition of bovine serum albumin (BSA, 1 mg/mL) as antibody stabilizer to the incubation buffer was required to achieve a stable biosensor response throughout each sample set. The calibration curve gave a mean IC50 of 4.7 ± 0.21 ng/mL (n = 7). Over sample concentrations from 2.5 to 10 ng/mL recoveries were typically approximately 100-110% while inter and intra assay reproducibility (% CV) were usually less than 10% and 6% respectively. Comparison of biosensor results with results obtained from high performance liquid chromatography (HPLC) and enzyme linked immunosorbent assays (ELISA) using enzyme-hydrolyzed urine (to convert ractopamine conjugates to free ractopamine) gave correlation coefficients of 0.94 for sheep and 0.86 for cattle. Slopes of the lines, with zero intercepts, equaled 0.80 for sheep and 0.74 for cattle. For untreated (non-hydrolyzed) urine samples, the correlation between biosensor and HPLC results was 0.95 for sheep or 0.72 for cattle with a slope of 1.18 (sheep) or 1.69 (cattle). The slopes greater than unity indicate that the biosensor responded to ractopamine metabolites in addition to free ractopamine. The biosensor assay is an excellent analytical tool to determine ractopamine residues in sheep or cattle urine and the results should be extendible to other species with suitable validation.