Immunoaffinity Column as Sample Cleanup Method for Determination of the Beta-Adrenergic Agonist Ractopamine and Its Metabolites

Abstract

A monoclonal antibody-based immunoaffinity column (RAC-IAC) was developed as a cleanup method for the determination of ractopamine and ractopamine glucuronides. [14C]Ractopamine (5 microg) and [14C]ractopamine glucuronides (5 microg) were fortified into 10 mL cattle urine, and loaded onto an RAC-IAC (5 mg IgG/mL) column. The column was washed and the bound analytes were eluted. In the initial loading and washing, 22% of the radioactivity was washed off and the subsequent elution step recovered 78%. A blank column prepared from nonspecific IgG retained <10% of the radioactivity. The RAC-IACs were damaged by high methanol concentrations, preventing reuse. Elution of the analytes with 50mM glycine buffer, pH 2.8, prevented damage, and the columns could be reused at least 20 times with no change in performance. They were stored >3 months in phosphate-buffered saline with 0.02% sodium azide at 4 degrees C. The method was used with fortified cattle muscle, liver, and kidney samples with recoveries of 82.1+/-7.6, 87.8+/-1.9, and 92.5+/-0.4%, respectively (n = 3). Similar studies with sheep muscle, liver, and kidney samples gave recoveries of 91.8+/-0.2, 91.7+/-0.3, and 92.3+/-0.3, respectively (n = 3). Liver and kidney samples were diluted to prevent column plugging, but all of the eluants were suitable for liquid chromatography analysis. This IAC is a selective, efficient, and economical cleanup method in a variety of matrixes for ractopamine determination.