Simultaneous Determination of Multiple Tetracycline Residues in Milk by Metal Chelate Affinity Chromatography: Collaborative Study

Abstract

To meet federal and state regulatory needs, a liquid chromatographic (LC) method with ultraviolet (UV) detection was developed for determination of 7 tetracyclines at 30 ng/ml in milk. Raw milk samples are defatted, acidified, and centrifuged to remove proteins, and tetracyclines are specifically absorbed from the milk by chelation with metal ions bound to small Chelating Sepharose Fast Flow columns. Tetracyclines are removed from these columns with EDTA-containing buffer, and extracts are further cleaned by ultrafiltration. Finally, extracts are concentrated and analyzed simultaneously by using on-line concentration. This method was validated in a collaborative study that involved 11 laboratories, including the authors' laboratory. Each laboratory was asked to prepare and analyze known control and fortified milk samples, as well as 18 coded blind samples. Eight laboratories completed all analyses. Average interlaboratory recoveries for the known fortified samples ranged from 59% (methacycline at 15 ng/ml) to 78% (oxytetracycline at 60 ng/ml). Average recovery for each of 7 residues at 30 ng/ml were between 60 and 110%, meeting single-residue guidelines for accuracy set by the U.S. Food and Drug Administration. Reproducibility relative standard deviation (RSDR) for the known fortified samples varied from 11 to 39%, with 6 of 7 residues at the 30 ng/ml level having RSDR values at or below 20%. Seven of 8 laboratories correctly identified blind control milk samples and all 28 residues present in blind samples. The metal chelate affinity-LC method for determination of multiple tetracycline residues in milk has been adopted first action by AOAC INTERNATIONAL.