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Background Antibiotics are used in ethanol production to discourage the growth of bacteria that would lower the yield of the product. Any antibiotic residues remaining in distillers grain (DG) co-product could lead to antimicrobial resistance, which is a public health concern. The U.S. Food and Drug Administration (FDA) Center for Veterinary Medicine (CVM) previously developed an LC-MS/MS analytical method to detect residues of erythromycin A, penicillin G, virginiamycin M1, and virginiamycin S1 in DG to enable regulatory decision making. Objective The objective of this study was to ensure the method’s robustness by carrying out a multi-laboratory validation of the method. Method Test portions were extracted with a mixture of acetonitrile and buffer. The extract was cleaned by solid phase extraction. The concentrated eluant was reconstituted and analyzed by LC-MS/MS. Eight laboratories participated in the study. Results Average accuracies for the combined three matrixes for all four compounds at all fortification levels ranged from 83–&amp;gt;109% with repeatability relative standard deviation (RSDr; within laboratory) ≤17% and reproducibility relative standard deviation (RSDR; between laboratory) ≤21%. The Horwitz ration (HorRat) values ranged 0.4–1.0 indicating that method reproducibility is acceptable. Conclusions An interlaboratory study was successfully conducted to evaluate an LC-MS/MS method for the determination of the drugs of interest in DG. The results demonstrate that the method is fit for purpose to determine the drugs in DG and could serve as a regulatory method capable of being used for compliance actions for DG containing these antibiotic contaminants. Highlights The method was posted to the FDA/Foods Program Compendium of Analytical Laboratory Methods.

An LC-MS/MS Method for the Determination of Antibiotic Residues in Distillers Grains: Collaborative Study