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

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 co-product could lead to antimicrobial resistance, which is a public health concern. US. Food and Drug Administration (FDA)/Center for Veterinary Medicine (CVM) previously developed a LC-MS/MS analytical method to detect residues of erythromycin A, penicillin G, virginiamycin M1 & virginiamycin S1 in distillers grain to enable regulatory decision-making. Objective The objective of this study was to ensure method's robustness by carrying out a multi-laboratory validation of the method. Methods 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 combined three matrices for all four compounds at all fortification levels ranged from 83 to 109% with repeatability RSDr (within laboratory) ≤17% and reproducibility RSDR (between laboratory) ≤21%. The HorRat values ranged 0.4-1.0 indicating that method reproducibility is acceptable. Conclusions A collaborative study was successfully conducted to evaluate an LC-MS/MS method for the determination of the drugs of interest in distillers grains. The results demonstrate that the method is fit-for-purpose to determine the drugs in distillers grain (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. Distillers grain, a major co-product of dry-grind ethanol distillation, is an excellent animal feed supplement. However, any antimicrobial processing aids (i.e., antibiotics) remaining from the fermentation process could be a cause for regulatory concern if distillers grain is fed to animals. Only limited data were available to the U.S. Food and Drug Administration (FDA) on the extent and levels of antibiotics in distillers grain that were produced by either domestic or foreign ethanol facilities and marketed as animal feed ingredients in the U.S. Because of this limited data and because of the marked increase in the amount of distillers grain used in animal feed, regulators at the FDA intended to conduct a survey to determine the extent and levels of antibiotic residues in distillers grain. To enable this survey, an analytical method (1) was developed for thirteen antibiotics that belonged to several chemical classes. The surveys conducted by the FDA (2 & 3) using this method revealed several antibiotic residues in the distillers grain by-product used as an animal feed ingredient. This finding caused FDA to look more closely into the situation and prompted further investigations as low levels of antibiotic residues in DG may lead to antimicrobial resistance (AMR) development and become a public health concern. To quantitate antimicrobials in distillers grain at the low levels that could potentially lead to AMR development, FDA needed an analytical method that was significantly more sensitive than the previous screening method. Accordingly, a method was developed and single-laboratory validated that quantifies residues of erythromycin, penicillin G, virginiamycin M1 & virginiamycin S1 (Figure 1) in DG in the range 5 to 1200 ng/g (4) with a LOQ of 5 ng/g for all drugs. This range encompasses the 50-1000 ng/g concentration range FDA/Center for Veterinary Medicine (CVM) was targeting for. The distillers grain matrix used was dry distillers grains with solubles (referred to as DG, henceforth), the most prevalent type of distillers coproduct in the marketplace. Subsequently, the method was approved for a multi-laboratory collaborative validation (MLV) to adequately verify it to be robust and reliable for FDA's compliance and regulatory actions. The objective of this study was to carry out a collaborative MLV of the LC-MS/MS analytical method developed in FDA/CVM laboratory (4) to determine residues of erythromycin, penicillin G, virginiamycin M1 and virginiamycin S1 in DG.