LC-MS/MS Determination of Antibiotic Residues in Distillers Grains: Method Modification.

BACKGROUND: Antibiotics are used in ethanol production to discourage undesirable bacteria growth. To determine if antibiotic residues remain in the distillers grain (DG) byproduct, which is used as an animal food ingredient, the U.S. Food and Drug Administration/Center for Veterinary Medicine previously developed an LC-MS/MS method to detect residues of erythromycin A, penicillin G, virginiamycin M1, and virginiamycin S1 in DG to enable regulatory decision-making. OBJECTIVE: Erythromycin and penicillin G were quantitated using the stable isotope dilution technique with their isotopically labeled compounds, which are considered optimal internal standards (ISTDs) for quantitative mass spectrometry. With the commercial availability of virginiamycin M1-d2 since then, the objectives of this study were to evaluate the feasibility of its use as it is only doubly deuterated, and to incorporate it in the method to enhance method performance. METHOD: Antibiotic residues were solvent-extracted from DG; the extract was cleaned up by a hexane wash and solid phase extraction (SPE) and analyzed by LC-MS/MS. RESULTS: We established suitability of virginiamycin M1-d2 as an ISTD and incorporated it in the method. For all analytes, accuracy and precision ranged 90 to 102% and 3.8 to 6.8, respectively. CONCLUSIONS: We modified a previously developed LC-MS/MS method that uses virginiamycin M1-d2 as an ISTD to support surveillance studies to determine several drugs in DG. HIGHLIGHTS: Virginiamycin M1-d2 was successfully incorporated into the method for better virginiamycin M1 quantitation. This addition also allowed calibration curves for all analytes to be constructed in solvent, thereby simplifying the method.

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 (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->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.

Antibiotics are used in ethanol production to discourage the growth of bacteria that would result in lower ethanol content and a lower quality product. A survey conducted by the FDA (FY 2010 Nationwide Survey of Distillers Grains for Antibiotic Residues, 2009 [1]) revealed that the residues of these antibiotics can remain in the distillers grains (DG) by-product, which is used as an animal feed ingredient. The low levels of antibiotic residues in DG could be a public health concern, as they could lead to antimicrobial resistance. To enable the quantitative determination of these antibiotics (erythromycin, penicillin G, virginiamycin M1 and virginiamycin S1), we developed a sensitive LC-MS/MS method. The residues were extracted from distillers grains with a mixture of acetonitrile and buffer followed by acetonitrile. The combined extract was diluted with water and washed with hexane. An aliquot was cleaned up on an Oasis HLB solid phase extraction cartridge. Extracts were analyzed by LC-tandem mass spectrometry. The method was successfully validated using a variety of different matrices such as corn DG, corn & milo DG, and deoiled corn DG. Absolute recoveries of the analytes ranged from 53 to 106%. Accuracy ranged from 90 to 101% based on calibration by matrix standards. The limits of quantitation and relative standard deviation were all satisfactory to support future surveillance studies.

Screening for toxic phorbol esters in jerky pet treat products using LC-MS.

Since 2007, the U.S. FDA's Center for Veterinary Medicine (CVM) has been investigating reports of pets becoming ill after consuming jerky pet treats. Jerky used in pet treats contains glycerin, which can be made from vegetable oil or as a byproduct of biodiesel production. Because some biodiesel is produced using oil from Jatropha curcas, a plant that contains toxic compounds including phorbol esters, CVM developed a liquid chromatography-mass spectrometry (LC-MS) screening method to evaluate investigational jerky samples for the presence of these toxins. Results indicated that the samples analyzed with the new method did not contain Jatropha toxins at or above the lowest concentration tested.

Determination of erythromycin in medicated salmonid fish feed by liquid chromatography and UV spectroscopy.

A simple, robust LC-UV method was developed to assay erythromycin in medicated salmonid feed. In this method, erythromycin was extracted from feed with acetonitrile and water, cleaned up by SPE, evaporated to dryness, reconstituted, and analyzed by LC-UV. The resulting method produced high accuracy, 82-90%, for both salmon and trout feed that represented varied pellet sizes and ingredient amounts. The intraday and interday precisions, at < or = 6 and 5%, respectively, indicated the method's good repeatability. Calibration was linear over the range of drug concentrations typically used in medicated feed. An independent analyst validation further demonstrated the repeatability of the method. The developed method could support the drug approval process for erythromycin in medicated salmonid fish feed.

Multiclass, multiresidue method for the detection of antibiotic residues in distillers grains by liquid chromatography and ion trap tandem mass spectrometry.

The increased production of ethanol in the US has resulted in large amounts of distillers grains (DG) which is an excellent feed supplement for livestock. However, the use of antimicrobials during ethanol fermentation has led to a growing concern over the possibility of their residues remaining in DG. To enable the detection of antimicrobial residues, a robust LC-MS/MS method was developed that included 13 antibiotics of diverse chemistries, ampicillin, penicillin G, tetracycline, oxytetracycline, chlortetracycline, bacitracin A, virginiamycin M1, chloramphenicol, erythromycin A, clarithromycin, tylosin A, monensin A and streptomycin. The residues were extracted with an aqueous solution of EDTA and trichloroacetic acid followed by methanol. The combined extract was subjected to a two-track cleanup and concentration on either hydrophilic polymeric or weak cation exchange solid phase extraction cartridges. The extracts are analyzed by LC/ion trap tandem mass spectrometry. The method was validated in dry DG matrix. Absolute recoveries of the analytes ranged from 50 to 100%. Accuracy ranged from 89 to 111% based on calibration by processed standards. The limits of detection and relative standard deviation are satisfactory to support future surveillance studies. The method was subsequently tested in three different end-products of DG: distillers dry grains, distillers wet grains and distillers grains solubles.