An LC-MS/MS method to determine antibiotic residues in distillers grains

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1 An LC-MS/MS method to determine antibiotic residues in distillers grains Hemakanthi de Alwis FDA Center for Veterinary Medicine Office of Research

2 Distillers grain (DG) q DG is a major co-product of the corn ethanol industry q DG production has increased exponentially over the last decade q 2016/17 year: A record ~41 million tons of DG q Product of dry-grind milling process (wet milling process is only ~10% fuel ethanol production) 0 U.S. DG production U.S. Fuel ethanol co-products 45,000 Thousand Metric tons Thousand Metric tons 40,000 Corn Gluten Meal Corn Gluten Feed DG 0 Reference: U.S. Grains Council, DDGS User Handbook, 4th Edition 2

3 Dry-Grind Ethanol Production Corn Grind Distillation Ethanol Whole stillage Cook Centrifuge Liquefy Thin stillage Enzyme Centrifuge Saccharify Yeast Fermentation CO2 Wet DG 30% DDGS 46% Distillers Wet Grains Corn Oil Evaporator Distillers Solubles Dryer DDGS Reduced-oil DDGS Co-products Reference: U.S. Grains Council, DDGS User Handbook, 4th Edition 3

4 Distillers grain as animal feed q DG is rich in proteins, fats and minerals. Hence, an excellent feed supplement for livestock. q Majority is fed to beef and dairy cattle, swine, and poultry, in the US. 100% U.S. DG Consumption Poultry Swine Dairy 0% Thousand Metric tons 40,000 Beef Export and Domestic DG Use Export Domes,c q About 70% of DG is used domestically. Remainder is exported. 0 Reference: U.S. Grains Council, DDGS User Handbook, 4th Edition 4

5 Antibiotics in DG? q Antibiotics are used to control bacterial contamination during the ethanol fermentation process 5

6 Dry-Grind Ethanol Production Corn Grind Distillation Ethanol Whole stillage Cook Centrifuge Liquefy Thin stillage Enzyme Centrifuge Saccharify Yeast Fermentation CO2 Wet DG 30% DDGS 46% Distillers Wet Grains Corn Oil Evaporator Distillers Solubles Dryer DDGS Reduced-oil DDGS Co-products Reference: U.S. Grains Council, DDGS User Handbook, 4th Edition 6

7 Antibiotics in DG? q Antibiotics are used to control bacterial contamination during the ethanol fermentation process q FDA/CVM s concerns: Are there any antibiotic residues remaining in DG that is fed to food-animals? Do they pose any potential risks to animals and humans? What antibiotics are being used? CVM needed: To conduct surveillance on antibiotics in DG An analytical method for surveillance 7

8 We developed an LC-MS/MS Method q Developed in 2008 q Used an ion trap tandem mass spectrometer q Compounds: Penicillin G, ampicillin, virginiamycin M1, streptomycin, erythromycin, chloramphenicol, tetracycline, oxytetracycline, tylosin, chlortetracycline, bacitracin A, clarithromycin and monensin De Alwis, H., Heller, D.N., Multiclass, Multiresidue Method for the Determination of Antibiotic Residues in Distillers Grains by Liquid Chromatography and Ion Trap Tandem Mass Spectrometry, Journal of Chromatography A., vol 1217, ,

9 Nationwide Surveys q In 2008 & 2010, CVM coordinated two nationwide surveys using our method. Ref: Any antibiotic residues in DG? 9

10 CVM s concern on antimicrobial resistance development (AMR) q Survey reported detecting several residues in DG: erythromycin, virginiamycin & penicillin G q Questions: Possible rise of antimicrobial-resistant bacteria due to exposure to these antibiotics What antibiotic levels would be relevant for any AMR development? 10

11 Microbiological work q CVM microbiologists examined the effects of these antibiotics on bacterial resistance development in vitro. q Low concentrations (0.1, 0.25, and 0.5 µg/ml) of erythromycin, penicillin, and virginiamycin can select for resistant Campylobacter & Enterococcus variants. Effects of low concentrations of erythromycin, penicillin, and virginiamycin on bacterial resistance development in vitro, B. Ge, K. J. Domesle, Q. Yang, S. R. Young, C. L. Rice-Trujillo, S. M. Bodeis Jones, S. A. Gaines, M. W. Keller, X. Li, S. A. Piñeiro, B. M. Whitney, H. C. Harbottle & J. M. Gilbert, Scientific Reports, Vol 7, p 1-11 (2017) Need a more sensitive & quantitative method to detect residues at low levels important for AMR 11

12 Analytical method needed q A quantitative and more sensitive method than the previous screening method q Penicillin G, erythromycin, virginiamycin M1 & virginiamycin S1 q Variety of DG matrices: DDGS Reduced-oil DDGS Corn-based or corn/milo-based DDGS Corn Distillers Oil Mllion kg 1,

13 Developed LC-MS/MS method Distillers grain 1. Extract with buffer & acetonitrile, centrifuge & transfer supernatant 2. Repeat extraction with ACN & centrifuge 3. Combine supernatants & dilute with water Clean-up extract: Hexane wash & solid phase extraction Analysis: Liquid chromatography -Tandem mass spectrometry (LC-MS/MS) 13

14 Single-Lab Validation q Shimadzu LC/AB Sciex API 4000 triple quadrupole tandem mass spectrometer q Method validated using FDA Food and Veterinary Medicine Program Guidelines for the Validation of Chemical Methods h8ps:// Kaleb J. Duelge, Upul Nishshanka, Hemakanthi G. De Alwis, An LC-MS/MS method for the determination of antibiotic residues in distillers grains at levels of concern for antimicrobial resistance development, Journal of Chromatography B., vol 1053, 81-86,

15 Validated method A representative chromatogram of a DG sample fortified with the drugs at 10.0 ng/g (ppb) 15

16 Method Attributes Compound Inter-day Accuracy LOQ (ng/g) Inter-day Accuracy by different DG matrices 10 ng/g 100 ng/ g 1000 ng/g All levels (n=8) (n=8) (n=8) (n=24) Corn DG (n=6) Corn & Milo DG (n=6) Reducedoil DG, Source 1 (n=6) Reducedoil DG, Source 2 (n=6) Erythromycin A Penicillin G Virginiamycin M Virginiamycin S q Accuracy, precision, limit of quantitation (LOQ), recovery, and correlation coefficient for all matrices are all acceptable per guidelines 16

17 Multi-Laboratory Validation q Multi-lab validation of the method is underway q Once successfully validated, this method could serve as a regulatory method capable of being used for compliance actions for distillers grains containing the contaminants 17

18 Summary q We developed a screening LC-MS/MS method that helped conduct two nationwide surveys for the presence of antibiotics in DG q Survey findings led to concerns on antimicrobial resistance development q CVM microbiologists examined the effects and levels of the antibiotics on AMR development q Erythromycin, penicillin, and virginiamycin can select for resistant Campylobacter & Enterococcus variants. q To detect residues at low concentrations important for AMR, we developed and validated a quantitative and more sensitive method q Method is now in the process of multi-laboratory validation 18

19 Acknowledgments David Heller Kaleb Duelge Upul Nishshanka Cristina Nochetto Philip Kijak 19

20 Thank you Questions? 20

21 21

22 Analyte r1 LOQ2 (µg/ g) Recovery Accuracy & precision (in parenthesis) 0.01 µg/g 1.0 µg/g 0.01 µg/ 0.10 µg/g (n= 1.0 µg/g (n= 8) g (n= 8) 8) All levels (n=24) Erythromycin A ± ± (3.3) 100 (1.9) 103 (3.6) 100 (3.9) Penicillin G ± ± (4.2) 100 (2.3) 96 (2.1) 101 (5.4) Virginiamycin M ± ± (6.9) 91 (4.8) 94 (8.9) 91 (7.4) Virginiamycin S ± ± (4.9) 87 (8.7) 97 (9.3) 90 (9.9) 22

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