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

Hemakanthi de Alwis FDA Center for Veterinary Medicine Office of Research 07-31-2018 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)

U.S. DG production U.S. Fuel ethanol co-products 40,000 45,000 Corn Gluten Meal Corn Gluten Feed DG Metric tons Metric tons Thousand Thousand 0 0

2 Reference: U.S. Grains Council, DDGS User Handbook, 4th Edition Dry-Grind Ethanol Production

Corn Grind Distillation Ethanol

Whole stillage Cook Centrifuge Thin stillage Liquefy Enzyme Saccharify Centrifuge Corn Oil

Evaporator Yeast Fermentation Distillers Wet Grains Distillers Solubles CO2 DDGS Dryer DDGS Reduced-oil DDGS Wet DG 46% 30%

Co-products Reference: U.S. Grains Council, DDGS User Handbook, 4th Edition 3 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. U.S. DG Consumption 100% Swine Poultry

Dairy

Beef 0% 40,000 Export and Domestic DG Use Export q About 70% of DG is used Metric tons domestically. Remainder is Domesc exported. Thousand 0

4 Reference: U.S. Grains Council, DDGS User Handbook, 4th Edition in DG? q Antibiotics are used to control bacterial contamination during the ethanol fermentation process

5 Dry-Grind Ethanol Production

Corn Grind Distillation Ethanol

Whole stillage Cook Centrifuge Thin stillage Liquefy Enzyme Saccharify Centrifuge Corn Oil

Evaporator Yeast Fermentation Distillers Wet Grains Distillers Solubles CO2 DDGS Dryer DDGS Reduced-oil DDGS Wet DG 46% 30%

Co-products Reference: U.S. Grains Council, DDGS User Handbook, 4th Edition 6 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 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, , , , , , , , A, 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, 3076-3084, 2010 8 Nationwide Surveys q In 2008 & 2010, CVM coordinated two nationwide surveys using our method.

Ref: www.fda.gov

Any antibiotic residues in DG? 9 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 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, “Effects of low concentrations of erythromycin, penicillin, and virginiamycin penicillin, and virginiamycin can on bacterial resistance development in select for resistant Campylobacter vitro”, B. Ge, K. J. Domesle, Q. Yang, S. R. Young, C. L. Rice-Trujillo, S. M. Bodeis & Enterococcus variants. 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 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

1,600 Corn Distillers Oil kg Mllion

0

12 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 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 hps://www.fda.gov/downloads/ScienceResearch/FieldScience/UCM273418.pdf

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, 2017 14 Validated method

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

LOQ Inter-day Accuracy by different DG Compound Inter-day Accuracy (ng/g) matrices Reduced- Reduced- 100 ng/ 1000 All Corn Corn & 10 ng/g oil DG, oil DG, g ng/g levels DG Milo DG Source 1 Source 2 (n=6) (n=6) (n=8) (n=8) (n=8) (n=24) (n=6) (n=6)

Erythromycin A 97 100 103 100 5 102 98 100 100

Penicillin G 107 100 96 101 5 100 103 102 98

Virginiamycin M1 88 91 94 91 5 92 90 97 85

Virginiamycin S1 85 87 97 90 5 88 86 100 87

q Accuracy, precision, limit of quantitation (LOQ), recovery, and correlation coefficient for all matrices are all acceptable per guidelines 16 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 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 Acknowledgments

David Heller Kaleb Duelge Upul Nishshanka Cristina Nochetto Philip Kijak

19 Thank you

Questions?

20 21 Analyte Recovery Accuracy & precision (in parenthesis) LOQ2 (µg/ r1 g) 0.01 µg/ 0.10 µg/g (n= 0.01 µg/g 1.0 µg/g 1.0 µg/g (n= 8) All levels (n=24) g (n= 8) 8)

Erythromycin A 0.999 0.0025 57 ± 7.8 57 ± 1.9 97 (3.3) 100 (1.9) 103 (3.6) 100 (3.9)

Penicillin G 0.998 0.0025 53 ± 6.6 59 ± 1.4 107 (4.2) 100 (2.3) 96 (2.1) 101 (5.4)

Virginiamycin M1 0.999 0.0025 106 ± 13.3 78 ± 1.7 88 (6.9) 91 (4.8) 94 (8.9) 91 (7.4)

Virginiamycin S1 0.998 0.005 78 ± 13.5 69 ± 0.7 85 (4.9) 87 (8.7) 97 (9.3) 90 (9.9)

www.fda.gov 22