Hemakanthi de AlwisFDA Center for Veterinary Medicine

Office of Research07-31-2018

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

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Distillers grain (DG)q DG is a major co-product of the corn ethanol industry q DG production has increased exponentially over the last

decadeq  2016/17 year: A record ~41 million tons of DG

Reference: U.S. Grains Council, DDGS User Handbook, 4th Edition

U.S. Fuel ethanol co-products

Thou

sand

Met

ric to

ns

45,000 Corn Gluten MealCorn Gluten FeedDG

0

Thou

sand

Met

ric to

ns

40,000U.S. DG production

q Product of dry-grind milling process (wet milling process is only ~10% fuel ethanol production)

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Grind

Cook

Liquefy

Saccharify

Distillation

Centrifuge

Evaporator

Dryer

Whole stillage

Thin stillage

Ethanol

DDGS Reduced-oil DDGS

CO2

Distillers Solubles

Distillers Wet Grains

Corn

Yeast

Enzyme

Dry-Grind Ethanol Production

DDGS46%Wet DG

30%

Co-products

Fermentation

Corn OilCentrifuge

Reference: U.S. Grains Council, DDGS User Handbook, 4th Edition

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U.S. DG Consumption

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.

Beef

Dairy

Swine Poultry100%

0%

Export and Domestic DG Use

0

Thou

sand

Met

ric to

ns

40,000

Reference: U.S. Grains Council, DDGS User Handbook, 4th Edition

q About 70% of DG is used domestically. Remainder is exported.

Export

Domes,c

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Antibiotics in DG?q Antibiotics are used to control bacterial

contamination during the ethanol fermentation process

6

Grind

Cook

Liquefy

Saccharify

Distillation

Centrifuge

Evaporator

Dryer

Whole stillage

Thin stillage

Ethanol

DDGS Reduced-oil DDGS

CO2

Distillers Solubles

Distillers Wet Grains

Corn

Yeast

Enzyme

Dry-Grind Ethanol Production

DDGS46%Wet DG

30%

Co-products

Fermentation

Corn OilCentrifuge

Reference: U.S. Grains Council, DDGS User Handbook, 4th Edition

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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

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We developed an LC-MS/MS Method

q Developed in 2008q Used an ion trap tandem mass

spectrometerq 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, 3076-3084, 2010

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Nationwide Surveysq  In 2008 & 2010, CVM coordinated two nationwide surveys

using our method.

Any antibiotic residues in DG?

Ref:www.fda.gov

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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?

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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.

Need a more sensitive & quantitative method to detect residues at low levels important for AMR

“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)

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Analytical method neededq A quantitative and more sensitive method

than the previous screening methodq Penicillin G, erythromycin, virginiamycin

M1 & virginiamycin S1q Variety of DG matrices:

DDGSReduced-oil DDGS Corn-based or corn/milo-based DDGS

Corn Distillers Oil

0

Mlli

on k

g

1,600

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Developed LC-MS/MS methodDistillers grain

1.  Extract with buffer & acetonitrile, centrifuge & transfer supernatant

2.  Repeat extraction with ACN & centrifuge3.  Combine supernatants & dilute with water

Clean-up extract: Hexane wash & solid phase extraction

Analysis: Liquid chromatography-Tandem mass spectrometry

(LC-MS/MS)

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Single-Lab Validationq Shimadzu LC/AB Sciex API 4000 triple quadrupole tandem

mass spectrometerq Method validated using FDA Food and Veterinary Medicine

Program Guidelines for the Validation of Chemical Methods

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

h8ps://www.fda.gov/downloads/ScienceResearch/FieldScience/UCM273418.pdf

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Validated method

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

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Method Attributes

Compound   Inter-day Accuracy   LOQ

(ng/g)  Inter-day Accuracy by different DG

matrices

   10 ng/g 100 ng/

g1000 ng/g

All levels      

Corn DG

(n=6)

Corn & Milo DG

(n=6)

Reduced-oil DG,

Source 1 (n=6)

Reduced-oil DG,

Source 2 (n=6)(n=8) (n=8) (n=8) (n=24)

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

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Multi-Laboratory Validation

q Multi-lab validation of the method is underwayq Once successfully validated, this method could

serve as a regulatory method capable of being used for compliance actions for distillers grains containing the contaminants

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Summaryq 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

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Acknowledgments

David HellerKaleb DuelgeUpul NishshankaCristina NochettoPhilip Kijak

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Thank you

Questions?

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22www.fda.gov

Analyte

r1 LOQ2 (µg/g)

Recovery Accuracy & precision (in parenthesis)

0.01 µg/g 1.0 µg/g 0.01 µg/g (n= 8)

0.10 µg/g (n= 8) 1.0 µg/g (n= 8) All levels (n=24)

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)