An LC-MS/MS method to determine antibiotic …...Analysis: Liquid chromatography-Tandem mass...
Transcript of An LC-MS/MS method to determine antibiotic …...Analysis: Liquid chromatography-Tandem mass...
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
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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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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)