We think you have liked this presentation. If you wish to download it, please recommend it to your friends in any social system. Share buttons are a little bit lower. Thank you!
Presentation is loading. Please wait.
Published byAiyana Lilly
Modified about 1 year ago
©2011 Waters Corporation 1 The use of Liquid Chromatography (LC) methods for the analysis of Mycotoxins in Ethanol Coproducts Mr. Lanny Smith National Sales Manager, VICAM
©2011 Waters Corporation 2 Mycotoxins of economic, health and agricultural significance Mycotoxins are metabolic products of food spoilage fungi that induce toxic responses when consumed by animals or people. Hundreds of mycotoxins have been identified; They will fall into many different chemical classes, and induce a wide variety of toxic responses.
©2011 Waters Corporation 3 (FOA Food and Nutrition Paper #81) Mycotoxins regulated in Foods Worldwide MycotoxinNumber of countries Total Aflatoxin76 Aflatoxin B161 Aflatoxin M160 Patulin48 Ochratoxin A37 Deoxynivalenol37 Zearalenone17 Fumonisin6
©2011 Waters Corporation 4 Major Mycotoxins of Interest Aflatoxin Ochratoxin Deoxynivalenol Fumonisin Zearalenone T-2/HT-2 Citrinin NIV
©2011 Waters Corporation 5 AnimalFeedAflatoxin level Finishing beef cattle Corn and peanut product 300 ppb Beef cattle, swine or poultry Cottonseed meal300 ppb Finishing swine of 100 lbs. or greater Corn and peanut products 200 ppb Breeding beef cattle, breeding swine, or mature poultry Corn and peanut products 100 ppb Immature animalsAnimal feeds and ingredients, excluding cottonseed meal 20 ppb Dairy animals, for animal species or uses not specified above, or when the intended use is not known Animal feeds and ingredients 20 ppb FDA Acceptable Limits for Aflatoxin in Animal Feed/Ingredients
©2011 Waters Corporation 6 FDA Acceptable Limits for Fumonisin in Animal Feed/Ingredients Animal or ClassLevels in Corn & Corn By-products Feed Factor (Fraction of corn or corn by-product mixed into the total ration) Fumonisin Levels in Finished Feeds Horse (includes asses, zebras and onagers) Rabbit50.21 Catfish Swine Ruminant, Poultry & Mink Breeding Stock (includes laying hens, roosters, lactating dairy cows and bulls) Ruminants (cattle, sheep, goats and other ruminants that are >3 months old and fed for slaughter Mink (fed for pelt production) Poultry (turkeys, checkens, ducklings and other poultry fed for slaughter) All others (includes dogs and cats)
©2011 Waters Corporation 7 FDA Recommended Maximum Vomitoxin (DON) Description of Destination or End-Use 1 ppm Finished wheat products for human consumption. 5 ppm Grain and grain by-products destined for swine diets, but not to exceed 20% of the final diet and for other animals (except cattle and chickens), but not to exceed 40% of the final diet. 10 ppm Grain and grain by-products destined for ruminating beef and feedlot cattle older than 4 months and for chickens, but not to exceed 50% of the final diet. FDA Acceptable Limits for Vomitoxin (DON) in Animal Feed/Ingredients
©2009 Waters Corporation | COMPANY CONFIDENTIAL 8 Corn to Ethanol Co-Products – Mycotoxins concentrate up to 3x in coproducts Distillers Dried Grains with Solubles (DDGS) Distillers Dried Grain (DDG) Condensed Distillers Solubles (CDS) What happens when inbound corn contains 5 ppb aflatoxin contamination? Inbound Corn Ethanol Fermentation DDG DDGS CDG 15 +ppb 5 ppb
©2009 Waters Corporation | COMPANY CONFIDENTIAL 9 VICAM VICAM AflaTest® introduced, patented in 1987 Immunoaffinity column chromatography —Versatile – used with fluorometer, HPLC, UPLC & LC/MS/MS —Single or multiple mycotoxin detection —Simple, safe methods for field or laboratory
©2011 Waters Corporation 10 Vicam Solution for Testing Mycotoxins AflaTest AflaTest WB Afla WB SR Afla M1 HPLC AflaOchra HPLC AOZ HPLC CitriTest HPLC DONtest HPLC DONtest WB DON-NIV WB FumoniTest FumoniTest WB Fluorometeric Tests AflaTest Afla B Afla M1 FL+ FumoniTest FumoniTest 200 OchraTest ZearalaTest LC/MS/MS Myco6in1 Qualitative Strips AflaCheck DONCheck Quantitative Strips Afla-V DON-V Fumo-V HPLC/UPLC/MS OchraTest OchraTest WB T-2Test HPLC T-2/HT-2 HPLC ZearalaTest ZearalaTest WB Myco6in1+
©2009 Waters Corporation | COMPANY CONFIDENTIAL 11 Sample Analysis Step by Step Process for fluorometry, HPLC, UPLC or LC/MS/MS detection FluorometerHPLC or UPLCLC/MS/MS Extract Filter & Dilute Pour Over Column Rinse & Elute
©2009 Waters Corporation | COMPANY CONFIDENTIAL 12 Mycotoxin Analysis Using LC Greater sensitivity Able to deal with complex sample types Exisitng methods supporting mycotoxin detection Ethanol plant laboratory already understands and uses LC Good fit for routine detection of toxins in corn, DDG, DDGS and CDS
©2011 Waters Corporation 13 High Performance Liquid Chromatography (HPLC)
©2011 Waters Corporation 14 Samples preparation the same as for fluorometer Blend a sample with extraction solution Filter through fluted filter paper Dilute with water or buffer solution Filter through microfiber filter paper Pass 10mL extract through IAC Make sure to pass through drop by drop (about 1-2 drops/second Wash column with water or buffer Elute with Methanol or other Eluting solution at rate of 1 drop per 2 seconds or slower
©2011 Waters Corporation 15 Difference from fluorometer methods Collect the eluate, but do NOT add developers. Eluate can be diluted with water or dried down and reconstituted in the mobile phase. Then sample is injected onto the HPLC or UPLC
©2011 Waters Corporation 16 Making Your HPLC System Ready for Mycotoxin Testing Aflatoxins -Post column chemical or photochemical reactor and detector -PHRED Ochratoxin -Fluorescence detector Fumonisin -Post column derivatization with OPA and 2ME -Fluorescence detector Vomitoxin (DON) -Absorbance detector (DON has no natural fluorescence) T2/HT2 -Derivatize with 1-AN -Fluorescence or absorbance detection
©2011 Waters Corporation 17 AflaTest The aflatoxin molecule is naturally fluorescent, but must be derivatized to enhance the fluorescence of aflatoxin B1 and G1 with one of the following: (1) post column iodine or bromine (2) Photochemical reactor (PHRED, PhCR), (3) KOBRA cell (4) TFA 4.3 ppb naturally contaminated aflatoxin corn sample
©2011 Waters Corporation 18 Post-Column PHRED for Aflatoxin Detection with LC Example ： The following HPLC chromatogram shows quantification of total aflatoxin (G 2 、 G 1 、 B 2 、 B 1) with PhCR post column Derivatization following AflaTest® immunoaffinity column for sample clean up. HPLC Conditions: Column: Biosil 5u Pro-ODS-U 4.6mm x 150mm Mobile Phase: Methanol : H2O = 45 : 55 Flow Rate: 1ml/min Fluorescence Detector: EX 440nm EM365nm Post Column derivatization: PhCR Standard Solution: Aflatoxin G2 、 G1 、 B2 、 B1 = 10, 3, 10, 3 ppb in Methanol Injection: ul Temp: 30 ℃
©2011 Waters Corporation 19 PHRED Post Column Photochemical Reactor PhCR photochemical reactor PhCR is a UV light that reacts with mycotoxins as a post HPLC column derivatization method for aflatoxin. It is easy to setup and operate. The sample passes through the PHRED unit prior to going through the fluorescence detector.
©2011 Waters Corporation 20 Preparation of aflatoxin standards for HPLC To make standards for HPLC for 10ml (1g equivalent) AflaTest methods only: 2.6ppb (1B1:0.3B2:1G1:0.3G2ng/g) X 1g = 2.6ng ÷ 0.026ng/µl standard = 100µl standard added to 900µl methanol 26ppb (10B1:3B2:10G1:3G2ng/g) X 1g = 26ng ÷ 0.26ng/µl standard = 100µl standard added to 900µl methanol 52ppb (20B1:6B2:20G1:6G2ng/g) X 1g = 52ng ÷ 2.6ng/µl standard = 20µl standard added to 980µl methanol 104ppb (40B1:12B2:40G1:12G2ng/g) X 1g = 104ng ÷ 2.6ng/µl standard = 40µl standard added to 960µl methanol We add 1ml water to all our standards and samples before injecting onto the HPLC to make the solvent for the standards and samples similar to the mobile phase. Instructions for making standards for other mycotoxin products is in each of the respective HPLC instruction manuals.
©2011 Waters Corporation 21 OchraTest Dilute extract with PBS based buffers First column wash with PBS buffer Ochratoxin molecule is naturally fluorescent 20ppb Ochratoxin A spiked corn sample
©2011 Waters Corporation 22 Fluorescence Detector w/LC
©2011 Waters Corporation 23 FumoniTest Dilute extract with PBS buffers Fumonisin does NOT naturally fluoresce or absorb Derivatized with OPA and 2ME to make fluorescent 1.73 ppm fumonisin contaminated corn sample Fumonisins B1, B3 and B2
©2011 Waters Corporation 24 DONtest DON molecule is not fluorescent Detected by absorbance Samples extracted with water 1.11ppm Naturally Contaminated Wheat
©2011 Waters Corporation 25 ZearalaTest Zearalenone naturally fluoresces and absorbs Cross reactive with zearalenone, zearalanone, alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol 1.0 ppm spiked zearalenone sample
©2011 Waters Corporation 26 Multiple Mycotoxin Detection Simultaneous detection of multiple mycotoxins by LC or LC/MS/MS AflaOchra HPLC AOZ DON/NIV T2/HT-2 Myco 6in1+ -Aflatoxin -Ochratoxin -Fumonisin -Zearalenone -Vomitoxin (DON) -Nivalenol -T-2 -HT-2
©2011 Waters Corporation 27 Summary HPLC systems at the ethanol facility may be outfitted for mycotoxin detection Increased productivity of LC unit Decreased overall cost of mycotoxin monitoring Better overall mycotoxin monitoring with single and multiple mycotoxin detection capabilities.
©2011 Waters Corporation 28 Questions???
©2011 Waters Corporation 29
ETHANOL.. Ethanol (ethyl alcohol, grain alcohol) is a clear, colorless liquid with a characteristic, agreeable odor. In dilute aqueous solution, it has.
A practical approach to metabolomics Rob Linforth Food Sciences – Biosciences University of Nottingham.
Animal Science Agriscience Applications. Objective Investigate careers related to the field of animal science Investigate careers related to the field.
Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings PowerPoint ® Slides prepared by Jay Withgott and Heidi Marcum Copyright © 2008.
Enzyme Activity Lab 13 AP Biology (Peroxidase + Hydrogen Peroxide Complex Peroxidase + Water + Oxygen) 2H 2 O 2 2H 2 O + O 2 (gas)
3 Main Factors that affect show animal performance 1.) Genetics – Inherited performance potential the prospect animal has from parents at birth. Ex. Growth.
INDUSTRIAL HYGIENE - SAMPLING OF GASES AND VAPORS UNIVERSITY OF HOUSTON - CLEAR LAKE.
The Importance of Veterinary Toxicology for Animal Agriculture and the Food Supply Birgit Puschner DVM, PhD, Dipl. ABVT California Animal Health and Food.
INDUSTRIAL HYGIENE DIRECT-READING INSTRUMENTS FOR GASES, VAPORS, AND PARTICULATES UNIVERSITY OF HOUSTON - DOWNTOWN.
DINATEC Diversified Nutri-Agri Technologies Inc., Presented by Dr. Martin Moreira Ph. D. A Systems Approach to beneficial yeast inclusion technology.
GAS CHROMATOGRAPHY. Classification based on Mobile Phase Gas Chromatography Gas - solid Gas - liquid Stationary Phase Sample MUST be volatile at temperatures.
Biodiesel Cold Flow Basics Information for Petroleum Distributors, Blenders, and End-Users on Issues Affecting Biodiesel in the Winter Months Prepared.
Chapter 6 Livestock Nutrition Vitamins, Feed Additives and Water.
Can you see?. I like him. When will we go? All or some.
SANITATION THE FOUNDATION OF FOOD SAFETY Retail Meat & Poultry Processing Training Modules Training Modules.
High-Frequency Phrases First 100 Words. The people.
Feeding BioFuels Co-products to Livestock: Challenges to Animal Health NIAA, Sacramento, 4/2/07 Gavin L Meerdink, DVM, D.ABVT.
Activity 48 Follow-up Discuss in your groups the difference in results for each neutralization between pairs. Lack of consistency in drop size Error in.
Environmental Monitoring Considerations Nancy Roscioli, Ph.D. Don Hill and Associates, Inc.
An Introduction to Quality Assurance in Analytical Science Dr Irene Mueller-Harvey Mr Richard Baker Mr Brian Woodget.
Biomass role in Energy consumption. Figure 4-1: Biomass Role in U.S. Energy Consumption.
BL TIER 3 TIER 3 Identify suitable experimental procedures for measuring rates of reactions Identify the factors affecting the rate of a reaction Calculate.
Lets build fluency! The people By the water You and I.
Controls proportionate to the Risks for the European meat Industry Dream or Reality? Controls proportionate to the Risks for the European meat Industry.
REGULATIONS ON AFLATOXIN IN LIVESTOCK FEEDS AND CURRENT STATUS OF ANIMAL FEED REGULATION IN NIGERIA JOSEPH NYAGER (DVM, MSC) DIRECTOR/CVO FEDERAL DEPARTMENT.
Sampling and monitoring the environment Marian Scott Sept 2006.
MCA Test Preparation Part 2. #1: 2/2 = 1 pt. #2: /3 = 1 pt. 26 Points Total.
Animal Science II Principles of Animal Nutrition.
Unit-V -SOFTWARE QUALITY. To develop and deliver robust system, we need a high level of confidence that Each component will behave correctly Collective.
© 2016 SlidePlayer.com Inc. All rights reserved.