Presentation on theme: "Mr. Lanny Smith National Sales Manager, VICAM"— Presentation transcript:
1 Mr. Lanny Smith National Sales Manager, VICAM The use of Liquid Chromatography (LC) methods for the analysis of Mycotoxins in Ethanol CoproductsMr. Lanny SmithNational Sales Manager, VICAM
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.2
3 Mycotoxins regulated in Foods Worldwide Mycotoxin Number of countriesTotal Aflatoxin 76Aflatoxin B1 61Aflatoxin M1 60Patulin 48Ochratoxin A 37Deoxynivalenol 37Zearalenone 17Fumonisin 6(FOA Food and Nutrition Paper #81)
4 Major Mycotoxins of Interest AflatoxinOchratoxinDeoxynivalenolFumonisinZearalenoneT-2/HT-2CitrininNIV4
5 FDA Acceptable Limits for Aflatoxin in Animal Feed/Ingredients Aflatoxin levelFinishing beef cattleCorn and peanut product300 ppbBeef cattle, swine or poultryCottonseed mealFinishing swine of 100 lbs. or greaterCorn and peanut products200 ppbBreeding beef cattle, breeding swine, or mature poultry100 ppbImmature animalsAnimal feeds and ingredients, excluding cottonseed meal20 ppbDairy animals, for animal species or uses not specified above, or when the intended use is not knownAnimal feeds and ingredients
6 FDA Acceptable Limits for Fumonisin in Animal Feed/Ingredients Animal or ClassLevels in Corn & Corn By-productsFeed Factor (Fraction of corn or corn by-product mixed into the total ration)Fumonisin Levels in Finished FeedsHorse (includes asses, zebras and onagers)50.21RabbitCatfish200.510SwineRuminant, Poultry & Mink Breeding Stock (includes laying hens, roosters, lactating dairy cows and bulls)3015Ruminants (cattle, sheep, goats and other ruminants that are >3 months old and fed for slaughter60Mink (fed for pelt production)Poultry (turkeys, checkens, ducklings and other poultry fed for slaughter)10050All others (includes dogs and cats)
7 FDA Recommended Maximum FDA Acceptable Limits for Vomitoxin (DON) in Animal Feed/IngredientsFDA Recommended MaximumVomitoxin (DON) Description of Destination or End-Use1 ppmFinished wheat products for human consumption.5 ppmGrain 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 ppmGrain 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.
8 What happens when inbound corn contains 5 ppb aflatoxin contamination? Corn to Ethanol Co-Products – Mycotoxins concentrate up to 3x in coproductsDistillers Dried Grains with Solubles (DDGS)Distillers Dried Grain (DDG)Condensed Distillers Solubles (CDS)What happens when inbound corn contains 5 ppb aflatoxin contamination?Inbound CornEthanolFermentationDDGDDGSCDG15 +ppb5 ppb
9 VICAM VICAM AflaTest® introduced, patented in 1987 Immunoaffinity column chromatographyVersatile – used with fluorometer, HPLC, UPLC & LC/MS/MSSingle or multiple mycotoxin detectionSimple, safe methods for field or laboratory
11 Sample Analysis Step by Step Process for fluorometry, HPLC, UPLC or LC/MS/MS detection ExtractFilter & DilutePour Over ColumnRinse & EluteFluorometerHPLC or UPLCLC/MS/MS
12 Mycotoxin Analysis Using LC Greater sensitivityAble to deal with complex sample typesExisitng methods supporting mycotoxin detectionEthanol plant laboratory already understands and uses LCGood fit for routine detection of toxins in corn, DDG, DDGS and CDS
14 Samples preparation the same as for fluorometer Blend a sample with extraction solutionFilter through fluted filter paperDilute with water or buffer solutionFilter through microfiber filter paperPass 10mL extract through IACMake sure to pass through drop by drop (about 1-2 drops/secondWash column with water or bufferElute with Methanol or other Eluting solution at rate of 1 drop per 2 seconds or slowerThe more sample that is passed through the column, the larger peak you will see on the HPLC and lower limits of detection will be possible.
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
16 Making Your HPLC System Ready for Mycotoxin Testing Aflatoxins-Post column chemical or photochemical reactor and detector-PHREDOchratoxinFluorescence detectorFumonisinPost column derivatization with OPA and 2MEVomitoxin (DON)-Absorbance detector (DON has no natural fluorescence)T2/HT2-Derivatize with 1-AN-Fluorescence or absorbance detection
17 AflaTestThe 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) TFA4.3 ppb naturally contaminated aflatoxin corn sample
18 Post-Column PHRED for Aflatoxin Detection with LC Example：The following HPLC chromatogram shows quantification of total aflatoxin (G2、G1、 B2、B1) with PhCR post columnDerivatization following AflaTest® immunoaffinity column for sample clean up.HPLC Conditions:Column: Biosil 5u Pro-ODS-U4.6mm x 150mmMobile Phase: Methanol : H2O = 45 : 55Flow Rate: 1ml/minFluorescence Detector: EX 440nm EM365nmPost Column derivatization: PhCR-02525Standard Solution: Aflatoxin G2、G1、B2、B1= 10, 3, 10, 3 ppb in MethanolInjection: ulTemp: 30℃
19 PHRED Post Column Photochemical Reactor PhCR photochemical reactorPhCR 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.
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 methanol26ppb (10B1:3B2:10G1:3G2ng/g) X 1g = 26ng ÷ 0.26ng/µl standard =52ppb (20B1:6B2:20G1:6G2ng/g) X 1g = 52ng ÷ 2.6ng/µl standard =20µl standard added to 980µl methanol104ppb (40B1:12B2:40G1:12G2ng/g) X 1g = 104ng ÷ 2.6ng/µl standard =40µl standard added to 960µl methanolWe 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.
21 OchraTest Dilute extract with PBS based buffers First column wash with PBS bufferOchratoxin molecule is naturally fluorescent20ppb Ochratoxin A spiked corn sample
23 FumoniTest Dilute extract with PBS buffers Fumonisin does NOT naturally fluoresce or absorbDerivatized with OPA and 2ME to make fluorescentFumonisins B1, B3 and B21.73 ppm fumonisin contaminated corn sample
24 1.11ppm Naturally Contaminated Wheat DONtestDON molecule is not fluorescentDetected by absorbanceSamples extracted with water1.11ppm Naturally Contaminated Wheat
25 ZearalaTest Zearalenone naturally fluoresces and absorbs Cross reactive with zearalenone, zearalanone, alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol1.0 ppm spiked zearalenone sample
26 Multiple Mycotoxin Detection Simultaneous detection of multiple mycotoxins by LC or LC/MS/MSAflaOchra HPLCAOZDON/NIVT2/HT-2Myco 6in1+AflatoxinOchratoxinFumonisinZearalenoneVomitoxin (DON)NivalenolT-2HT-2
27 SummaryHPLC systems at the ethanol facility may be outfitted for mycotoxin detectionIncreased productivity of LC unitDecreased overall cost of mycotoxin monitoringBetter overall mycotoxin monitoring with single and multiple mycotoxin detection capabilities.