Mr. Lanny Smith National Sales Manager, VICAM The use of Liquid Chromatography (LC) methods for the analysis of Mycotoxins in Ethanol Coproducts Mr. Lanny Smith National Sales Manager, VICAM

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

Mycotoxins regulated in Foods Worldwide Mycotoxin Number of countries Total Aflatoxin 76 Aflatoxin B1 61 Aflatoxin M1 60 Patulin 48 Ochratoxin A 37 Deoxynivalenol 37 Zearalenone 17 Fumonisin 6 (FOA Food and Nutrition Paper #81)

Major Mycotoxins of Interest Aflatoxin Ochratoxin Deoxynivalenol Fumonisin Zearalenone T-2/HT-2 Citrinin NIV 4

FDA Acceptable Limits for Aflatoxin in Animal Feed/Ingredients Aflatoxin level Finishing beef cattle Corn and peanut product 300 ppb Beef cattle, swine or poultry Cottonseed meal Finishing swine of 100 lbs. or greater Corn and peanut products 200 ppb Breeding beef cattle, breeding swine, or mature poultry 100 ppb Immature animals Animal 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

FDA Acceptable Limits for Fumonisin in Animal Feed/Ingredients Animal or Class Levels 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) 5 0.2 1 Rabbit Catfish 20 0.5 10 Swine Ruminant, Poultry & Mink Breeding Stock (includes laying hens, roosters, lactating dairy cows and bulls) 30 15 Ruminants (cattle, sheep, goats and other ruminants that are >3 months old and fed for slaughter 60 Mink (fed for pelt production) Poultry (turkeys, checkens, ducklings and other poultry fed for slaughter) 100 50 All others (includes dogs and cats)

FDA Recommended Maximum   FDA Acceptable Limits for Vomitoxin (DON) in Animal Feed/Ingredients 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.

What happens when inbound corn contains 5 ppb aflatoxin contamination? 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

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

Vicam Solution for Testing Mycotoxins LC/MS/MS Myco6in1 HPLC/UPLC/MS Fluorometeric Tests AflaTest Afla B Afla M1 FL+ FumoniTest FumoniTest 200 OchraTest ZearalaTest OchraTest OchraTest WB T-2Test HPLC T-2/HT-2 HPLC ZearalaTest ZearalaTest WB Myco6in1+ 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 Remark on wide-bore 3mL Qualitative Strips AflaCheck DONCheck Quantitative Strips Afla-V DON-V Fumo-V 10

Sample Analysis Step by Step Process for fluorometry, HPLC, UPLC or LC/MS/MS detection Extract Filter & Dilute Pour Over Column Rinse & Elute Fluorometer HPLC or UPLC LC/MS/MS

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

High Performance Liquid Chromatography (HPLC)

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 The 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.

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

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 Vomitoxin (DON) -Absorbance detector (DON has no natural fluorescence) T2/HT2 -Derivatize with 1-AN -Fluorescence or absorbance detection

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

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 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-02525 Standard Solution: Aflatoxin G2、G1、B2、B1 = 10, 3, 10, 3 ppb in Methanol Injection: 20-100ul Temp: 30℃

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.

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 = 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.

OchraTest Dilute extract with PBS based buffers First column wash with PBS buffer Ochratoxin molecule is naturally fluorescent 20ppb Ochratoxin A spiked corn sample

Fluorescence Detector w/LC

FumoniTest Dilute extract with PBS buffers Fumonisin does NOT naturally fluoresce or absorb Derivatized with OPA and 2ME to make fluorescent Fumonisins B1, B3 and B2 1.73 ppm fumonisin contaminated corn sample

1.11ppm Naturally Contaminated Wheat DONtest DON molecule is not fluorescent Detected by absorbance Samples extracted with water 1.11ppm Naturally Contaminated Wheat

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

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

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.

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