1. 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

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 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)

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

5. 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

6. 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)

7. 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.

8. 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

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

10. 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

11. 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

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

13. High Performance Liquid Chromatography (HPLC)

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
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.

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
-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

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

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 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: ul
Temp: 30℃

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.

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 =
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.

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

22. Fluorescence Detector w/LC

23. 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

24. 1.11ppm Naturally Contaminated Wheat
DONtest
DON molecule is not fluorescent
Detected by absorbance
Samples extracted with water
1.11ppm Naturally Contaminated Wheat

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

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

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.

28. Questions???