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
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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
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Mycotoxins regulated in Foods
Worldwide
Mycotoxin
Number of countries
Total Aflatoxin
Aflatoxin B1
Aflatoxin M1
Patulin
Ochratoxin A
Deoxynivalenol
Zearalenone
Fumonisin
76
61
60
48
37
37
17
6
(FOA Food and Nutrition Paper #81)
©2011 Waters Corporation
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Major Mycotoxins of Interest








Aflatoxin
Ochratoxin
Deoxynivalenol
Fumonisin
Zearalenone
T-2/HT-2
Citrinin
NIV
©2011 Waters Corporation
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FDA Acceptable Limits for Aflatoxin in
Animal Feed/Ingredients
Animal
Finishing beef
cattle
Beef cattle, swine
or poultry
Finishing swine of
100 lbs. or
greater
Breeding beef
cattle, breeding
swine, or mature
poultry
Feed
Corn and peanut
product
Cottonseed meal
Aflatoxin level
300 ppb
Corn and peanut
products
200 ppb
Corn and peanut
products
100 ppb
300 ppb
Immature animals Animal feeds and 20 ppb
ingredients,
excluding
cottonseed meal
Dairy animals, for Animal feeds and 20 ppb
animal species or ingredients
uses not specified
above, or when
the intended use
is not known
©2011 Waters Corporation
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FDA Acceptable Limits for Fumonisin in
Animal Feed/Ingredients
Animal or Class
Horse (includes
asses, zebras and
onagers)
Feed Factor
Fumonisin Levels
(Fraction of corn in Finished Feeds
or corn by-product
mixed into the
total ration)
5
0.2
1
Rabbit
Catfish
Swine
Ruminant, Poultry &
Mink Breeding Stock
(includes laying
hens, roosters,
lactating dairy cows
and bulls)
5
20
20
30
0.2
0.5
0.5
0.5
1
10
10
15
Ruminants (cattle,
sheep, goats and
other ruminants that
are >3 months old
and fed for slaughter
60
0.5
30
Mink (fed for pelt
production)
60
0.5
30
100
0.5
50
10
0.5
5
Poultry (turkeys,
checkens, ducklings
and other poultry fed
for slaughter)
All others (includes
dogs and cats)
©2011 Waters Corporation
Levels in Corn &
Corn By-products
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FDA Acceptable Limits for Vomitoxin
(DON) in Animal Feed/Ingredients
FDA Recommended
Maximum
©2011 Waters Corporation
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.
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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?
DDG
5 ppb
DDGS
Inbound Corn
Fermentation
15 +ppb
CDG
Ethanol
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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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
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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Vicam Solution for Testing Mycotoxins
LC/MS/MS
Myco6in1
HPLC/UPLC/MS
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
©2011 Waters Corporation
OchraTest
OchraTest WB
T-2Test HPLC
T-2/HT-2 HPLC
ZearalaTest
ZearalaTest WB
Myco6in1+
Fluorometeric
Tests
AflaTest
Afla B
Afla M1 FL+
FumoniTest
FumoniTestStrips
200
Qualitative
OchraTest
AflaCheck
ZearalaTest
DONCheck
Quantitative Strips
Afla-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
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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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
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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High Performance Liquid Chromatography
(HPLC)
©2011 Waters Corporation
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Samples preparation the same as for
fluorometer
Blend a sample
with extraction
solution
Pass 10mL
extract
through IAC
©2011 Waters Corporation
Filter through
fluted filter paper
Dilute with water
or buffer solution
Filter through
microfiber filter
paper
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
14
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
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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
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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
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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 c
Derivatization following AflaTest® immunoaffinity column for sample clean up.
HPLC Conditions:
Column: Biosil 5u Pro-ODS-U
4.6mm x 150mm
13.105 5403234
150
Mobile Phase: Methanol : H2O = 45 : 55
mv
Flow Rate: 1ml/min
11.193 3717477
100
9.692 2034926
Fluorescence Detector: EX 440nm EM365nm
Post Column derivatization: PhCR-02525
Standard Solution: Aflatoxin G2、G1、B2、B1
= 10, 3, 10, 3 ppb in Methanol
8.542 1637163
50
7.703
67154
6.575
59095
6.020
82910
7.232
34709
0
0
5
10
minutes
15
20
Injection: 20-100ul
Temp: 30℃
©2011 Waters Corporation
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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
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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
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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
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Fluorescence Detector w/LC
©2011 Waters Corporation
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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
©2011 Waters Corporation
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DONtest

DON molecule is not fluorescent

Detected by absorbance

Samples extracted with water
1.11ppm Naturally Contaminated Wheat
©2011 Waters Corporation
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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
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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
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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
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Questions???
©2011 Waters Corporation
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©2011 Waters Corporation
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