©2011 waters corporation 1 the use of liquid chromatography (lc) methods for the analysis of...
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©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
Mycotoxin Number of countries
Total Aflatoxin 76Aflatoxin B1 61Aflatoxin M1 60Patulin 48Ochratoxin A 37Deoxynivalenol 37Zearalenone 17Fumonisin 6
©2011 Waters Corporation 4
Major Mycotoxins of Interest
Aflatoxin Ochratoxin Deoxynivalenol Fumonisin Zearalenone T-2/HT-2 Citrinin NIV
©2011 Waters Corporation 5
Animal Feed Aflatoxin levelFinishing beef cattle
Corn and peanut product
300 ppb
Beef cattle, swine or poultry
Cottonseed meal 300 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 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
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 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 5 0.2 1Catfish 20 0.5 10Swine 20 0.5 10Ruminant, Poultry & Mink Breeding Stock (includes laying hens, roosters, lactating dairy cows and bulls)
30 0.5 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
Poultry (turkeys, checkens, ducklings and other poultry fed for slaughter)
100 0.5 50
All others (includes dogs and cats)
10 0.5 5
©2011 Waters Corporation 7
FDA Recommended Maximum
Vomitoxin (DON) Description of Destination or End-Use
1 ppmFinished 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 TestsAflaTestAfla BAfla M1 FL+FumoniTestFumoniTest 200OchraTestZearalaTest
LC/MS/MSMyco6in1
Qualitative StripsAflaCheckDONCheck
Quantitative Strips
Afla-VDON-VFumo-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
Fluorometer HPLC or UPLC LC/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 (G2 、 G1、 B2 、 B1) with PhCR post column Derivatization following AflaTest® immunoaffinity column for sample clean up.
mv
minutes0 5 10 15 20
0
50
100
150
6.020 82910 6.575 59095 7.232 34709 7.703 67154
8.542 1637163
9.692 2034926
11.193 3717477
13.105 5403234
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℃
©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
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