©2011 waters corporation 1 the use of liquid chromatography (lc) methods for the analysis of...

©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


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.


(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


Major Mycotoxins of Interest

Aflatoxin Ochratoxin Deoxynivalenol Fumonisin Zearalenone T-2/HT-2 Citrinin NIV


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


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


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


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

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+


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


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


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-Fluorescence detector

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.

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℃


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 = 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.


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

1.73 ppm fumonisin contaminated corn sample

Fumonisins B1, B3 and B2


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.


Questions???

©2011 waters corporation 1 the use of liquid chromatography (lc) methods for the analysis of...

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