1. Midwest AOAC, St. Paul, MN, June 10, 2003
Development of a Multi-Residue Method for Mycotoxin Analysis in Feeds and Grains
--Multi-residue mycotoxin separation and detection by HPLC and Post-Column Derivatization
--although Aspergillus and generally associated with peanuts
these fungi and those that produce otehr toxins are not host selective and so can cross plant species.
The situation is complicated by microscopic mold may not be visible to the naked eye
also, when infected grains are processed, and visible mold is lost but the toxic metabolites carry over into the finished products.
multi-residue analytical screens for toxins in grain and finished goods are a wiser choice than single-family protocols
Midwest AOAC, St. Paul, MN, June 10, 2003

2. Introduction and background
Two basic methods can screen for or quantitate 5 classes of toxins
Multi-residue method needed for convenience and increased sensitivity
The ruggedness of separation and detection of the mycotoxins has been well-established
Easy to use with current post-column system
Uses a reliable and affordable sample cleanup for feeds, grains
-Current methods either use a separate sample prep and detection method or ELISA for screening (not very sensitive, can lead to false positives)
--Pickering has developed a rugged method for the separation and detection of the mycotoxins
--A cleanup with immunoaffinity columns has been well-demonstrated by Vicam and ATF on beer, wine and sake
--Pickering’s goal was to take this method and adapt a sample prep for grains and feeds
-Many feeds, foods can contain several mycotoxins because of mixing of plant species and resultant combination of mycotoxins
-Samples can range from wheat to beer, from corn to feeds, from rice to sake
-Have several options as to how to prep. the samples. Can use silica clean-up column, or immunoaffinity column
Multi-residue method needed for ease of analyzing several mycotoxins in one run and increased sensitivity
Two basic methods can screen for or quantitate 5 classes of toxins
The ruggedness of separation and detection of the mycotoxins has been well-established
Produce a reliable and affordable sample cleanup for feeds, grains
Easy to use with current post-column system
Many different types of samples can be run on the same method

3. 2 Methods to Analyze 5 classes of Mycotoxins
Trichothecene (DON)
Aflatoxins
(incl. M1 & M2)
Zearalenone
Ochratoxin A
Method 2:
Trichothecene (DON)
Aflatoxins
(incl. M1 & M2)
Fumonisins
(FB1, FB2, FB3)
–alumina eats ochratoxin and fumonisins (not 100%)

4. Post Column Set Up PCX 5200 with duplex pump, 2 ml reactor
Simplex pump will also be sufficient, but requires more conversion time
PCX Control software
Photochemical reactor (Needed for Aflatoxins Only)
UV detector
Fluorescence detector
-- Duplex system with 2 OPA panels is ideal so there is no contamination between the Iodine reagent and the OPA reagent
-However, simplex system will work fine, but requires excessive flushing to remove Iodine from panel for OPA method and vice-versa
--Also, an Aflatoxin system should work fine. This contains a 1.4 ml reactor. The smaller reactor should work with no problem. Most likely the 70°C listed in the literature will not be sufficient, however 90°C should probably work well.
-- PCX Control software connected to PCX via serial cable to facilitate pump control

5. Flow Paths of Post-Column Systems
Method 1
Method 2

6. Concentration (animal feed) Concentration (humans)
Maximum Levels
Analyte
Species of Fungus
Concentration (animal feed)
Concentration (humans)
DON
Fusarium
5 ppm, 10 ppm for chickens
1 ppm
Fumonisin
5 ppm – horses
2 ppm (FB1+FB2+FB3)
Aflatoxin
Aspergilllus
50 ppb – dairy feed
2 ppb for B1
4 ppb for B1+B2+G1+G2
Ochratoxin A
Aspergillus
3 ppb
Zearalenone
N/A
--Can easily detect these values with post-column method
--Took lowest values listed on FDA’s Guidance for Industry “Fumonisin Levels in Human Foods and Animal Feeds”, November 2001
--Took lowest values listed on USDA (GIPSA)’s Fact Sheet on Vomitoxin in wheat, October 1996
--Took lowest Aflatoxin values for humans from EC’s Official Journal of the European Communities “Commission Regulation (EC) No 1525/98, July 1998
--Took lowest Ochratoxin A values for humans from EC’s Official Journal of the European Communities “Commission Regulation (EC) No 472/2002, March 2002
--N/A values for Zearalenone are per Darsa Siantar, ATTB, no established limits

7. Sample Preparation and Cleanup Method 1
Aflatoxin, DON, Zearalenone
ACN : H2O (84:16) 100 mL
Add 25 g of ground sample
C18 : Alumina (1:1)
Fill column with 1.5g
Filter 6 mL of extract
Inject
Note: for increased DON response, evaporate and reconstitute in MeOH
-- Ochratoxin A eaten up by alumina, but immunoaffinity column works well.
-- Take chromatograms of Darsa’s samples with Vicam’s column
-- Make a solution of 84:16 ACN:Water
-- Add ACN/H2O to 25 g of sample and shake for one hour to extract mycotoxins
-- For fatty samples (like feeds) freeze the sample so that any fats coagulate.
--Centrifuge to separate fat
--Pass the extract through the column
--Then inject
--After passing the extract through the C18/Alumina column, evaporate and reconstitute in MeOH
--Evaporate and reconstitute in MeOH
--This will result in a larger response from DON
--But does not decrease response for other mycotoxins
--The MeOH is not strong enough to wash mycotoxins from Al/C18 column with the same recoveries as ACN/H2O (SEE RECOVERY NOTES )
--If it is know that there is no DON in the sample, this step can be skipped

8. Method 1 Conditions Mobile Phase
H2O, ACN, MeOH, Phosphate Buffer (pH3.3):MeOH (90:10)
HPLC Flow Rate
1.0 mL/min
Column
Reverse-phase C18, 4.6x250mm
Column Temperature
40o C
Post-column Reagent
Iodine (100mg/L)
Reagent Flow Rate
0.3 mL/min
Reactor Volume
2.0 mL
Reactor Temperature
90o C

9. Method 1 Detection

10. Standard, method 1
Note that the pump is turned off at 30 minutes, but drop in absorbance takes time before you notice it on the detector
DON is collected using the UV detector, and is on a different channel

11. Corn, m1 -- Large B1 goes off scale on spiked sample
-- Only spikes reconstituted in MeOH. Natural samples not reconstituted.
SAMPLE 2

12. Wheat, m1
SAMPLE 6

13. Pig Feed, m1
SAMPLE 1

14. Feed sample, m1
SAMPLE 4 (Range land cake)

15. Corn 2, m1
SAMPLE 8

16. Hay, m1
** low density sample = light weight sample, used 4x more extraction solution. Sample was not concentrated after cleanup (prior to injection)
SAMPLE 9

17. Conclusions for Method 1
Sample prep works well for most samples
Certain matrices have interferences with portions of the chromatogram
Good sensitivity on the detection. Can see well below recommended amounts
Method can easily match the allowed limits

18. Sample Preparation and Cleanup, Method 2
DON, Aflatoxins, Fumonisins
MeOH : H2O (80:20) 100 mL
Add 50 g of ground sample
C18 only
Fill column with 1.5 g
Filter 6 mL of extract
Note: No alumina – the Fumonisins stick to the alumina
-- ACN has low response for fumonisin and DON
-- Reconstituting in straight MeOH could help increase DON and fumonisin response, but hasn’t been tried yet
--to increase the response even more, reconstitute in a lower volume

19. Method 2 Conditions Mobile Phase
H2O, ACN, MeOH, Phosphate Buffer (pH 3.3): MeOH (90:10)
HPLC Flow Rate
1.0 mL/min
Column Type
Reverse-phase C18, 4.6x250 mm
Column Temperature
40° C
Photochemical Reactor (Needed for Aflatoxins only)
ambient temp
Post-column Reagent
OPA, Thiofluor in GA104
Reagent Flow Rate
0.3 mL/min
Reactor Volume
2.0 mL
Reactor Temperature
60° C
--Phosphate buffer: NaOH (0.025 M) in phosphoric acid, pH 3.3

20. Method 2 Detection

21. Standard for Method 2 -Note increase in background – due to OPA
-Separation has been optimized so that Fumonisins elute after the baseline has stabilized

22. Corn Sample Expanded, m2
SAMPLE 2

23. Corn Samples SAMPLE 8 AND 5 --Corn contaminate with FB 3
--Sample spiked with FB1 and FB2, but not FB3

24. Feed Samples, m2
--Pig feed labeled as simply feeds – one is pig, one is “feed”
--Bottom chromatogram is pig feed
SAMPLE 3 (on top)
SAMPLE 1 (on bottom)

25. Conclusions for Method 2
Reliable extraction for Fumonisins requires more investigation
Some interferences with Aflatoxins as with Method 1
Can easily detect below allowed limits
- Fumonisin extraction can always be achieved using immunoaffinity columns, or with strong anion exchange (SAX) and C18

26. Future work Improve sample preparation and cleanup for Fumonisins
Examine interferences in some samples
Try reconstituting in MeOH
Investigate recoveries

27. Acknowledgements Nancy Thiex, South Dakota State University
Beth Tacke, North Dakota State University
Maria Ofitserova, Ph.D., Pickering Laboratories, Inc.
Darsa Siantar, Ph.D., ATTB

28. Questions and Discussion
Sareeta Nerkar & Maria Ofitserova