1. Mycotoxins in Botanical Materials
Mary W. Trucksess, Ph.D.
Food and Drug Administration
Center for Food Safety and Applied Nutrition
College Park, MD

2. Subjects of Discussion
Mycotoxins
Method development and method validation for mycotoxins in botanicals
Pure substance reference materials and certified matrix for mycotoxin determination

3. Mycotoxins
Secondary metabolites produced by certain fungi: Aspergillus, Fusarium and Penicillium
More than 300 mycotoxins have been identified, major mycotoxins: aflatoxins, ochratoxin A, deoxynivalenol, fumonisins, zearalenone
Toxicological problems, including teratogenic, carcinogenic, estrogenic or immunosuppressive effects

4. Aflatoxins Produced by Aspergillus flavus and A. parasiticus
Found in corn, cottonseed, peanuts, tree nuts, copra, figs, and milk
Bioactivated aflatoxin B1 can bind to DNA, RNA and proteins
IARC has classified as a probable human carcinogen

5. Chemical structures of aflatoxins B1, B2, G1 and G2
AFG1
AFB1
AFG2
AFB2
Chemical structures of aflatoxins B1, B2, G1 and G2

6. US Exporter 20 T Canada 15 T EU 4 T / 2 B1 Japan 10 B1 Mexico 12% 50%
30% 4%
Whitaker 2008

7. Ochratoxin A
Produced by Aspergillus ochraceus and related species and Penicillium spp.
Found in corn, barley, wheat, oats, and green coffee beans, raisins, figs
Ochratoxin A causes kidney damage in animals, and may be the cause for Balkan kidney disease
IARC has determined ochratoxin A to be an animal carcinogen

8. Chemical structure of ochratoxin A

9. Subjects of Discussion
Mycotoxins
Method development and method validation for mycotoxins in botanicals
Pure substance reference materials and certified matrix for mycotoxin determination

11. 6-12
2-3
4-14
1-8
23-71
2-8

12. Method Development and Method Evaluation for Mycotoxins in Botanicals
Sampling and sample preparation
Multi-toxins in botanicals
International collaborative study
Single laboratory validation study
On-going research multi-toxins in foods

13. Conclusions of Sampling Studies
Sampling study from Bulk (1 lb bag)
Demonstrated that 5 g sample size is sufficient for
AF and OTA analysis in powdered roots. It is
advisable to collect 4 bag of powdered ginger and
analyze two 5 g test samples and average results.
Sampling study from ginger capsules
Demonstrated that in order to reduce the total
variability of the test procedure, it is advisable to
collect 4 bottles of capsules and analyze four 5 g
test samples of the mixed composite and average
results.

14. Dilute with water, post-or pre-column derivatization,
Determination of Aflatoxins B1, B2, G1 and G2 and Ochratoxin A in Ginseng and Ginger
25 g sample +
Extraction solvent
IAC cleanup
Wash with water
elute with MeOH
Blend 3 Minutes, filter,
Dilute, filter
Dilute with water, post-or
pre-column derivatization,
LC analysis

15. Post-column Derivatization (PHRED/UV cell) for Aflatoxins

16. Post-column Derivatization (PHRED/UV cell) for Aflatoxins
Figure 1. LC chromatograms of AFL standard and AFL in ginger product.
AFB2a
AFB2
AFG2a
AFG2 -2 3 8 13 18 23 5 10 15 20 25 30 35
minute
Ginger product
Aflatoxin standard mV

17. LC Chromatogram of Ochratoxin A

18. LC/MS/MS MRM chromatograms (total ions) AFL & OTA
10000
20000
30000
40000
50000
60000
70000 5 10 15 20 25 30 35 40
Minutes
STD: AFL + OTA
Ginger Sample
LC/MS/MS MRM chromatograms (total ions) AFL & OTA
In standard and in ginger
AFB1
AFG1
AFB2
AFG2
OTA
Response

19. Preparation of Homogenous Mycotoxin Naturally Contaminated Materials for Collaborative Study
Materials include a variety of different matrices such as grains, nuts, feeds, foods
Identify a large amount of naturally contaminated material
Grind grains or nuts with a proper mill to result a product of proper particle size.
Pass particles through a number 20 sieve
Mix with mixer
For juice or finely ground powders, only mixing is necessary
Determine concentration of specific mycotoxin.

20. Preparation of Homogenous Mycotoxin Naturally Contaminated Powdered Ginger for Collaborative Study
Identify a lot of powdered ginger naturally contaminated with aflatoxins and ochratoxin A
Mix by tumbling for 8 hours
Analyze using our validated method
Conduct multiple replications (30) over a few days to verify homogeneity of the material.
Store at room temperature and analyze weekly (replicates of 4 analyses) for four weeks.
Obtain mean value and standard deviation range

21. Results of Collaborative Study
Results met AOAC Official Method Performance criteria. This collaboratively studied multi- mycotoxin method has been adopted as AOAC Official First Action Method
This work was partially supported by the Office of Dietary Supplements, National Institutes of Health.

22. Results of Single Laboratory Validation Study (SLV)
Single Laboratory Validation Study Protocol: 4 spiking levels, 10 test sample/botanical/level, 3 day duration
Mycotoxins: aflatoxins, ochratoxin A, fumonisin B1
Results: Recoveries >75% for 4-7 botanicals, manuscripts in preparation

23. Subjects of Discussion
Mycotoxins
Method development and method validation for mycotoxins in botanicals
Pure substance reference materials and certified matrix for mycotoxin determination

24. Some Suppliers of Reference Materials (These are only examples, not a complete list)
Pure substance reference materials:
Sigma-Aldrich, Supelco:
Aflatoxins, deoxynivalenol, ochratoxin A, zearalenone, fumonisins, patulin
Certified matrix reference materials:
Europe: Joint Research Centre (JRC)
Institute for Reference Materials and Measurements (IRMM)
USA: American Oil Chemist Society (AOCS)
National Institute of Science and Technology (NIST)
Trilogy Labs
Romer Labs

25. Available Certified matrix reference materials
Mycotoxins
Certified Reference Matrix
Aflatoxins
Corn gluten, mixed feeds, peanut paste, peanut butter, peanut meal
Aflatoxin M1
Milk powder
Deoxynivalenol
Wheat, barley, corn
Fumonisins
Corn, mixed feeds
Ochratoxin A
Wheat, corn, wine, roasted coffee, green coffee

26. THANK YOU!
谢谢!
谢谢!