Mycotoxins in Botanical Materials: Study, Analysis, and Evaluation

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

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

6. Canada 15 T Japan 10 B1 30% EU 4 T / 2 B1 4% 50% US Exporter 20 T 12% Mexico 20 T 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 2-8 23-71

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. Determination of Aflatoxins B1, B2, G1 and G2 and Ochratoxin A in Ginseng and Ginger IAC cleanup 25 g sample + Extraction solvent 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. 23 Ginger product AFB2a Aflatoxin standard 18 AFB2a AFB2 AFB2 13 AFG2 AFG2 mV 8 AFG2a AFG2a 3 -2 0 5 10 15 20 25 30 35 minute Figure 1. LC chromatograms of AFL standard and AFL in ginger product. Post-column Derivatization (PHRED/UV cell) for Aflatoxins

17. LC Chromatogram of Ochratoxin A

18. STD: AFL + OTA Ginger Sample 70000 60000 50000 OTA 40000 AFB1 30000 AFG1 20000 AFG2 AFB2 10000 0 0 5 10 15 20 25 30 35 40 Minutes LC/MS/MS MRM chromatograms (total ions) AFL & OTA In standard and in ginger 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 2008.02. • 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

26. THANK YOU! 谢谢! 谢谢!