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

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

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

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

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

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

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

Chemical structure of ochratoxin A

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

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

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

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.

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

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

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

LC Chromatogram of Ochratoxin A

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

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.

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

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.

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

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

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

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

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