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Analysis of Mycotoxins in Cereals

Analysis of Mycotoxins in Cereals. ‘On the road to Innovation Union: European food legislation’. Dr Martin Danaher, Head of National Reference Laboratory, Food Safety Department, Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland. FOODstars. Table of contents.

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Analysis of Mycotoxins in Cereals

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  1. Analysis of Mycotoxins in Cereals ‘On the road to Innovation Union: European food legislation’ Dr Martin Danaher, Head of National Reference Laboratory, Food Safety Department, Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland. FOODstars

  2. Table of contents • What are mycotoxins? • Legislation • Mycotoxin Reduction Project (MycoRed) • Results to Date and Ongoing

  3. What are mycotoxins?

  4. What are mycotoxins? Mykes = Fungus/MoldToxikon = Poison Mycotoxins are toxic secondary metabolites produced by fungi that readily colonise crops in the field or during storage. Toxins are especially produced under certain conditions •Host plant stress in the field •Harvesting: late harvesting, dry crops, slow storage filling •Storage: wet grain, incorrect fermentation of ensiled products •Finished feeds and forages: poor hygiene, exposure to air/moisture, incorrect storage (temperature/moisture)

  5. Common mycotoxins Aflatoxins (AFs) • Produced by Aspergillusflavusand Aspergillusparasiticus, especially found in areas with hot, humid climates • AflatoxinB1is the most toxic; It is one of the most powerful carcinogenic currently known. • International Agency for Research on Cancer (IARC): naturally occurring aflatoxins are carcinogenic to humans (group 1). • Main target organ is the liver • Also cytotoxic & immunosuppressive AFB1 AFB2 AFG2 AFG1 AFM1

  6. Common mycotoxins Aflatoxins (AFs) • Most commonly associated with: • Cereals (especially maize) • Groundnuts • Tree nuts • Dried fruit • Figs • Spices • Vegetable oils • Cocoa beans • Cottonseed • Copra • Dairy products (indirectly)

  7. Common mycotoxins Ochratoxin A (OTA) • Secondary metabolite primarily produced by Aspergillusochraceus and Aspergillus carbonarius • IARC classified OTA as possibly carcinogenic to humans (group 2B) • Main effects: nephrotoxic and teratogenic action, immunosuppression, altered metabolism of Phe, lipid peroxidation • Naturally occurs in a variety of products: cereals, coffee beans, pulses, fruit, vegetables, beverages and meat

  8. Common mycotoxins • Produced by several fungi including Fusariumgraminearum (Gibberellazeae), F. culmorum, F. cerealis, F. equiseti and F. semitectum. • Oestrogenic activity: decreases in fertility, reproductive tract alterations, increases in embryonic resorptions, reductions in litter size, changes in weight of adrenal, thyroid and pituitary glands, and changes in serum levels of progesterone and 17-β-oestradiol. • Products susceptible of contamination include maize, barley, oats, wheat, rye, sorghum, soybeans

  9. Common mycotoxins FB1 • Formed mainly by Fusariumverticillioides (syn.Fusariummoniliforme) and Fusariumproliferatum. • ≥12 fumonisin known analogues, the most important are the B series (FB1and FB2, FB3 and FB4lesstoxic). • IARC classified FB1 as possibly carcinogenic to humans (group 2B). • Biological effects: impairment of cell growth and differentiation, cellular oxidative stress, apoptosis and necrosis • Mainly associated with maize crops grown in warmer regions. FB2

  10. Common mycotoxins A and B Trichothecenes • Large family of chemically related mycotoxins produced by various species of Fusarium, Trichoderma e Trichothecium. • Sesquiterpenes with alcohol and ester groups and an epoxy group responsible for the high toxicity. • The most important compounds are DAS, T-2 and HT-2 toxins (type A) and NIV, DON, 3-ADON, 15-ADON (type B).

  11. Common mycotoxins A and B Trichothecenes • T-2 and HT-2: potent inhibitors of protein synthesis, immune suppression, apoptosis of hematopoietic cells, lipid peroxidation; interfere with the receptors for steroid hormones. • DON (aka vomitoxin): toxicity occurs mainly with sudden vomiting (hence the name), nausea and diarrhea. • The most commonly contaminated products are cereals: maize, barley, oats, wheat, sorghum, rice.

  12. Analysis of mycotoxins

  13. Analysis of mycotoxins Official methods

  14. “Emerging” mycotoxins ENNIATINS • Fusarium genre also produces the so-called “emerging” mycotoxins, such as Beauvericin (BEA) and Enniatins (ENNs) • Permitted maximum levels have not been established yet. • Studies (most in vitro) revealed the possible toxicity of these compounds, increased by the synergetic interaction with mixtures of mycotoxins present in food. BEA

  15. “Masked” mycotoxins The possible hydrolysis of masked mycotoxins during mammalian digestion can quantitatively contribute to the total amount of mycotoxins. Some examples: DON T-2 toxin Plant Metabolism Mammalian Digestive Syst. Mammalian Digestive Syst. Plant Metabolism T-2-Glucoside D3G

  16. II. Legislation

  17. EU legislation on mycotoxins Maximum levels in food COMMISSION REGULATION (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. • Regarding to cereals, the regulation sets a maximum level for the following mycotoxins: • AFB1, AFB2, AFG1, AFG2 • FB1, FB2 • DON • ZON • OTA

  18. EU legislation on mycotoxins Monitoring T-2 and HT-2 toxins COMMISSION RECOMMENDATION (2013/165/EU) of 27 March 2013 on the presence of T-2 and HT-2 toxin in cereals and cereal products “Member States (MS) should” …“perform monitoring for the presence of T-2 and HT-2 toxins in cereals and cereal products. MS should encourage that samples are simultaneously analysed for the presence of T-2 and HT-2 and “… “also the masked mycotoxins in particular the mono- and di- glycosylated conjugates of T-2 and HT-2 toxins.”

  19. III. Mycotoxin Reduction Project (MycoRed)

  20. MycoRed project “Development and validation of multi-residue methods for the analysis of mycotoxins and their masked metabolites in cereals and beverages using UHPLC-MS/MS” Development of improved analytical UHPLC-MS/MS methods, including regulated, emerging and masked mycotoxins, as they are synthetized by project partners at QUB The overall aim of our project is to reduce mycotoxins levels in cereals and alcoholic beverages. Application of the method(s) to samples generated by project partners at UCC and Teagasc Oak Park, allowing the evaluation of different decontamination strategies Investigation of mycotoxins in Irish food and beverages

  21. IV. Results to Date

  22. Results to date LC-MS/MS conditions Electrospray ionisation parameters (cone voltage, capillary voltage, temperatures, gas flows, negative/positive ionisation modes, etc.) were optimised to achieve best ionisation and detection for 52 analytes. Chromatographic expedients such as solvents (methanol/acetonitrile), acids (formic/acetic acids) and salts (ammonium acetate or ammonium formate) and their effects on ionisation have been evaluated. Mob. Phase A:5mM amm. acetate in H2O:MeOH (90:10, v/v). Mob. Phase B:5mM amm. acetate + 0.2% CH3COOH in MeOH

  23. Results to date Chromatographic Separation • Chromatographic separation for 52 toxins (plus 7 I.S.) was achieved. • However, 10 analytes were excluded as peak shapes and sensitivity were not satisfactory in matrix under the developed conditions (total = 42 mycotoxins in the method) • Chromatography includes the emerging Enniatins (A, A1, B, B1) and Beauvericin as well as the masked mycotoxins D3G and T2G

  24. Results to date Sample preparation A QuEChERS-based sample prep has been developed and fully validated for the analysis of 42 mycotoxins in unprocessed oat samples.

  25. Results to date Validation studies (evaluation of method performances) • Selectivity (inj. of single Std/IS and analysis of 5 different negative oats) • Linearity (7 levels in matrix, > 5 runs, R2 ≥ 0.98) • Trueness* (as apparent recovery, RA) • Precision* (as RSD%, within laboratory repeatability “WLr” and within laboratory reproducibility “WLR”) • Matrix effect (signal suppression/enhancement, SSE) – Use of IS • Limits of quantification (LOQs), from 0.5 ppb (AFB1) to 100 ppb (NIV, DON,ENB1, ENB) *three levels, low (LVL), medium (MVL) and high (HVL), corresponding to level 2, 4 and 6 of the calibration curve, in quintuplicate (WLr, n = 15). Procedure repeated in five separate days, each level analysed in duplicate (WLR, n = 30) possibly by three different analysts.

  26. Results to date Validation studies – Trueness and precision Very good precision < 4% Trueness (% of recovery) and precision (RSD%, shown by error bars) of the method under reproducibility conditions (WRL, n= 10) at the medium validation level (L4)

  27. Ongoing – method for Beer Sample preparation The QuEChERS-based sample prep has been adapted to the beer matrix. Method includes 41 mycotoxins; validation in progress.

  28. Ongoing – method for Beer Validation studies •  Selectivity (inj. of single Std/IS and analysis of 27 different beers) •  Linearity (7 levels in matrix, > 5 runs, R2 ≥ 0.98) • Trueness* (as apparent recovery, RA) • Precision* (as RSD%, within laboratory repeatability “WLr” and within laboratory reproducibility “WLR”) •  Matrix effect (signal suppression/enhancement, SSE) – Use of IS •  Limits of quantification (LOQs), from 0.1 ppb (OTA) to 5 ppb (NIV, DON) *three levels, low (LVL), medium (MVL) and high (HVL), corresponding to level 2, 4 and 6 of the calibration curve, in quintuplicate (WLr, n = 15). Procedure repeated in five separate days, each level analysed in duplicate (WLR, n = 30) possibly by three different analysts.

  29. Ongoing - 3 year survey The survey plan • The complete survey includes a total of > 750 Irish oat samples harvested over a 3 year period (2015, 2016, 2017), plus a pilot study of 71 samples • The samples are generated under different conditions (i.e. some have a fungicidal treatment, some others are from land not previously planted with cereals, some are with and without the hull) • All the samples have been carefully milled (using a 0.5 mm screen) and homogenised, before being analysed • Each sample was given a lab identification code

  30. Results of pilot study Overall Results HT-2 + T-2 + T2G ENNs

  31. Preliminary Survey T-2 and HT-2 Results Recommended ML for the sum of T-2 and HT-2: 1000 µg/kg (oat with husk) NOTE: Results marked with * are above the upper calibration range of the method and will have to be retested

  32. Acknowledgments This research was funded by the FIRM programme administered by the Irish Department of Agriculture, Food and the Marine (Contract 13F497) Lorenzo De Colli (Teagasc) Prof. Chris Elliott (QUB)

  33. THANK YOU FOR YOUR KIND ATTENTION

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