Detection of 27 secondary fungal metabolites in maize silage by rapid extraction and LC-MS/MS. Ida ML Drejer Storm and Rie R Rasmussen

Transcription

1 Detection of 27 secondary fungal metabolites in maize silage by rapid extraction and LC-MS/MS Ida ML Drejer Storm and Rie R Rasmussen

2 Overview Introduction Maize silage Project background Method Application and results Hot-spots Survey Conclusions 2

3 Introduction maize silage O 2 < 1-2% CO % ph 3.8 3

4 Tonnes Introduction maize silage Unexplained health Increased maize silage problems at dairy farms production in Denmark Illness Decrease in milk yield Dead cows Mycotoxins Year O O OH O OH O? C H 3 O H OH O N CH S 3 O N CH 3 S OH CH 3 HO OH 4

5 DFFE Project: Mycotoxin carry-over from maize silage via cattle into dairy products Photo: Shutterstock.com 5

6 Fungi in maize and silage L Fusarium Alternaria Epicoccum Pho oto: Fangeat Photo: Søren nsen JL Penicillium roqueforti Penicillium paneum Zygomycetes (Mucor and Rhizopus) : Kalkrup H Photo Phoma Aspergillus fumigatus Aspergillus flavus Byssochlamys nivea Aspergillus parasiticus ii Monascus ruber Storm et al. 2008: Stewart Postharvest Review 6(4) 6

7 Selected mycotoxins and other secondary metabolites ESI- Alternariol Alternariol monomethyl ether Altersetin Andrastin A Citreoisocoumarin Deoxynivalenol Gliotoxin Mycophenolic acid Nivalenol Ochratoxin A Patulin Penitrem A Roquefortine C Tenuazonic acid Zearalenone ESI+ Citrinin Cyclopiazonic acid Enniatin B Fumigaclavine A Fumigaclavine B Fumigaclavine C Fumitremorgin A Fumitremorgin C Fumonisin B 1 Fumonisin B 2 Marcfortine A Marcfortine B Mevinolin Ochratoxin A PR-toxin Roquefortine A Sterigmatocystin T-2 toxin 7

8 IDS12 Physical and chemical properties Estimated by ACDLabs software 8

9 Dias nummer 8 IDS12 Lav en version der kun har nogle få grafer med. Start med koordinatsystem uden grafer. Ida Storm, 12/10/2009

10 Method characteristics Wanted: Both pre- and post-harvest fungal metabolites One extract Minimal clean-up Buffering of ph Suitable for a dirty plant-based matrix Found: Quick, Easy, Cheap, Effective, Rugged, Safe (QuEChERS) method (Anastassiades et al. (2003) J. AOAC Int. 86, ) GC- and LC-MS 9

11 Extraction: Modified QuEChERS H 2 O 1% HAc in MeCN MgSO 4 Silage NaAc Shake 2 min Shake 1 min Centrifuge 10 min MeCN MeCN for LC-MS/MS Silage H 2 O MgSO 4 10

12 Mycotoxin detection: Validation results Rasmussen RR et al., Anal Bioanal Chem (2010) 397: metabolites in maize silage: 18 quantitatively 9 qualitatively Recoveries: 37 to 201% (majority: 60 to 115%) Repeatabilities: 5 to 27% Reproducibilities: 7 to 35% LOD (1-739 μg/kg) Cleaning between runs: Injections of 5% formic acid, methanol and water. 11

13 Method Multi-mycotoxin analysis of maize silage by LC-MS/MS Rasmussen RR et al., Anal Bioanal Chem (2010) 397: 765 Spiked Blank silage Spiked min 12

14 Application: Hot-spots roquefortine C: µg kg g -1 gliotoxin: 906 µg kg g -1 mycophenolic acid 1646 µg kg g -1 andrastine A: 8811 µg kg -1 mycophenolic acid: 407 µg kg -1 citreoisocoumarine fumitremorgin C fumigaclavine B and C andrastin A 211 µg kg -1 citreoisocoumarine roquefortine A roquefortine marcfortine A 13

15 Application Survey Fresh and ensiled maize collected from all over Denmark 17 whole fresh maize plants taken at field level 82 ensiled maize samples collected as grab samples from the cutting face of the silage stack to form a composite sample. in full depth with a silage drill approximately 1 meter behind the cutting face of the silage stack. Results Common pre- and post-harvest fungal metabolites detected 14

16 Application Survey Penicillum B. nivea µg/kg fresh weight n pos Analyte mean pos max Roquefortine C Andrastin A Mycophenolic acid Alternaria Quantitative Fusarium Penicillum Alternariol Alternariol monomethyl ether Nivalenol Deoxynivalenol Zearalenone Enniatin B* Roquefortine A 9 Marcfortine B 1 Qualitative Marcfortine A 6 Phoma Citreoisocoumarin 8 * Significantly higher conc./occurrence in fresh maize compared to ensiled 15

17 Recent Danish results Survey of 99 Danish silages High occurrence of Penicillum B. nivea µg/kg fresh weight n pos Analyte mean pos max Roquefortine C Andrastin A Mycophenolic acid Alternaria Quantitative Fusarium Penicillum Alternariol Alternariol monomethyl ether Nivalenol Deoxynivalenol Zearalenone Enniatin B* Roquefortine A 9 Marcfortine B 1 Qualitative Marcfortine A 6 Phoma Citreoisocoumarin 8 * Significantly higher conc. in fresh maize compared to ensiled 16

18 Recent Danish results Survey of 99 Danish silages Only in hot-spots Penicillum B. nivea A. fumigatus Alternaria Quantitative Fusarium Penicillum µg/kg fresh weight n pos Analyte mean pos max Roquefortine C Andrastin A Mycophenolic acid Gliotoxin 0 Alternariol Alternariol monomethyl ether Nivalenol Deoxynivalenol Zearalenone Enniatin B* Roquefortine A 9 Marcfortine B 1 Qualitative Marcfortine A 6 Phoma Citreoisocoumarin 8 A. fumigatus Fumigaclavine A 0 * Significantly higher conc./occurrence in fresh maize compared to ensiled 17

19 Application Survey o. of sa amples N No. of analytes detected in samples 18

20 Conclusions High content of fungal metabolites in hot-spots with visible fungal growth High occurrence of Fusarium toxins in maize silage Fusarium toxins were below values recommended by the European Commission Gliotoxin, PR-toxin and patulin were not detected in field samples These studies do not indicate that mycotoxins in maize silage have caused the general health problems observed at Danish dairy cattle farms The possibility of synergistic effects and effects of long-term exposure calls for more research 19

21 Acknowledgements Supervisors/advisors Kristian F Nielsen Peter H Rasmussen Jørn Smedsgaard External co-operators Niels B Kristensen, DJF Aarhus University Danish Plant Directorate Danish Agricultural Advisory Service Funding DFFE Danish Cattle Association Research School FOOD DTU 20

22 Thank you for your attention! r from maize silage via cattle into dairy products 21