Detection of 27 secondary fungal metabolites in maize ...orbit.dtu.dk/files/2369635/2010-08-17_18...

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

Overview• Introduction• Introduction

– Maize silage– Project background

Method• Method• Application and results

– Hot-spotsS– Survey

• Conclusions

2DanSAK 2010 18th August 2010

IntroductionIntroductionmaize silage

•O2 < 1-2% •CO2 ≈ 20-90% •pH ≈ 3.8


Introductionmaize silage

Increased maize silage

maize silage

Unexplained health problems at dairy farms

production in Denmark

6 000 000

Tonnes

problems at dairy farms

Illness

Decrease in milk yield

5.000.000

6.000.000 Decrease in milk yield

Dead cows

3.000.000

4.000.000

1.000.000

2.000.000

O

Mycotoxins

01992 1996 2000 2004 2008

Y

?OOH CH3 H

N

NOS

OS

CH3

OH

OHCH3

CH3O

O OH


YearO

OH OH

DFFE Project:Mycotoxin carry over from maize silage via Mycotoxin carry-over from maize silage via cattle into dairy products


Photo: Shutterstock.com

Fungi in maize and silage

Loto

: Fa

ngea

t

nse

n J

LFusarium :

Kal

krup

H

Penicillium roqueforti

Pho

Photo

: Søre

nFusarium

Alternaria

Epicoccum

Photo

Penicillium roqueforti

Penicillium paneum

Zygomycetes (Mucor and Rhizopus)

P

Phoma

Aspergillus flavus

A ill i i

Aspergillus fumigatus

Byssochlamys nivea

M b


Aspergillus parasiticus Monascus ruber

Storm et al. 2008: Stewart Postharvest Review 6(4)

Selected mycotoxins and other secondary Selected mycotoxins and other secondary metabolites

ESI-

Alternariol

Alternariol monomethyl ether

ESI+

Citrinin

Cyclopiazonic acid

Roquefortine A

Sterigmatocystin

Altersetin

Andrastin A

Citreoisocoumarin

Enniatin B

Fumigaclavine A

Fumigaclavine B

T-2 toxin

Deoxynivalenol

Gliotoxin

Mycophenolic acid

Fumigaclavine C

Fumitremorgin A

Fumitremorgin C

Nivalenol

Ochratoxin A

Patulin

Fumonisin B1

Fumonisin B2

Marcfortine A

M f ti BPenitrem A

Roquefortine C

Tenuazonic acid

Z l

Marcfortine B

Mevinolin

Ochratoxin A

PR toxin


Zearalenone PR-toxin

Physical and chemical properties

Estimated by ACDLabs software


IDS12

Dias nummer 8

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

Method characteristicsWanted:Wanted:• Both pre- and post-harvest fungal metabolites • One extract

Minimal clean up• 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, 412-431)

• GC- and LC-MS


Extraction: Modified QuEChERSExtraction: Modified QuEChERS

H2O

1% HAc

in MeCNMgSO4

NaAcSilageSilage

MeCN

Silage

Shake 2 min Centrifuge 10 minShake 1 min MeCN for

LC-MS/MS

H2O

MgSO4


Mycotoxin detection:

Validation results• Rasmussen RR et al Anal Bioanal Chem (2010) 397: 765• Rasmussen RR et al., Anal Bioanal Chem (2010) 397: 765

– 27 metabolites in maize silage: • 18 quantitatively• 9 qualitatively• 9 qualitatively

– Recoveries: 37 to 201% (majority: 60 to 115%) – Repeatabilities: 5 to 27%

Reproducibilities: 7 to 35%– Reproducibilities: 7 to 35%– LOD (1-739 μg/kg)

Cleaning between runs:

Injections of 5% formic acid, methanol and water.


MethodMethod

Multi-mycotoxin analysis of maize silage by LC-MS/MSRasmussen RR et al., Anal Bioanal Chem (2010) 397: 765

SpikedSpiked

’Blank’ silage

Spiked


min

Application:

Hot-spots

roquefortine C: 33660 µg·kg-1 gliotoxin: 906 µg·kg-1 mycophenolic acid 1646 µg·kg-1q µg g

andrastine A: 8811 µg·kg-1

mycophenolic acid: 407 µg·kg-1

citreoisocoumarine

g µg g

fumitremorgin C

fumigaclavine B and C

y p µg g

andrastin A 211 µg·kg-1

citreoisocoumarine

roquefortine Acitreoisocoumarine

roquefortine

roquefortine A

marcfortine A


Application

SurveyFresh and ensiled maize collected 2007 – 2009Fresh and ensiled maize collected 2007 – 2009from 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 p g pp y

the cutting face of the silage stack.

R ltResultsCommon pre- and post-harvest fungal metabolites detected


ApplicationSurveySurvey

nµg/kg fresh weight

Analytenpos

meanpos maxRoquefortine C 2 173 189Andrastin A 15 169 691PenicillumPenicillum

B B niveanivea

Quantitative

Mycophenolic acid 2 43 52

Alternariol 2 18 24Alternariol monomethyl ether 3 9 11

B. B. niveanivea

AlternariaAlternaria Alternariol monomethyl ether 3 9 11Nivalenol 16 263 758Deoxynivalenol 7 1841 2974Zearalenone 34 71 666

FusariumFusarium

Enniatin B* 28 75 365Roquefortine A 9Marcfortine B 1

PenicillumPenicillum

Qualitative Marcfortine A 6Citreoisocoumarin 8PhomaPhoma


* Significantly higher conc./occurrence in fresh maize compared to ensiled

Recent Danish results Survey of 99 Danish silages Survey of 99 Danish silages


High occurrence of

Analytenpos


B B niveanivea

Quantitative

Mycophenolic acid 2 43 52

Alternariol 2 18 24Alternariol monomethyl ether 3 9 11

B. B. niveanivea


FusariumFusarium



Qualitative Marcfortine A 6Citreoisocoumarin 8PhomaPhoma


* Significantly higher conc. in fresh maize compared to ensiled

Recent Danish results Survey of 99 Danish silages Survey of 99 Danish silages


Only in hot-spots

Analytenpos


B B niveanivea

Quantitative

Mycophenolic acid 2 43 52Gliotoxin 0Alternariol 2 18 24Alternariol monomethyl ether 3 9 11

B. B. niveaniveaA. A. fumigatusfumigatus


FusariumFusarium



Qualitative Marcfortine A 6Citreoisocoumarin 8Fumigaclavine A 0

PhomaPhomaA. A. fumigatusfumigatus


* Significantly higher conc./occurrence in fresh maize compared to ensiled

ApplicationSSurvey

30

35

20

25

am

ple

s

10

15

o.

of

sa

35 30 11 …

0

5

10

N

0

0 1 2 3 4 5 6 7

No. of analytes detected in samples


No. of analytes detected in samples

ConclusionsConclusions

• High content of fungal metabolites in hot spots with visible fungal growth• 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 y pCommission

• Gliotoxin, PR-toxin and patulin were not detected in field samples

• These studies do not indicate that mycotoxins in maize silage have • 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 • The possibility of synergistic effects and effects of long-term exposure calls for more research


AcknowledgementsAcknowledgements

• Supervisors/advisors– Kristian F Nielsen– Kristian F Nielsen– Peter H Rasmussen– Jørn Smedsgaard

• External co-operators– Niels B Kristensen, DJF Aarhus

UniversityUniversity– Danish Plant Directorate– Danish Agricultural Advisory

ServiceService

• Funding– DFFEDFFE– Danish Cattle Association– Research School FOOD– DTU


– DTU

Thank you for your attention!y y”r from maize silage via cattle into dairy products”


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