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
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IntroductionIntroductionmaize silage
•O2 < 1-2% •CO2 ≈ 20-90% •pH ≈ 3.8
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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
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YearO
OH OH
DFFE Project:Mycotoxin carry over from maize silage via Mycotoxin carry-over from maize silage via cattle into dairy products
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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
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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
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Zearalenone PR-toxin
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
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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
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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.
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MethodMethod
Multi-mycotoxin analysis of maize silage by LC-MS/MSRasmussen RR et al., Anal Bioanal Chem (2010) 397: 765
SpikedSpiked
’Blank’ silage
Spiked
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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
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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
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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
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* Significantly higher conc./occurrence in fresh maize compared to ensiled
Recent Danish results Survey of 99 Danish silages Survey of 99 Danish silages
nµg/kg fresh weight
High occurrence of
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
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* Significantly higher conc. in fresh maize compared to ensiled
Recent Danish results Survey of 99 Danish silages Survey of 99 Danish silages
nµg/kg fresh weight
Only in hot-spots
Analytenpos
meanpos maxRoquefortine C 2 173 189Andrastin A 15 169 691PenicillumPenicillum
B B niveanivea
Quantitative
Mycophenolic acid 2 43 52Gliotoxin 0Alternariol 2 18 24Alternariol monomethyl ether 3 9 11
B. B. niveaniveaA. A. fumigatusfumigatus
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 8Fumigaclavine A 0
PhomaPhomaA. A. fumigatusfumigatus
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* 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
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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
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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
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– DTU