VIP Session ASMS 2015
1
The new PCDL for mycotoxins and related metabolites
Content and integration in accurate mass screening workflows
Dr. Thomas GlaunerSenior LC/MS Applications ScientistEMEA Market Development Team
June 22, 2015
Mycotoxins are low molecular weight, secondary metabolites of fungi
Acute toxic, carcinogenic, mutagenic, estrogenic, and immunotoxic effects
Fusarium, Aspergillus, Penicillium, Alternaria spp.
>25% of all agricultural goods are contaminated
annual losses of several hundred million tons of food worldwide
annual economical losses: 1 billion USD (US only)
100+ countries have regulations for the control ofmycotoxins in food and feed
VIP Session ASMS 2015
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MycotoxinsBackground
June 22, 2015
MycotoxinsBackground
• Regulated mycotoxins• Aflatoxins• Ochratoxins (OTA)• Trichothecenes• Zearalenone• Fumonisins• Patulin
• Further mycotoxins• Ergot alkaloids• Citrinin• Cyclopiazonic acid• Sterigmatocystin• Monoliformin• Gliotoxin• Citreoviridin• Tremorgenic mycotoxins• Penicillic acid• Roquefortine• 3-Nitropropionic acid• Fusaproliferin
VIP Session ASMS 2015
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• … in addition there are masked mycotoxinsJune 22, 2015
MycotoxinsChemical diversity – a challenge not only for sample prep
VIP Session ASMS 2015
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ON
ON
NO
O
OO
O O
O
Enniatins
beauvericinenniatin A, A1, B, B1
APOLAR
beauvericin
O
O
[COOH][COOH]
O
O [COOH]
[COOH]
CH3 CH3 OH
OH
CH3
OH
NH2
Fumonisins
fumonisin B1, FB2, FB3, hydrolyzed FB1
POLAR, ACIDIC
FB1
Ergot alkaloids
ergotamin, ergocornin, ergovalin, dihydroergosin
POLAR, BASIC
ergovalin
June 22, 2015
Regulations for MycotoxinsEuropean Commission Regulation 1881/2006 (EC)
VIP Session ASMS 2015
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AnalytesMLs µg/kg
(EC Reg. No 1881/2006)
Commodities
Aflatoxin B1
0.1 processed cereal-based baby food2.0-12
sum of aflatoxins: 4.0-15.0
nuts and cereals
Deoxynivalenol 200500 - 1750
processed cereal-based baby foodprocessed / unprocessed cereals,
bread, pasta, breakfast cerealsFumonisin B1 200
8001000 / 4000
processed maize-based baby foodmaize-based breakfast cereals
maize / unprocessed maizeFumonisin B2
Patulin 10-50fruit juices, apple products, baby food
other than processed cereal-based foods
Ochratoxin A
0.53.0 / 5.0
10.015 / 20 / 80
processed cereal-based baby foodprocessed / unprocessed cereals
dried vine fruitspices / liquorice root / extract
Zearalenone2050
75-350
processed cereal-based baby foodbread, biscuits, breakfast cerealsprocessed / unprocessed cereals
June 22, 2015
Regulations for MycotoxinsFDA Regulatory guidelines
VIP Session ASMS 2015
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Analytes Limit µg/kg Commodities
Aflatoxins, sum 20 All foods except milk
Patulin 50 Apple juice, apple juice concentrate, apple components in processed food
Deoxynivalenol 1000 Finished wheat products
Fumonisins sum of B1, B2, B3
2000degermed dry milled corn products
(e.g. flaking grits, corn grits, corn meal, corn flour)
3000 cleaned corn intended for popcorn
4000whole of partially degermed dry milled corn products (e.g. flaking grits, corn
grits, corn meal, corn flour
Aflatoxin M1 0.5 milk
June 22, 2015
LC/MS Multi-mycotoxin Methods
Reasons• Unified methods save time and costs• Screening of wide range of analytes/matrix combinations for
- evaluation of synergistic effects due to co-exposure of mycotoxins- discovery of unexpected contamination patterns
(e.g. fumonisins in grapes, aflatoxins in Swiss maize)- monitoring of changes in regional fungal spread due to climate change- analysis of food and feed mixtures of several commodities
Possible Setup• UHPLC/Q-TOF/MS combined with an accurate
mass database and library for mycotoxins
VIP Session ASMS 2015
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June 22, 2015
Multi-Mycotoxin methodsGeneral considerations
Common separation and detection principle
• Liquid chromatography / mass spectrometry
- Wide range of relevant concentrations:2 µg/kg (AFB1) to 1750 µg/kg (DON)
- Use of high end analytical equipment (sensitivity, linearity)
Generic sample preparation
• Careful optimization of extraction solvents
• Common clean-up (difficult)
- Multifunctional columns restrict method to the most important analytes
- QuEChERS looks promising but very polar analytes are lost
• Injection of (diluted) crude extracts
- Matrix effects / signal suppression
June 22, 2015
VIP Session ASMS 2015
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Accurate mass screening
Advantages• Very high selectivity• Virtually unlimited number of analytes• Easy acquisition method setup• Non-target acquisition with targeted data analysis
Requirements• High resolution accurate mass Q-TOF LC/MS• Databases and libraries
• Innovative data mining tools
• Efficient software workflows for Qual and Quant
VIP Session ASMS 2015
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June 22, 2015
The new Mycotoxin PCDL
Database- For more than 450 compounds
• Mycotoxins, fungal and bacterialmetabolites
• Relevant for food and theenvironment
- Includes names, commonidentifiers (CAS, ChemSpider),formulas, structures (.mol files),monoisotopic masses
- Database can be easily extended or split in sub-databases- RTs are updated from experimental data or .csv files- Fully searchable and fully integrated in data mining algorithms
VIP Session ASMS 2015
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June 22, 2015
The new Mycotoxin PCDL
Library- Accurate mass spectra for more
than 300 compounds- Including all relevant precursor
ions ([M+H]+, [M+NH4]+, [M-H]-,[M+CHOO]-) and severalcollision energies
- Data acquisition performed bythe Institute for Analytical Chemistry, IFA Tulln
- Fragment ions were corrected for their theoreticalformula weight
VIP Session ASMS 2015
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June 22, 2015
How do we acquire library spectra ?
• Experimental setup- FIA of pure standards or purified isolates- Chromatographic separation for mixtures or isomeric compounds (e.g. ergot
alkaloids)- Acidified ammonium formate buffer
• Qualifty control and data treatment- Specific procedure for calibration of instrument
- Minimum abundance filter- Mass accuracy filter for precursors and products- Correction of fragment masses compared to theoretical fragments- Noise filter and filtering on not assigned masses (optional)
- All implemented in MassHunter Qual B.07.00.
VIP Session ASMS 2015
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June 22, 2015
Acquisition of library data for Ochratoxin A
13
5x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
Cpd 7: OTA / Ochratoxin A: +ESI EIC(404.08954, 406.08748, 426.07149, 428.06943) Scan Frag=380.0V Waldbronn_3618_OTA_
0.677 950406
1 1
Counts vs. Acquisition Time (min)0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4
VIP Session ASMS 2015
3x10
0
0.25
0.5
0.75
1
1.25
1.5
Cpd 7: C20 H18 Cl N O6: (M+H)+: +ESI Product Ion (rt: 0.573, 0.601, 0.630, 0.659 ... min, 15 scans) Frag=380.0V [email protected] (40
239.01009 [C11 H8 Cl O4]+ 358.08348 [C19 H17 Cl N O4]+
120.08035 [C8 H10 N]+
3x10
0
0.5
1
1.5
2
2.5
3
Cpd 7: C20 H18 Cl N O6: (M+H)+: +ESI Product Ion (rt: 0.578, 0.607, 0.636, 0.665 ... min, 15 scans) Frag=380.0V [email protected] (40
239.00993 [C11 H8 Cl O4]+
358.08247 [C19 H17 Cl N O4]+ 120.08039 [C8 H10 N]+ 284.26425
3x10
0
0.25
0.5
0.75
1
1.25
Cpd 7: C20 H18 Cl N O6: (M+H)+: +ESI Product Ion (rt: 0.584, 0.613, 0.641, 0.670 ... min, 15 scans) Frag=380.0V [email protected] (40
220.99975 [C11 H6 Cl O3]+
137.01518 [C8 H6 Cl]+
341.05627 [C19 H14 Cl O4]+
Counts vs. Mass-to-Charge (m/z)40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
FIA chromatogram- Compound identification using Find-by-Formula- Automatic extraction of MS and MS/MS spectra
Annotation of fragment formulas
CE 10 eV
CE 20 eV
CE 40 eV
Export of spectra to PCDL- Abundance filter- Only annotated fragments- Calculated m/z
June 22, 2015
LC/MS Application
Kits
Method
Test Mixes
LC Columns
I&F and On-site Training
Databases and
Libraries
Fast-Track to Productivity and High Quality Results
June 22, 2015
VIP Session ASMS 2015
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LC/MS Application Kits & Standalone Databases
Pesticides
Veterinary Drugs
Forensic Toxicology
Mycotoxins (standalone PCDL only)
For TOF, Q-TOF LC/MS platforms (PCDL)
Complete application kits provide fast & simplified startup
Standalone Databases/Libraries
Screening by target MS/MS and library searching
• Acquisition using comprehensive target MS/MS method with RTs, precursor masses, and CEs specified
• Data mining using Find-by-Formula with PCDL as formula source
VIP Session ASMS 2015
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7x10
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1
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1.6
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2
2.2
2.4
2.6
2.8
+ESI TIC Scan Frag=150.0V Maize_Spike-r002.d
1 1
Counts vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Corn extract spiked with mycotoxins MassHunter Qual Method Editor
June 22, 2015
Compound results for T2-toxin in corn sample
VIP Session ASMS 2015
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MS1 target score 98.5 (out of 100)
5x10
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0.1
0.2
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0.9
1
1.1
1.2
1.3
1.4Cpd 36: T-2 Toxin: +ESI EIC(467.22756, 484.25411) p_tMSMS_Maize_30ppb.d
7.813948467
Counts vs. Acquisition Time (min)6.8 7 7.2 7.4 7.6 7.8 8 8.2 8.4 8.6 8.8 9
4x10
00.5
11.5
22.5
33.5
44.5
5
5.56
6.57
7.58
Cpd 36: T-2 Toxin: +FBF Spectrum (rt: 7.813-7.881 min) p_tMSMS_Maize_30ppb.d
489.20950(M+Na)+
484.25401(M+NH4)+
485.25696(M+NH4)+ 490.21204
(M+Na)+
486.25870(M+NH4)+
491.21437(M+Na)+488.26962
(M+NH4)+
Counts vs. Mass-to-Charge (m/z)483 484 485 486 487 488 489 490 491 492 493 494 495
Library score 91.8 (out of 100)
x102
0
0.2
0.4
0.6
0.8
1
+ESI Product Ion (rt: 7.766, 7.801, 7.835, 7.869 ... min, 10 scans) Frag=380.0V [email protected]
185.09637
245.11674 305.13691
105.06965365.15824
x102
-1
-0.5
0
0.5
1
+ESI Product Ion (rt: 7.766, 7.801, 7.835, 7.869 ... min, 10 scans) Frag=380.0V [email protected]
185.09637
245.11674 305.13691105.06965
365.15824
x102
0
0.2
0.4
0.6
0.8
1
T2 / T-2 Toxin C24H34O9 + Product Ion Frag=120.0V [email protected] Mycotoxins_AM_PCDL
215.10666185.09609
245.11722 305.13835
105.06988
365.15948
Counts vs. Mass-to-Charge (m/z)40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
215.10631
215.10631
June 22, 2015
Easy method setup using All Ions MS/MS
• Fragmentation without precursor isolation- Low energy trace (no collision energy, full spectrum acquisition)- At least one high energy trace (e.g. CE = 20 eV, fragment ion spectra)
- Extremely fast- Non-target acquisition including product ions
VIP Session ASMS 2015
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June 22, 2015
Easy data mining and unambiguous identification
• PCDL loads inside MassHunter Workstation Software –just run samples and then press search- Find-by-Formula database search- Automatic extraction of major
fragment ions from library
- Coelution scoring of precursorand fragment ions
VIP Session ASMS 2015
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June 22, 2015
Quantitation of mycotoxins using accurate massBatch review process using Compounds at a Glance
Blankextract
Spike 1
Spike 2
VIP Session ASMS 2015
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June 22, 2015
Cross-Validation
Standards + 3 matrices (maize, hazelnut and red wine)Subset of analytes (45)• ionising in pos. or neg. mode• polar to apolar compounds• small and large mycotoxins• (de-)protonated ions or adduct ions
8 levels (0.1 to 1000 µg/L)Procedure: • selection of blank samples• spiking after extraction• application and evaluation
- Target MS/MS screening- All Ions approach
VIP Session ASMS 2015
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June 22, 2015
Multi-mycotoxin screeningSample preparation
Sampling
Milling
Extraction
Analysis
Evaluation
blender (household mill)
representative sample
acetonitrile:water:formic acid; 79:20:0.1, v:v:v90 min at room temp. on rotary shaker
Filtration
Clean-up
June 22, 2015
VIP Session ASMS 2015
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Results Cross-Validation
• Compounds detected for scores > 75
• Library score > 60 or co-elution score > 90 for verification
VIP Session ASMS 2015
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Target MS/MS Compound name All Ions MS/MS Red wine Maize Hazelnut Red wine Maize Hazelnut 15-Monoacetoxyscirpenol 16-Keto-Aspergillimide 3-Acetyldeoxynivalenol Aflatoxin B1 Aflatoxin B2 Aflatoxin G1 Aflatoxin G2 Aflatoxin M1 Agroclavine Alternariol Alternariolmethylether Aspergillimide Beauvericin Brevianamid F Curvularin Cyclopiazonic acid Cyclosporin A Diacetoxyscirpenol Deoxynivalenol Emodin Enniatin B Ergosine Ergosinine
Ergosinine Ergotaminine/Ergotamine Fumonisin B1 Fumonisin B2 Fusarenon-X HT-2 toxin Macrosporin Moniliformin Mycophenolic acid Nivalenol Ochratoxin A Paraherquamide A Patulin Roquefortine C Skyrin Stachybotrylactam Sulochrin T-2 Toxin Tenuazonic acid Terphenyllin Viridicatin Zearalenone Compound found by FBF and verified by
library matching or co-elution of
Compound found by FBF, not verified
Target MS/MS Compound name All Ions MS/MS Red wine Maize Hazelnut Red wine Maize Hazelnut
June 22, 2015
Screen, identify, and quantitate
• Mycotoxin detects in a contaminated hazelnut sample
• Brevianamid F (<LLOQ), • Alternariol (310 µg/kg)• Mycophenolic acid (6100 µg/kg)• Zearalenone (21 µg/kg)• Alternariolmethylether (220 µg/kg), • Macrosporin (520 µg/kg)
VIP Session ASMS 2015
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Mass deviation < 5 ppm forprecursor and fragment
Perfect coelution Consistent ion ratios
June 22, 2015
Take home message
• HR Q-TOF LC/MS screening with an Agilent Q-TOF and the new accurate mass MS/MS library for mycotoxins and related metabolites allows the unambiguous identification for a practically unlimited number of compounds with a single injection, even without standards and in retrospective data analysis.
VIP Session ASMS 2015
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Acknowledgements
• Elisabeth Varga• Franz Berthiller• Michael Sulyok, Rudolf Krska
• Emma Rennie
See me at poster WP052 for more results.June 22, 2015