the new pcdl for mycotoxins and related metabolites and... · the new pcdl for mycotoxins and...

VIP Session ASMS 2015

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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


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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


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• … in addition there are masked mycotoxinsJune 22, 2015

MycotoxinsChemical diversity – a challenge not only for sample prep


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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)


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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


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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


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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


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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


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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


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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


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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.


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June 22, 2015

Acquisition of library data for Ochratoxin A

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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


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


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


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


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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


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7x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

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


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MS1 target score 98.5 (out of 100)

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

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

Compound results for T2-toxin in corn sample


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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


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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


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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


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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


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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


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Results Cross-Validation

• Compounds detected for scores > 75

• Library score > 60 or co-elution score > 90 for verification


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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)


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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.


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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

Screening and verifying mycotoxins using the PCDL


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June 22, 2015

Publication No.5991-5667EN

the new pcdl for mycotoxins and related metabolites and... · the new pcdl for mycotoxins and...

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