Mycotoxins are are secondary metabolites produced by fungi and are dangerous for feed and food chains as they can create contamination in pre- and post-harvest processes. Many are highly toxic and as such levels in food products are regulated in Europe, the US, Japan and other countries. This presentation is an overview of the application of ultra-performance liquid chromatography combined with tandem quadrupole mass spectrometry to analyse various food products for mycotoxins in line with regulatory requirements.

1. ©2015 Waters Corporation 1
Analysis of Mycotoxins by Tandem
Quadrupole Mass Spectrometry

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Overview
 Mycotoxins and their significance?
– Mycotoxins & phytotoxins
 Mycotoxin occurrence data
 Analytical strategies for mycotoxins
– Vicam rapid screening methods
– Aflatoxin analysis with FL
– H-Class Xevo TQD
o Analysis of patulin in apple juice
o Multi-toxin method
– Xevo TQ-S
o Analysis of complex matrices (reduction of matrix effects)
 Summary

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What are mycotoxins & phytotoxins?
 Mycotoxins are secondary metabolites produced by fungi that are toxic to
humans & animals consuming the products
 Phytotoxins are substances produced by plants e.g. alkaloids, terpenes,
phenolics, herbicides
“They represent one of the most important and sensitive problems for our world and
our life, as various many products we normally use in our diet are exposed to their
contamination” MycoRed FP7 Project http://www.mycored.eu/
 Mycotoxins are dangerous for feed and food chains as they can create
contamination in pre- and post-harvest processes
 Resistant to decomposition, digestion high or low temperature degradation &
remain in the food
Toxic Effects
– Aflatoxin B1 is a carcinogen. It is immunotoxic and causes stunted growth in children
and growth retardation in animals
– Fusarium toxins, especially fumonisins are neurotoxic and possible carcinogens,
trichothecenes are immunotoxic and zearalenone is estrogenic
– Ochratoxin A is a nephrotoxin, possibly carcinogenic to humans and associated with
Balkan Endemic Nephropathy

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Foodstuffs effected by mycotoxin &
phytotoxin contamination
• Tree nuts
• Peanuts
• Grains
• Wine
• Coffee
• Cereals
• Feed
• Fruits and
vegetables
• Fruit juices
• Honey
• Oats
• Ethanol
• Dairy
• Rice
• Botanicals
• Spices
• Snack Foods
• Pet Food

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Global occurrence & significance of
mycotoxins?
The occurrence of mycotoxins in food and feed typically shows a geographical
pattern but can be affected by climatic changes…

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Analytical requirements for mycotoxins &
volume of testing
HR-MS
LC-
MS/MS
(QqQ)
LC +core detector
Fluorometer
ELISA
Strip Tests
Research and metabolomic based analysis
Academic, government, commercial
laboratories
Finished food products, biological
samples, speciation, conjugated
metabolites
High volume testing - field tests, short
turnaround times
Analysis conducted at receiving
points, transit points, processors
sites, commercial labs
Quantitative laboratory based testing,
Commercial & official control laboratories
finished foods & feeds, complex matrices,
multi-toxin analysis

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VICAM rapid screening solutions -
Immunoaffinity columns and strip tests
• AflaTest
• AflaTest WB
• Afla WB SR
• Afla M1 HPLC
• AflaOchra HPLC
• AOZ HPLC
• Myco6in1
• CitriTest HPLC
• DONtest
• DONtest WB HPLC
• DON-NIV WB
• FumoniTest
• FumoniTest WB
Fluorometeric Tests
AflaTest
Afla B
Afla M1 FL+
FumoniTest
FumoniTest 200
OchraTest
ZearalaTest
HPLC/UPLC/LC/MS Tests
using IAC
Aflatoxins, DON, NIV,
T-2, HT-2, OTA,
fumonisins,
zearalenone
New 6 in 1 IAC
Qualitative Strips
AflaCheck
DONCheck
Quantitative Strips
Afla-V
DON-V
Fumo-V
http://vicam.com/products

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Semi-quantitative test kits-
IAC columns with fluorometer detection

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PBS SAMPLE EXTRACTION
Extract sample with PBS
Centrifuge and remove 35ml PBS supernatant
70% METHANOL EXTRACTION
Add 35ml methanol to make a 70% solution
Extract sample, centrifuge and remove supernatant.
Dilute supernatant and filter through glass
microfibre filter
(Extract B)
AFFINITY CHROMATOGRAPHY STEP 1
Pass 50 mL Extract B over affinity column.
Wash column with PBS
AFFINITY CHROMATOGRAPHY STEP 2
Pass 5 mL Extract A over column.
Wash column with water.
Elute toxins.
FILTRATION
Filter through glass
microfibre filter
(Extract A)
Meets CEN
criteria
Can be used
with LC detectors
or LC-MS/MS
Myco6in1” Extraction Procedure

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Aflatoxins analysis in foodstuffs
ACQUITY UPLC H-Class with direct
Fluorescence Detection
Collateral number: 720003286en

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Introduction – aflatoxin analysis
 Routinely analyzed using RP HPLC with FL detection
• Reverse phase eluents quench the fluorescence of aflatoxins
B1& G1
• Derivitization is needed to enhance the response
 Derivitization methods for aflatoxins include;
• Post-column iodine addition
• Electrochemically generated bromine using a Kobra Cell®
• Photochemical Reaction for Enhanced Detection (PhCR)
 Post-column derivatisation can interfere with FL detection of
other mycotoxins in multi-toxin analysis!
 Limits sample throughput

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Aflatoxin Analysis Kit
 Vicam AflaTest® WB provides selective extraction for aflatoxins
using wide-bore immunoaffinity columns (IACs)
 Waters UPLC method uses the ACQUITY™ Fluorescence Detector
 Uses a specialized flow cell and mercury/xenon lamp, avoids
requirement for post-column derivatization
 Provides higher sensitivity than HPLC methods
 Use of UPLC ternary mixing allows chromatographic separation to be
optimized (analysis time reduced 12 to 4 min)

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Aflatoxin analysis kit – chromatographic
separation, AF spiked milk powder
Minutes
1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80
5
4
3
1
2
Aflatoxins
1 Aflatoxin M1
2 Aflatoxin G2
3 Aflatoxin G1
4 Aflatoxin B2
5 Aflatoxin B1
ACQUITY FLR Detector with large
volume flow cell
FL detection; Ex 365 nm and Em 429
AF B1 & G1
signal quenching
 Allows detection of the
aflatoxins at <EU permitted limits
without need for derivatisation
Improved separation, sensitivity and speed

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Aflatoxin analysis kit – performance in
spiked matrices
Spiking levels: 4 μg kg-1 total G and B2, 1.5 μg kg-1 B1 and 0.05 μg kg-
1 M1 (cereal only)

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Rapid MS/MS analysis of patulin
in apple juice
UPLC with TQD

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Patulin - Introduction
 Patulin is produced by Aspergillus and Penicillium fungi species on apples
• Damaged fruit more susceptible to infection
• Suspected genotoxicity
 5-hydroxymethylfurfural (HMF) is formed from sugars during thermal
processing and shares UV chromophore with patulin at λ276 nm
 Legislative limits in EU, US FDA and Japan for apple juice are 50 µg L-1
,
infant apple juice 10 µg L-1 and for solid apple products 25 µg kg-1
Patulin and HMF

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Why choose UPLC-MS/MS?
UPLC uses sub 2 μM particles, mobile phases
at higher linear velocities and operating at
higher pressures than HPLC =
 Improved resolution for complex mixture
analysis
 Enhanced resolution reduces ion suppression
by separating species that co-elute in HPLC
 Shorter run times without compromising
chromatographic resolution, increasing sample
throughput
 Narrower chromatographic peaks increase
concentration of analytes entering the MS source,
increasing signal intensity and improving LoDs
Speed Resolution
Sensitivity

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MS/MS Detection
 TQ Detector (TQD) designed to be;
• UPLC compatible (short inter scan &channel delays)
• Small foot print
• Multi-mode (ionisation /acquisition)
• Easier to operate
• IntelliStart tools
• Set up checks
• LC & MS method
• Monitor performance
34cm (~13”) of linear bench space

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TQD Schematic
T-wave enabled collision cell allows short ion
residence times =
ensures sufficient data points across narrow UPLC peaks
&allows multiple analytes to be monitored in parallel
MRM mode: MS1 and MS2 are both set to a static value
giving enhanced selectivity for the analytes of interest

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Experimental conditions
Extraction Protocol
 SPE: Waters Oasis HLB 3cc /60mg
 Condition step: 3 mL methanol
3 mL water
 Load: 2.5 mL sample
 Wash 1: 3 mL 1% NaHCO3 (1g/100mL)
 Wash 2: 1 mL 0.1% acetic acid
Dry under vacuum
 Elute: 2 x 1.5 mL 10% ethyl acetate in
methyl t-butyl ether (MTBE)
 Reconstitute: 500 µL water

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Instrument parameters
UPLC Conditions
Mobile phase: H2O – MeCN
gradient (pH 10)
Analytical column: BEH shield
RP18 2.1 X 100 mm 1.7 μm
Flow rate: 0.6 mL/min
Run time: 4.5 min
Injection volume: 20 µL (full
loop mode)
Acquisition: Multiple Reaction
Monitoring (MRM)
Collision gas: argon
Software: MassLynx v4.1 for
acquisition and TargetLynx for
data processing
MS/MS Conditions

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Chromatographic performance –apple
juice
Time
0.60 0.80 1.00 1.20 1.40 1.60 1.80
%
0
100
0.60 0.80 1.00 1.20 1.40 1.60 1.80
%
0
100
0.60 0.80 1.00 1.20 1.40 1.60 1.80
%
0
100
1161
541
7528
HMF
Patulin
Patulin: 153>109
Patulin: 153>81
HMF: 129>95
16 data points across the peak
Reporting level 50 μg L-1
Rt = 1.1 min
Rt = 1.4 min

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Linearity – apple juice
TargetLynx
is used for
quantitation,
QC checks and
ion
confirmation
Linear range 1 – 1000 μg L-1

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Multi-mycotoxin analysis in pistachio,
almond and cashew nuts
Collaboration with
• André de Kok
• Peter Rensen
• Martien Spanjer
UPLC with TQD

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Aim
 To evaluate the suitability of the ACQUITY TQD for the
simultaneous determination of multiple mycotoxins in
foodstuffs at EU legislative limits
 Multiple reaction monitoring (MRM)
 Mycotoxin extracts and solvent standards provided by the
Food and Consumer Product Safety Authority (VWA),
Amsterdam, Netherlands
• Pistachio
• Almond
• Cashew nut

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Automated MRM scheduling functionality
MRM Method Development
Streamlining the workflow – Quanpedia

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Total Ion Chromatogram (TIC)
Mycotoxins spiked in almond extract
Time
2.00 4.00 6.00 8.00 10.00 12.00
%
14
Alfatoxin B1
Alfatoxin B2
Alfatoxin G1
Aflatoxin G2
Ochratoxin A
Deoxynivalenol
Citrinin
Fumonisin B1
Fumonisin B2
Nivalenol
Diacetoxyscirpenol
H2 toxin
HT2 toxin
3-acetyl-DON
15-acetyl-DON
Zearalenone (Zen)
Penicillic acid
Fusarenon X
Ergotamine
Roquefortin
Β-Zearalanol
Α-Zearalanol
Cyclopiazonic acid
SterigimatocystinVarious dwell times and time windows employed to achieve
12 data points across each peak
Nivalenol
Cyclopiazonic
acid

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TargetLynx Browser

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Ion Ratio Reproducibility
0
0.2
0.4
0.6
0.8
1
1 3 5 7 9 11 13 15
Injection number
Ionratio
Fumonisin B1, 1.8% Fumonisin B2, 2.3%
Ergotamine, 2.1% Roquefortin, 3.5%

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Peak Area Reproducibility
0
500
1000
1500
2000
1 3 5 7 9 11 13 15
Injection number
Peakarea
Penicillic acid, 4.2% RSD
3-Acetyl-Don, 8.9% RSD
Ergotamine, 5.6% RSD

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Application of the Xevo TQ-S to
improve sensitivity and reduce matrix
effects

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Xevo TQXevo TQ-S
Ion source
sampling cone
How did we increase sensitivity?
Much larger
sampling orifice and
modified vacuum
system to deal with
increased gas load

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TQ-S Stepwave ion optics
Maximising signal
Minimising noise
Two types of T-wave enabled RF ion tunnels;
On axis and off axis relative to the MS analyser - narrow ion
tunnel conjoined to wide ion tunnel
Entrance to the Stepwave=captures all
ions in the disperse ion cloud

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Comparison of Xevo TQ-MS and TQ-S
Spiked barley AF1 0.1 μg kg-1
Xevo TQ Xevo TQ-S
Quantitative transition
Confirmatory transition
Better peak shape and increased sensitivity (x40) reduces the
number of manual integrations
Processed data becomes more consistent giving more accurate
ion ratios increasing confidence in compound identification
Response of the secondary ion is stronger
40x sensitivity
increase

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Reduction in ion suppression
Spiked feed extract
Matrix matched standard comparison to S/Std
Ability to inject a
smaller amount or
dilute the sample
helps reduce matrix
effects

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 Rapid electronics allow instrument to
switch between MRM and full scan (FS
takes 100ms)
– No loss of MRM data quality
– Added information can be gained from
full scan
o Monitor matrix
o Assess for possibility of matrix
effects
o Added information for sample prep
method development
o Search for significant non-targeted
compounds
RADAR functionality – Xevo TQ-S
Simultaneous full scan & MRM
MRM
Full
scan

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Animal feeds – the challenge matrix
complexity & co-contamination
Feed extract (neat) background BPI
RADAR scan
Simultaneously acquired MRM transitions for
enniatins B1, A1, A, B2

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Measured concentrations for mycotoxins identified in 12
different samples of animal feedingstuffs (diluted 1:10)
using TQ-S
U1 / cattle
feed
U2 / pig
feed
U3 / maize
gluten
U4 / Diva L
Vital pig feed
U5 /Alpha
Maximal pig feed
U6 /
Rye
U7 /
Barley
U8 /
Wheat
U9 /
Oats
U10 /
Maize
U11 /
Sunflower oil
U12 / Pig
feed
15-acetyl-
deoxynivalenol 0.5 nd nd 152.8 nd nd nd 13.2 33.4 nd nd nd nd
Aflatoxin B1 0.001 nd nd nd nd nd nd nd nd nd nd 0.2 nd
Aflatoxin B2 0.001 nd nd 0.8 nd nd nd nd nd nd nd 0.1 nd
Aflatoxin G1 0.001 nd nd nd nd nd nd nd nd nd nd 0.1 nd
Aflatoxin G2 0.001 0.3 nd nd nd nd nd nd nd nd nd nd nd
Alternariol 0.06 nd 3.2 nd nd nd 5.3 nd nd 7.6 2.6 10.0 nd
DON 0.13 nd 21.2 283.6 13.2 18.4 nd nd nd 4.8 nd 0.3 nd
Enniatin A 0.01 59.3 6.3 1.4 15.7 39.9 9.7 11.7 0.4 3.2 nd nd 50.5
Enniatin A1 0.01 148.6 17.1 3.2 40.1 19.0 14.2 34.1 0.5 4.9 nd nd 122.4
Enniatin B 0.01 125.2 43.3 5.8 65.3 53.3 92.8 52.9 0.4 9.0 nd nd 116.1
Enniatin B1 0.01 263.0 41.8 5.5 72.1 32.3 42.8 64.0 0.5 9.9 nd nd 238.2
Fumonisin B1 0.01 0.3 0.7 18.9 nd 4.0 nd nd nd 0.4 92.8 nd 1.7
Fumonisin B2 0.01 0.1 nd 3.1 nd 0.8 nd 0.2 nd nd 16.0 nd 0.3
HT-2 Toxin 0.25 nd nd nd nd nd nd nd nd 3.9 nd nd nd
Ochratoxin A 0.006 0.1 nd nd 0.1 nd 0.2 2.8 nd nd nd nd 0.1
Roquefortine 0.003 nd 0.3 0.3 0.2 0.1 nd nd nd nd nd nd nd
Sterigmatocystin 0.003 nd 0.1 0.4 0.2 nd 10.7 nd nd nd nd 0.1 0.2
Zearalenone 0.2 nd 1.6 84.0 nd 4.9 31.2 nd 6.1 nd nd nd nd
8 10 12 8 9 8 7 6 8 3 6 8
*Concentration determined against a solvent calibration series
Mycotoxin
Measured Concentration in animal feed extract diluted 1:10 (ng/g)*
Animal feed sample identity and typeLOD
(ng/g)
Number of mycotoxins found
Collaboration with University of Ghent
Matrix dilution and Labelled standards for quantitation

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Summary
 Natural toxins pose a significant threat to food security
– Stringent global regulations for contaminant control established
o New EU regulations expected (EFSA opinions 2012)
 Occurrence often linked to climatic conditions
– Highest incident of RASFF alerts in EU
 Wide variety of toxins & very different chemistries…
 Variety of analytical strategies required for adequate control
 Different customer testing requirements
– High volume (PoC) testing
– Semi/ quantitative testing (single toxin)
– Highly complex matrices & need for high sensitivity
– Multi-toxin testing
– Masked toxins and metabolites
 Portfolio of different solutions from Waters & Vicam