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©2015 Waters Corporation 1
Analysis of Mycotoxins by Tandem
Quadrupole Mass Spectrometry
©2015 Waters Corporation 2
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
©2015 Waters Corporation 3
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
©2015 Waters Corporation 4
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
©2015 Waters Corporation 5
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…
©2015 Waters Corporation 6
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
©2015 Waters Corporation 7
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
©2015 Waters Corporation 8
Semi-quantitative test kits- IAC columns with fluorometer detection
©2015 Waters Corporation 9
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
©2015 Waters Corporation 10
Aflatoxins analysis in foodstuffs
ACQUITY UPLC H-Class with direct
Fluorescence Detection
Collateral number: 720003286en
©2015 Waters Corporation 11
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
©2015 Waters Corporation 12
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)
©2015 Waters Corporation 13
Aflatoxin analysis kit – chromatographic
separation, AF spiked milk powder
Minutes1.70 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
©2015 Waters Corporation 14
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)
©2015 Waters Corporation 15
Rapid MS/MS analysis of patulin
in apple juice
UPLC with TQD
©2015 Waters Corporation 16
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
©2015 Waters Corporation 17
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
©2015 Waters Corporation 18
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
©2015 Waters Corporation 19
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
©2015 Waters Corporation 20
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
©2015 Waters Corporation 21
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
©2015 Waters Corporation 22
Chromatographic performance –apple
juice
Time0.60 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
1001161
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
©2015 Waters Corporation 23
Linearity – apple juice
TargetLynx is used for
quantitation, QC checks and
ion confirmation
Linear range 1 – 1000 μg L-1
©2015 Waters Corporation 24
Multi-mycotoxin analysis in pistachio,
almond and cashew nuts
Collaboration with
• André de Kok
• Peter Rensen
• Martien Spanjer
UPLC with TQD
©2015 Waters Corporation 25
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
©2015 Waters Corporation 26
Automated MRM scheduling functionality
MRM Method Development Streamlining the workflow – Quanpedia
©2015 Waters Corporation 27
Total Ion Chromatogram (TIC) Mycotoxins spiked in almond extract
Time2.00 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 Sterigimatocystin Various dwell times and time windows employed to achieve
12 data points across each peak
Nivalenol
Cyclopiazonic acid
©2015 Waters Corporation 28
TargetLynx Browser
©2015 Waters Corporation 29
Ion Ratio Reproducibility
0
0.2
0.4
0.6
0.8
1
1 3 5 7 9 11 13 15
Injection number
Ion
rati
o
Fumonisin B1, 1.8% Fumonisin B2, 2.3%
Ergotamine, 2.1% Roquefortin, 3.5%
©2015 Waters Corporation 30
Peak Area Reproducibility
0
500
1000
1500
2000
1 3 5 7 9 11 13 15
Injection number
Peak a
rea
Penicillic acid, 4.2% RSD
3-Acetyl-Don, 8.9% RSD
Ergotamine, 5.6% RSD
©2015 Waters Corporation 31
Application of the Xevo TQ-S to
improve sensitivity and reduce matrix
effects
©2015 Waters Corporation 32
Xevo TQ Xevo 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
©2015 Waters Corporation 33
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
©2015 Waters Corporation 34
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
©2015 Waters Corporation 35
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
©2015 Waters Corporation 36
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
©2015 Waters Corporation 37
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
©2015 Waters Corporation 38
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
©2015 Waters Corporation 39
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