An Integrated Strategy for Natural Biotoxin analysis - Waters Corporation - Food Safety

©2015 Waters Corporation 1
Integrated Screening & Confirmatory
Strategy for the Analysis of Natural
Biotoxins

Overview
 Natural biotoxins & their significance?
 Challenges & analytical requirements?
 Screening assays
 Routine quantitative method for 12 regulated mycotoxins
– Benefits of mass detection?
– ACQUITY QDa mass detector
 Advanced MS functionality
– Confirmatory analysis
– Xevo TQ-S for large scale multi-toxin analysis
– Dealing with complex matrices (feedingstuffs)
 LC-MS/MS based phycotoxin analysis (TQ-S)
 Summary

41%
11%6%
12%
6%
6%
6%
12%
Mycotoxin ContaminantionIncidents by
Commodity(2011 -2013) Nuts (Brazil, Cashew, Peanut,
Walnut, Chestnut, Almond)
Alfatoxins
Coffee beans Ochratoxin A
Animal feed Aflatoxins
Spices (paprika & chilli) Aflatoxins
& Ochratoxin A
Peanutbutter Aflatoxins
Apples Patulin
Dried figs Aflatoxins
Confectionary Aflatoxins
Natural toxins – significance?
Natural toxins are chemicals that are naturally produced
by living organisms. These toxins are not harmful to the
organisms themselves but they may be toxic to other
creatures, including humans, when eaten
“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/

What are mycotoxins?
 Mycotoxins are secondary metabolites produced by fungi that
are toxic to humans & animals consuming the products
 Mycotoxins are dangerous for feed & 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 known carcinogen and immunotoxic
– Fusarium toxins, especially fumonisins are neurotoxic and possible
carcinogens, trichothecenes (type A&B) are immunotoxic and zearalenone
is estrogenic
– Ochratoxin A is a nephrotoxin, possibly carcinogenic to humans and
associated with Balkan Endemic Nephropathy

Foodstuffs affected by mycotoxin &
contamination?
 Tree nuts
 Peanuts
 Grains
 Cereals
 Animal feeds
 Coffee & tea
 Fruits
 Vegetables
 Fruit juices
 Honey
 Beer
 Wine
 Dairy produce
 Preserved meat
 Farmed fish
 Rice
 Botanicals
 Spices
 Snack Foods
 Processed foods

Food Legislation – which mycotoxins are
regulated?
 Maximum permitted levels for the major mycotoxins, aﬂatoxins
(AFB1, AFB2, AFG1, AFG2), ochratoxin A (OTA), fumonisins
(FB1, FB2), deoxynivalenol (DON), zearalenone (ZEA) & patulin
are included in the European legislation
 1881/2006/EC, 1126/2007/EC
 Indicative maximum levels for the sum of T-2 & HT-2 have
been recently issued (Recommendation 2013/165/EU)
 Although not regulated yet, attention is paid to the occurrence of
nivalenol (NIV), another Fusarium toxin that frequently
contaminates cereals in combination with DON
 EFSA Opinions (emerging toxins e.g. enniatins, beauvericin)

Current mycotoxin screening strategies?
Performance
criteria
Technique
LC-UV/FL
Immuno-
diagnostic
(ELISA/LFD)
TLC
(old technology but still
relevant in some
geographies)
Ability for multi-
mycotoxin screening
Methods are available for a
large number of
mycotoxins.
Multiple detectors required
to detect all target
compounds
Diverse physiochemical
properties mean an array of
kits are required to cover all
the regulated mycotoxins
Methods are available for a
large number of
mycotoxins. Detection and
identification procedures
have been developed
making use of molecular
properties or reactions with
spray reagents
Detection capability
LODs vary by analyte
Post-column derivatisation
required to achieve
detection limits for
alfatoxins
Typically very sensitive
<<permitted limits
Typically sensitive
<permitted limits
Time-to-result
Longer turnaround times Quick turnaround times Rapid - quick turnaround
times
Flexibility (extension
to other toxins)
Reliant on UV/FL
chromophore
Depends on antibody cross-
reactivity?
Reliant on UV/FL
chromophore or
chromogenic reagent
Quantitative
performance?
Quantitative Semi-quantitative Quantitative

Immunodiagnostic assays for mycotoxins
IAC; LFD; strip tests

Immunoaffinity chromatography
 Immunoaffinity chromatography (IAC) is a type of LC in which the
stationary phase consists of an antibody (or antibody-related reagent)
 IAC represents a sub-category of affinity chromatography, in which a
biologically related binding agent is used for the selective purification
or analysis of a target compound
 The selectivity & affinity of antibodies for their given targets has
made these agents of great interest for many years as immobilized
ligands in affinity chromatography

Semi-quantitative test kits
IAC columns with fluorometer detection

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

Current analytical strategies
- 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

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
 Provides higher sensitivity than HPLC methods
 Uses a specialized flow cell and mercury/xenon lamp, avoids
requirement for post-column derivatization
 Use of UPLC ternary mixing allows chromatographic separation to be
optimized (analysis time reduced 12 to 4 min)

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 < permitted limits without need
for derivatisation
Improved separation, sensitivity and speed

 Wide range of analytes of interest: Data rich spectra
 Often complex matrixes: Superior selectivity offered by Single Ion
Recording (SIR)
 Regulatory requirements and limits: Increased sensitivity
 High throughput of routine samples: Increased analytical capability & scope
 Ease of method development: Increased peak capacity (mass resolution of
chromatographic co-elutions)
 Enhanced consumer safety
Benefits of Mass Detection for
screening analysis?

What is a Mass Spectrometer?
1. Sample
Introduction
2. Ion Source
3. Mass
Analyser
4. Detector
5.Data System
LC, GC etc. Mass Spectrometer

What is a Mass Spectrometer?
Ion Source Mass Analyser (quadrupole) Detector

Analysis of regulated mycotoxins
using single quadrupole MS
(ACQUITY QDa)
 Simple protocol & consolidated method
Potential for expansion of scope (emerging
natural toxins)
Collaboration with Veronica Lattanzio ISPA CNR, Bari, Italy

QDa
PDA
 Mass detection
− m/z 30 to 1250
− ESI positive & negative
 Modular, small footprint
 Minimal maintenance
− Pre-optimised source ESI ±
− Consumable cone aperture
 Minimal user- intervention
− Push button
− Fast warm up/ internal check
− Run samples
“If you can use a PDA, you can use a QDa”
ACQUITY QDa – routine screening tool

Extraction protocol and clean-up
procedure – wheat & maize
 10 g sample + 40 mL water
 Extraction by blending for 2 min
 Add 60 mL methanol
 Extraction by blending for 2 min
 Filter the extract through paper filter
 5 mL of extract & evaporate until reduce the volume to approx 2mL
 Add 5 mL phosphate buffer (pH 7.4)
 Pass the sample through the Myco6in1+ column
 Wash the column with 10 mL water
 Elute the toxins with 3 mL methanol followed by 2 mL water
 Dry the eluate
 Reconstitute the residue with an appropriate volume of LC mobile phase
sequential extraction
with water and
methanol
immunoaffinity
column clean up
sample
analysis

Experimental -1
Multi-mycotoxin screening method
 Sample preparation
 UPLC conditions
Time %A %B
Initial 99.0 1.0
7.00 50.0 50.0
10.0 1.0 99.0
11.5 1.0 99.0
11.6 99.0 1.0
14.00 99.0 1.0
Parameter Setting
UPLC Acquity I Class
Column Cortecs UPLC C18 1.6 μm, 2.1x100 mm
Temperature (oC) 40
Flow rate (ml/min) 0.4
Injection volume (μl) 10
Mobile phase A
composition
Aq 0.2% acetic acid & 1 mM ammonium
acetate
Mobile phase B
composition
MeOH 0.2% acetic acid & 1 mM
ammonium acetate
Run time (min) 14

Experimental -2
Multi-mycotoxin screening method
Parameter Settings
Mode Performance
(rotary pump)
Mass range (m/z) 150 to 800
Acquisition mode SIR
Ionisation mode (ESI) Pos & neg
Desolvation temperature
(oC)
600 (default)
Cone voltage 10 to 20 (analyte
dependent)
Source temperature (oC) 150 (default)
Capillary voltage (kV) 0.8 (default)
Sampling frequency
(scan/s)
5 (default)

Overlay SIR Chromatograms for aflatoxins at
permitted limits in wheat matrix
AFG2
5.85
16216
AFG1
6.19
25728
AFB2
6.54
44160
AFB1
6.86
49509
Aflatoxin Spiked conc
μg kg-1
AFB1 2
AFB2 1
AFG1 1
AFG2 1

Overlay SIR Chromatograms for 12 regulated
toxins at permitted limits in wheat matrix
1.NIV
2.DON
3.AFG2
4.AFG1
5.AFB2
6.AFB1
7. HT-2
8. FB1 9. T-2
Mycotoxin
R.T
(min)
S:N
Spiked
conc
μg kg-1
1. NIV 2.21 1188 750
2. DON 2.97 20955 750
3. AFG2 5.85 5149 1
4. AFG1 6.19 18272 1
5. AFB2 6.53 1967 1
6. AFB1 6.86 12766 2
7. HT2 8.16 430 50
8. FB1 8.38 2682 800
9. T-2+NH4 8.64 64641 50
10. OTA 8.81 993 3
11. Zer 8.86 6682 100
12. FB2 9.00 1967 200
10. OTA
11. Zer
12. FB2
Normalised view

Quantitative, confirmatory
method
 *Analyst familiarisation
 Specificity (analyte and matrix)
 Calibration curve
 Recovery or trueness
 Repeatability (r)
 Within-laboratory
reproducibility
 Reproducibility (R)
 Decision limit (CCα)
 Detection capability (CCβ)
 Ruggedness (applicability)
 6 month storage stability
(solution and matrix)
Qualitative screening method
 *Analyst familiarisation
 Specificity (analyte and
matrix)
 Decision limit (CCα)
 Detection capability (CCβ)
 Specificity (analyte and
matrix)
 Ruggedness (applicability)
 6 month storage stability
(solution and matrix)
Method Validation – requirements
under 2002/657/EC for VDRs
*Not a mandatory requirement

Experimental outline for this study
 % analyte recoveries at maximum permitted levels (ML)
 Repeatability & reproducibility
 Detection & quantification limits (from matrix assisted calibration
graphs)
 Evaluation of matrix effects by comparison of standard & matrix
assisted calibration curves
 Robustness of repeated injections (response stability)
 Compliance with acceptability criteria for MS detection (criteria
established for screening & confirmation cf CD 657/2002/EC & SANCO
12571/2013)

Example linearity in processed corn
matrix
Compound name: AFB1
Correlation coefficient: r = 0.998746, r^2 = 0.997494
Calibration curve: 764.675 * x + 22.6578
Response type: External Std, Area
Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
3
-0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
Response
-0
5000
10000
3
Residual
-5.0
0.0
Compound name: AFG1
Calibration curve: 398.47 * x + -57.5865
Conc
-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00
Response
-0
1000
2000
Conc
Residual
-5.0
0.0
5.0
Compound name: T2 + NH4
Conc
-0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375
Response
-0
50000
100000
Conc
Residual
-0.00
5.00
Compound name: DON
Conc
-0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500
Response
-0
100000
200000
Conc
Residual
-2.50
0.00
2.50

Matrix matched standards

Multi-mycotoxin method performance in
matrix spiked at permitted limits (n=3)
Cornflake matrix
Mycotoxin
Retention time
(min)
ESI Mode
Spiking
concentration
(ug/kg)
% Recovery & (%RSD)
spikes n=3
Correlation
coefficient
(R2)
Slope
Nivalenol 2.2 Pos 750 95 (6.3) 0.9945 63.0
Deoxynivalenol 3.0 Pos 750 104 (5.3) 0.9960 8062.3
Aflatoxin G2 5.8 Pos 2.5 87 (5.0) 0.9957 125.6
Aflatoxin G1 6.2 Pos 1 97 (2.8) 0.9921 62.0
Aflatoxin B2 6.5 Pos 1 104 (4.3) 0.9969 102.1
Aflatoxin B1 6.9 Pos 2 104 (1.8) 0.9946 68.8
HT-2 8.2 Pos 50 102 (6.4) 0.9900 12.8
T2* 8.6 Pos 50 108 (5.5) 0.9976 932.6
Fumonisin FB1 8.4 Pos 800 94 (4.3) 0.9937 3589.4
Ochratoxin A 8.8 Pos 3 60 (10.9) 0.9943 24.0
Zearalenone 8.9 Neg 100 93 (5.9) 0.9986 3118.0
Fumonisin FB2 9.0 Pos 200 66 (5.3) 0.9370 971.1
*NH4 adduct monitored
Maize snack matrix
Mycotoxin
Retention time
(min)
ESI Mode
Spiking
concentration
(ug/kg)
% Recovery & (%RSD)
spikes n=3
Correlation
coefficient
(R2)
Slope
Nivalenol 2.2 Pos 750 97 (5.0) 0.9993 58.8
Deoxynivalenol 3.0 Pos 750 98 (0.7) 0.9973 1107.9
Aflatoxin G2 5.8 Pos 2.5 95 (10) 0.9965 89.4
Aflatoxin G1 6.2 Pos 1 87 (1.2) 0.9981 57.6
Aflatoxin B2 6.5 Pos 1 95 (2.4) 0.9984 6.03
Aflatoxin B1 6.9 Pos 2 89 (1.3) 0.9975 22.7
HT-2 8.2 Pos 50 112 (2.0) 0.9954 19.9
T2* 8.6 Pos 50 106 (1.1) 0.9967 2861.7
Fumonisin FB1 8.4 Pos 800 85 (3.5) 0.9897 64899.6
Ochratoxin A 8.8 Pos 3 101 (4.5) 0.9690 12.7
Zearalenone 8.9 Neg 100 100 (0.6) 0.9830 823.9
Fumonisin FB2 9.0 Pos 200 101 (2.0) 0.9954 2994.5
*NH4 adduct monitored

Large scale multi-mycotoxin method
using tandem quadrupole MS
(Xevo TQ-S)
Regulatory compliance
Extended scope (>35 toxins +)
Enhanced sensitivity
Applicable for challenging matrices
Advanced MS functions (RADAR)

MRM – Multiple Reaction Monitoring
 More selective & sensitive than SIR
– Specific transition needed for response
– Less interference by background ions of
the same mass
 A selected ion is transmitted through the first quadrupole
(precursor ion), fragmented in the collision cell, and a
specified fragment ion is then
transmitted through the second
quadrupole (product ion).

Confirmatory Methods
 Majority of reference methods currently used for quality control
purposes are based on immunoaffinity columns (IAC) with UV, FL or
PDA detection
 Kobra cell in combination with FL is reference for aflatoxins
 For unequivocal confirmation of chemical identity mass
spectrometric detection is required
 Identification criteria established for other residue analysis (cf CD
2002/657/EC)
– Precursor ion (quasi molecular ion)
– Diagnostic fragments
– Ion ratio (q:Q)
– Ion ration tolerances
– Retention time tolerances
 Triple quadrupole MS in MRM mode can easily achieve this criteria
standards vs samples

CD 2002/657/EC
Identification Point (IP) System
 Requirement: mass fragments being measured using MS-MS
techniques e.g. Selected Reaction Monitoring (SRM)
 Group A of Annex I (96/23/EC) 4 POINTS
 Group B of Annex I (96/23/EC) 3 POINTS
 S/N ratio for each diagnostic ion >3:1
 A minimum of 1 ion ratio shall be measured
 Ion ratio tolerances (based on relative ion intensity)

Experimental
TQ-S multi-mycotoxin confirmatory method
A generic and simplified sample extraction protocol using 84:16 (v/v)
acetonitrile: acidified water
Parameter Setting
Instrument Xevo TQ-S
Ionisation mode ES
(pos/neg switching)
Capillary (kV) 3.4
Source temperature (o C) 150
Desolvation temperature
(o C)
400
Cone gas flow (L/hour) 150
Desolvation gas flow rate
(L/hour)
800

MRM Acquisition
TQ-S multi-mycotoxins confirmatory method
Time scheduled
MRM acquisition mode
ES (pos/neg)
switching
>12 data points
across the peak
Quanpedia database
of UPLC & MS
parameters
Ion ratio tolerances
automatically
calculated
(TargetLynx)

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 & time windows employed to achieve
12 data points across each peak
Nivalenol
Cyclopiazonic
acid

TQ-S Confirmatory method
Quantitative performance & linearity
Mycotoxin LoD (ng/ml)*
NIV 1
DON 1
AFB1 0.015
AFB2 0.015
AFG1 0.05
AFG2 0.05
T2 5
HT-2 8
ZEA 0.5
OTA 1
FB1 2
FB2 0.5
*LOD determined in feed matrix extracts

The challenge - matrix complexity &
co-contamination
Feed extract (neat) background BPI
full scan
Simultaneously acquired MRM transitions for
enniatins B1, A1, A, B2

TQ-S sensitivity - reduction in ion suppression
Mixed mycotoxin spiked feed extract
Matrix matched standard comparison to S/Std
Ability to inject a
smaller amount or
dilute the sample
helps reduce matrix
effects
=
 Ion
suppression is
effectively
reduced

TQ-S Analysis of naturally contaminated
feed samples
Extract dilution 1:10
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-2Toxin 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
Numberof mycotoxins found
Mycotoxin
LOD
(ng/g)
Measured Concentration in animal feed extract diluted 1:10 (ng/g)*
Animal feed sample identity and type

Analysis of marine biotoxins using
Xevo TQ-S

Marine Biotoxins (phycotoxins)
 Certain pytoplankton spp produce toxic
allelopathic secondary metabolites
 Under favourable conditions unicellular
algae can proliferate termed “blooms” and
toxins can bioaccumulate in filter-feeding
bivalve molluscs
 Ingestion of contaminated seafood is
estimated to cause 20% of all foodbourne
illness in the USA with around 1.5%
mortality rate globally
 Over the past 3 decades the frequency and
global distribution of toxic algal incidents
have increased & human intoxications from
novel algal sources have occurred

Classification of the toxins
 Toxins vary in hydrophilicity and are
classed by their effects:
– Amnesic Shellfish poisoning (ASP)
Domoic acid
– Paralytic Shellfish poisoning (PSP)
Saxitoxins
– Neurotoxic Shellfish poisoning (NSP)
Brevetoxins
Diarrhetic Shellfish poisoning (DSP)
Okadaic acid (OA) group,
dinophysistoxin (DTX)
Yessotoxins (YTX)
Pectenotoxins (PTX)
– Azaspiracid Shellfish poisoning (AZA)
Azaspiracids
HydrophilicLipophilic

Lipophilic Toxins
 Structures of a) EU regulated toxins and b) non-regulated cyclic imines
Toxin R1 R2
Okadaic acid CH3 H
Dinophysistoxin-1 CH3 CH3
Dinophysistoxin-2 H CH3
Toxin R1 R2
Azaspiracid-1 H CH3
Azaspiracid-2 CH3 CH3
Azaspiracid-3 H H
Toxin R1
Pectenotoxin-1 OH
Pectenotoxin-2 H
Toxin R1 n
Yessotoxin H 1
Homo Yessotoxin H 2
45OH Yessotoxin OH 1
45OH Homo Yessotoxin OH 2
a) b)
Toxin R1 R2 R3 R4
Pinnatoxin-E H OH CH3
Pinnatoxin-F H OH CH3
Pinnatoxin-G O
H
H H
13-desmethyl spirolide C
Gymnodimine

Worldwide Regulations / Procedures
Lipophilic Toxins
 European Union
– Most types of lipophilic marine toxins can be found in shellfish and as a result EU
legislation covers OA, DTXs, PTXs, YTXs and AZAs
 USA
– FDA –via the FDA no routine monitoring programs for these toxins have been established
yet, legislation exists for OA and DTX1
 Canada
– CFIA Regions must have in place a program to adequately monitor marine biotoxins to
ensure that shellfish areas are closed when toxin levels reached proscribed levels
 Chile
– The National Health Service is responsible for detecting toxicity using a bioassay at 40
stations using monthly samples
– The Fisheries Research Institute monitors toxicity in conjunction with universities
– Programmes include measures of phytoplankton to understand more than just toxicity
– PSP & DSP toxins have had the most severe public health and economic impact in Chile

EU methods for official control
purposes
 Pre-2011
 Official method of control was mouse or rat bioassay (Yasumoto et al 1978)
 ESFA have noted the following shortcomings
– 24 hour observation time
– Insufficient detection capability; high variability & limited specificity
– Sacrifice of a large number of animals is involved
 Other assays including LC-F, LC-MS, immunodiagnostic and functional assays
 New regulations established in 2011 (15/2011)
 Since July 2011, the official method for control of shellfish for the presence of
lipophilic marine biotoxins has been LC-MS/MS
– Fixed extraction procedure
– Separation using LC – either an acidic mobile phase or alkaline mobile phase
– Quantitative detection by tandem quadrupole MS

LC-MS/MS Method Development Aims
 Produce a faster method using alkaline
conditions
– HPLC = 15 mins
– UPLC = 5 mins
 Develop the method for regulated and
some non-regulated cyclic imines
compounds
 Optimise method for different matrices
 Generate single day lab validation data

Sample Extraction
 Homogenized whole flesh shellfish tissue (1 g) was
extracted with methanol
 Extract was vortex-mixed and centrifuged
 Supernatant was transferred to a 10 mL volumetric
flask and made up to 10 mL with methanol
 Filter crude shellfish extract prior to spiking / analysis
 For DTX3 (ester forms of OA, DTX1 and -2)
– Extracts also subjected to alkaline hydrolysis using 2.5 M sodium
hydroxide
– Heat alkaline mixture for 40 min at 76oC, cool to RT and neutralise
using 2.5 M HCl

ACQUITY UPLC Method
Alkali Method
 Routine analysis » » rapid
analysis
 Need good separation of
compounds » » high
resolution chromatography
Time
(min)
% A % B
0.00 75 25
4.50 0 100
6.00 0 100
6.10 25 25
8.00 25 25
Mobile Phase A 100% H2O + 2 mM NH4HCO3
(adjusted to pH 11 with NH4OH)
Mobile Phase B 90% MeCN:10% H2O + 2 mM NH4HCO3
(adjusted to pH 11 with NH4OH)
Flow 0.6 mL/min
Column ACQUITY BEH C18
100 x 2.1mm, 1.7μm
Colum temp 30ºC
Inject. volume 2.5 µL

MRM Transitions
ESI Negative
Compound name
Parent
(m/z)
Daughter
(m/z)
Ionisation
Dwell
(s)
Cone (V)
Collision
(eV)
Standard
available
trinor YTX
550.4 396.4 - 0.003 75 30
No
550.4 467.4 - 0.003 75 30
YTX
570.4 396.4 - 0.003 75 30
Yes
570.4 467.4 - 0.003 75 30
homoYTX
577.4 403.4 - 0.003 75 30
No
577.4 474.4 - 0.003 75 30
45OH YTX
578.4 396.4 - 0.003 75 30
No
578.4 467.4 - 0.003 75 30
45OH Homo YTX
585.4 403.4 - 0.003 75 30
No
585.4 474.4 - 0.003 75 30
COOH YTX
586.4 396.4 - 0.003 75 30
No
586.4 467.4 - 0.003 75 30
COOH OH YTX
593.4 396.4 - 0.003 75 30
No
593.4 403.4 - 0.003 75 30
COOH Homo YTX
593.4 467.4 - 0.003 75 30
No
593.4 474.4 - 0.003 75 30
OA/DTX2
803.5 113.1 - 0.003 80 60
Yes
803.5 255.2 - 0.003 80 45
DTX1
817.5 113.1 - 0.003 80 60
Yes
817.5 255.2 - 0.003 80 45

MRM Transitions
ESI Positive
Compound name
Parent
(m/z)
Daughter
(m/z)
Ionisation
Dwell
(s)
Cone (V)
Collision
(eV)
Standard
available
GYM
508.2 162.2 + 0.003 60 55
Yes
508.2 490.2 + 0.003 60 40
SPX1
692.5 164.3 + 0.003 60 55
Yes
692.5 444.2 + 0.003 60 40
PnTX-G
694.5 164.3 + 0.003 60 55
Yes
694.5 676.5 + 0.003 60 40
20-Me SPX G
706.5 164.3 + 0.003 60 55
No
706.5 346.2 + 0.003 60 40
PnTX-F
766.5 164.3 + 0.003 60 55
Yes
766.5 748.5 + 0.003 60 40
PnTX-E
784.5 164.3 + 0.003 60 55
Yes
784.5 766.5 + 0.003 60 40
AZA3
828.5 658.4 + 0.003 35 40
Yes
828.5 792.5 + 0.003 35 30
AZA6 842.5 658.4 + 0.003 35 40 Yes
AZA1 842.5 672.4 + 0.003 35 40 Yes
AZA1/6 842.5 824.5 + 0.003 35 30 Yes/No
AZA4 844.5 658.4 + 0.003 35 40 No
AZA5 844.5 674.4 + 0.003 35 40 No
AZA4/5 844.5 826.5 + 0.003 35 30 No
AZA2
856.5 672.4 + 0.003 35 40
Yes
856.5 838.5 + 0.003 35 30
PTX12
874.5 213.1 + 0.003 40 30
No
874.5 821.5 + 0.003 40 30
PTX2
876.5 213.1 + 0.003 40 30
Yes
876.5 823.5 + 0.003 40 30
PTX11
892.5 213.1 + 0.003 40 30
No
892.5 839.5 + 0.003 40 30
PTX2sa
894.5 213.1 + 0.003 40 30
No
894.5 805.2 + 0.003 40 30

MRMs of Matrix Matched Standard
Mussel extract

Single day validation results
Compound
Concentration
(µg/kg)
Recovery
(%)
RSDr
(%)
RSDrl
(%)
CCα
(µg/kg)
OA 160 99 2.7 4.1 171
DTX1 160 99 7.6 12.2 192
DTX2 160 102 2.6 4.1 171
YTX 1000 100 2.5 4.0 1070
AZA1 160 98 1.3 2.1 166
AZA2 160 98 1.9 3.0 168
AZA3 160 99 1.9 3.0 168
PTX2 160 103 8.7 13.9 197
GYM 200 99 3.9 6.3 221
SPX11
100 108 14.6 23.4* 141
SPX12
100 104 12.8 20.4 135
PinE 200 122 23.1 36.9* 347
PinF 200 91 5.1 8.1 224
PinG 50 102 3.9 4.8 54

Summary
 Complete solution for targeted natural toxin analysis applicable for
complex matrices
 Immunodiagnostic assays & core detectors
– Point-of-control testing
– Cost-effective
 ACQUITY QDa – accessible MS suited for routine screening
– Increased scope & selectivity
– Screening “plus” (in-source CID)
 Xevo TQ-S – ultimate sensitivity MS suited for robust confirmation
– Confirmatory technique (MRM)
– Enhanced sensitivity
– Overcome challenges? (matrix interferences; requirement for labelled
internal standards; low sample volume)

An Integrated Strategy for Natural Biotoxin analysis - Waters Corporation - Food Safety

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