Analysis of Marine Toxins in
Shellfish
Andrew Turner
Food Safety Group
Cefas, Weymouth
Overview
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Background to shellfish biotoxins
Context & legislation
Current shellfish monitoring
Method development – PSP toxins
Ongoing work
Introduction
Cefas
c525 Staff
• >100 yr fishery
research
• Executive agency
since 1997
Weymouth (135)
RV Endeavour
Lowestoft (360)
Groups at Weymouth
• Aquatic Animal Disease
– Fish, shellfish, amphibians
• Environment and Animal Health
– Ecotox, welfare, endocrine disruption, pollution
• Food Safety
– Shellfish contam: viruses, bacteria, biotoxins
The Problem: Algal Biotoxins
ASP
DSP
PSP
(Domoic/epi-domoic acid)
(Lipophilic toxins include:
(Saxitoxin, GTX and C toxins)
1989
OA, DTX. YTX, PTX and AZA
groups)
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Nausea
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Diarrhoea
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Vomiting
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Confusion
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Memory loss (may be
permanent)
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1980s
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Nausea
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Abdominal pains
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Vomiting
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Diarrhoea
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May be tumourigenic
Can be fatal
160 µg/kg
3.75 mg/kg
20 mg/kg
Prorocentrum lima
Pseudo-nitzschia spp.
1995
1920s
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Numbness/tingling
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Headaches
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Nausea, Vomiting
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Respiratory distress
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Paralysis
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Can be fatal
800 µg/kg
Dinophysis spp. Azadinium spinosum
Alexandrium spp.
OVERALL ~20 algal species &>35 toxins all present in UK waters and requiring monitoring
The Problem: Algal Biotoxins
ASP
PSP
Toxin levels known to reach
1920s
extreme 1980s
levels 1995
in shellfish
under certain conditions
(Domoic/epi-domoic acid)
(Lipophilic toxins include:
1989
OA, DTX. YTX, PTX and AZA
groups)
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Nausea
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Diarrhoea
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Vomiting
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Confusion
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Memory loss (may be
permanent)
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DSP
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Nausea
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Abdominal pains
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Vomiting
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Diarrhoea
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May be tumourigenic
(Saxitoxin, GTX and C toxins)
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Numbness/tingling
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Headaches
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Nausea, Vomiting
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Respiratory distress
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Paralysis
Toxins are 160
heat
stable: not
µg/kg
20 mg/kg
800 µg/kg
3.75 mg/kg
destroyed by cooking or
processing
Can be fatal
Prorocentrum lima
Pseudo-nitzschia spp.
•
Can be fatal
Dinophysis spp. Azadinium spinosum
Alexandrium spp.
OVERALL ~20 algal species &~33 toxins all present in UK waters and requiring monitoring
Distribution & Mitigation
• 3 toxin groups regulated in Europe:
– PSP, ASP and Lipophilic toxins
• UK and EU affected by all 3 groups
• This is a global problem – worldwide distribution
• Serious Health Risks
• Shellfish must be tested
• Use approved methods
• Compliance vs Reg limits
Context & Current Monitoring
EU Regulations
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Water and shellfish testing
All commercially active shellfish production areas
Min. testing frequency
Toxins to be quantified and methods
Actions to be taken
Validation of methods
Accreditation of OC labs
Responsibility of national competent authority
Requirement for reference laboratories
Programme Framework
ISO17025:2005 accreditation
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Quality system
Training and management
Quality control & proficiency testing
Suppliers
Calibration and validation
Equipment
Improvements
Animal (Scientific Procedures) Act
1984
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Testing methods if involving animals
WHAT, HOW, WHEN, HOW MANY
Training and management
Facilities (min. standard, maintenance)
Community Reference Laboratory
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Standard methods
Proficiency testing
Method development
Method validation
National Reference Laboratory
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Standard methods
Recommendations
Audits
Proficiency testing
Food Standards Agency
Cefas
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UK Competent Authority (CA)
Responsible for Official Controls on bivalve molluscs
Sub-contract testing to OC labs
Lead UK “Shellfish Partnership” for OC testing
Run biotoxin monitoring programmes
Conduct OC testing
Methods – use of animals
• Official Reference Method for PSP is still a
Mouse Bioassay (MBA)a
– Direct quantitative toxicity determination
• Until 2011 (with leeway until 2015), RM for DSP
also the MBAb
– Qualitative assay only
– New EU RM for LT = LC-MS/MS
• No issues with ASP – RM is HPLC-UV
aAOAC
959.08; bbased on Yasumoto et al., 1978
Implementation of “new” methods
Process is time-consuming:
• Method developed and single-lab validated:
– Must follow full EC / IUPAC guidelines
– Demonstrate “equivalence” with current ref methods
• Formal multi-lab collaborative study
– Following specific guidelines (e.g. AOAC)
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Publication as Official Method (e.g. AOAC, CEN)
Method acceptable within EU legislation
Approval by Competent Authority and COT
Accreditation to ISO17025
Implementation now may be possible
Challenges – Chemical Methods
Chemical diversity
of toxins
Large numbers of
toxins
Method
complexity
Practical issues
Using chemistry
to estimate
toxicity
Method performance
variability between
shellfish from different
sources
Species
differences
Requirement for
standards
(calibration & matrix)
Fast turnaround
requirements +
contingency
requirements
Challenges
Large number of
toxins
Poor perception
Species differences
Chemical diversity
Incomplete validation to date
Practical issues
Performance issues
Method complexity
Requirements for
method validation
Current monitoring
Monitoring programmes for shellfish
•>200 sites
Flesh monitoring programme
Samples of shellfish are collected
from pre-determined monitoring
points within commercially active,
classified production and relaying
areas
•>3,500
samples
•Covering all
of GB
Shellfish testing process
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Samples received at Cefas daily
Shellfish shucked, >100g tissue homogenised
Sub-samples for each of three testing methods
Extraction, clean-up, derivatisation
Analysis overnight
Results reported next day (customer requirement)
• Results >MPL = shellfish beds closed for
harvest
• Two consecutive <MPL to re-open
Analyse for regulated
toxins
PSP
Lipophilic
toxins
ASP
Biotoxin Detection Methods
ASP
• Domoic acid & epi-domoic acid – total content of
whole shellfish or edible part alone
• EU reference method: HPLC-UV
HPLC
• Shellfish + 50% Methanol
extraction
• With or without SPE clean-up
• Very simple, reproducible – no
major issues
HPLC Chromatogram
Matrix peaks
Domoic acid
Matrix peak
LC-MS/MS for Lipophilic Toxins
From 1st July 2011
• EU Reference Method
• EU-RL SOP specifies:
– Aims and scope
– Extraction and general conditions
– Performance characteristics
• Can use any instrumentation
as long as method is specific,
robust, linear and acceptable
performance verified in-house
•MBAs may still be used
until 31st December 2014
•Afterwards, the MBA shall
be only used during the
periodic monitoring of
production
LT method overview
Shellfish
homogenate
100% methanol
extraction
Alkaline hydrolysis for
OA/DTX esters
• Determination of toxins in following
groups:
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Okadaic acid (OA and DTXs)
Pectenotoxin (PTXs)
Azaspiracid (AZAs)
Yessotoxin (YTXs)
• Direct determination of toxins
available as reference standards
– Indirect determination of other toxins
Filtration
LC-MS/MS
• LOQ & MU: varies depending on
toxin and shellfish species
LT LC-MS/MS
• Proportion of OA/DTXs as acyl-esters
• Either low or high pH mobile phase – both
present chromatographic challenges
• +/- switching to encompass all groups
• Known response drift issues with negmode toxins (OA/DTX)
• Highly variable matrix effects
• Known TEFs enable calculation of sample
toxicities
• Now implemented in >10 countries
• PT results from Quasimeme still indicate
high variability in performance between
different labs
Lipophilic
toxins
Detection of PSP
PSP toxins
• Saxitoxins (>55 analogues)
• Sodium channel blocker
• Highly potent neurotoxin*
– Mice LD50 ~ 10 µg/kg (I.P injection) for STX
– Acute oral tox of STX in mice 209-588 µg/kg
– Severe illness in human reported at 5.6 – 2,058 µg/kg STX eq
• Listed in Chemical Weapons Convention
• Majority are hydrophilic – but analogues also exist with
hydrophobic substituents
• Stable in weakly acidic solutions
• MPL = 800 µg STX eq/kg shellfish tissue
*R. Munday (2014) Toxicology of Seafood Toxins: A critical review
PSP
toxins
Saxitoxin
derivatives
• N-hydroxyl
– Carbamate NEO, GTX1&4
– Decarbamoyl dcNEO, dcGTX1&4
– N-sulfocarbamate GTX6, C3&4
• Non N-hydroxyl
– STX, GTX2&3, dcSTX, dcGTX2&3, GTX5, C1&2
• Others
– M toxins, GC toxins and more…
• All have different toxicities; TEF of some still unknown
PSP
toxins
Saxitoxin
derivatives
Thankfully: PSTs commonly occurring
in naturally contaminated shellfish in
• N-hydroxyl
UK/EU are available as standards and
– Carbamate NEO, GTX1&4
mostdcNEO,
have dcGTX1&4
fairly well described TEFs
– Decarbamoyl
– N-sulfocarbamate GTX6, C3&4
• Non N-hydroxyl
– STX, GTX2&3, dcSTX, dcGTX2&3, GTX5, C1&2
• Others
– M toxins, GC toxins and more…
• All have different toxicities; TEF of some still unknown
PSP Methods
1. AOAC 959.08 MBA (Ref method)
2. Alternative methods authorised in EU under reg.
2074/2005:
– Includes: “Lawrence method” - HPLC (AOAC 2005.06
method)
3. Other methods authorised in US/Canada:
– PCOX LC-FLD (AOAC 2011.02)
– Receptor Binding Assay (AOAC 2011.27)
4. Until recently no validated LC-MS method
– None authorised in EU or US legislation
1. PSP MBA
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Until Oct 2006 – all PSP testing conducted by MBA
Shellfish homogenised & extracted (HCl)
Injected into multiple mice
Death time gives PSP toxicity
Assay calibrated using STX solution bimonthly
LOD: ~330 µg STX eq./kg flesh (<1/2 regulatory limit)
• Negative samples – no death – mice still euthanised
• Many thousands mice required for routine testing
2. PSP by LC-FLD – more complex
Taken from M. Boundy, 2015
PSP LC-FLD
(AOAC 2005.06 OM)
Shellfish homogenate
Extraction
(1% Acetic acid)
C18 SPE clean-up / pH adj
Periodate ox (screen)
HPLC-FLD
Ion exchange SPE
(fractionation)
Peroxide
oxidation
HPLC-FLD
Fraction1 (C
toxins)
Fraction2
(GTX1/4, GTX2/3,
GTX5, GTX6*)
STX, dcSTX, GTX2/3,
GTX5, C1/2, dcGTX2,3
Perox.
Period
C1/2
C3/4*
Perox.
GTX2/3,
GTX5
* No certified standards currently available
Period
HPLC-FLD
GTX1/4
GTX6*
Unoxidised
Non-toxic coextractives
Fraction3 (STX,
NEO, dcNEO,
dcSTX)
Perox.
Period
HPLC-FLD
STX,
dcSTX
NEO,
dcNEO
HPLC-FLD
PSP LC-FLD
(AOAC 2005.06
OM)
Today’s
event summary?
“This meeting aims to cover developments in
analytical instrumentation that make it possible
to simultaneously analyse numerous pollutants
in complex matrices using minimal sample
clean-up”
Shellfish homogenate
Extraction
(1% Acetic acid)
C18 SPE clean-up / pH adj
Periodate ox (screen)
HPLC-FLD
Ion exchange SPE
(fractionation)
Peroxide
oxidation
Other than the “complex matrices”, this method
misses this aim on many levels!
HPLC-FLD
Fraction1 (C
toxins)
Fraction2
(GTX1/4, GTX2/3,
GTX5, GTX6*)
STX, dcSTX, GTX2/3,
GTX5, C1/2, dcGTX2,3
Perox.
Period
C1/2
C3/4*
Fraction3 (STX,
NEO, dcNEO,
dcSTX)
Even so – highly effective for monitoring
Used for OC since 2008
Perox.
GTX2/3,
GTX5
* No certified standards currently available
Period
HPLC-FLD
GTX1/4
GTX6*
Perox.
Period
HPLC-FLD
STX,
dcSTX
NEO,
dcNEO
Consequences: Use of mice
16000
1400016000
1200014000
12000
Number of mice
10000
10000
8000
6000
4000
Number of mice used
8000
Projected
mice
Projected mice
used used
6000
4000
2000
2000
0
0
2001
2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012
2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012
YearYear
Total UK replacement of MBA for shellfish food safety
3. Other PSP methods
• AOAC 2011.02 – Post-column oxidation
LC-FLD:
– Not in EU legislation
– Requires at least 2 columns/systems to run
each sample
– Use of ion-pairing chromatography
– Very short column lifetime
– Prone to matrix effects – false +/-
4. LC-MS/MS options?
• Considered the “Holy Grail” for PSP
• HILIC-MS/MS approach first demonstrated
in 2007, but issues with:
– Sensitivity and run-time
– Huge matrix effects in shellfish extracts
– In-source fragmentation implications
• All 3 issues needed resolving…
• Cefas/Cawthron 2014 collaboration
a) Sensitivity & run-time
• Waters Acquity &
Xevo TQ-S
• 10-50 times sensitivity
of Xevo TQ
• Enabled very low
LOD/Q for standards
• Fast UPLC – ideal for
fast cycle time
b) In-source fragmentation mitigation
• Traditionally all PST
MRM acquired in +ve
mode
– 100% source fragment
– Shared parent ions
– Shared MRMs
– Specificity lost in source
Two solutions:
i) –ve mode for some GTX transitions
ii) Optimise HILIC separation
HILIC separation
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Waters BEH
Amide HILIC
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+/- switching
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Full separation of
critical pairs,
including epimers
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Total cycle time
of 8.5 min for all
PSTs
c) Matrix effects - salts
• 4 most common metals in ocean are Na, Mg, K & Ca
• Each observed as formate salt clusters in –ve ion
scans
• High salt conc =
Both
– Large PST suppression
– Retention time changes
– Poor chromatography
chromatography
AND ionisation is
compromised
-ve ESI 2D plot MS scan showing elution of the four major salt cations K+, Na+, Mg2+, Ca2+
c) Removal of matrix effects
• Will “dilute and
shoot” work for
PSTs in
shellfish?
Even with x20 or
higher dilution,
matrix effects
remain
Mean recoveries (n=8) and LOQ (µg STX.2HCl
eq/kg) for mussel extracts diluted 1/20 in
80:20:0.25 acetonitrile/water/acetic acid
c) Removal of matrix effects
• Supelclean ENVI-Carb 250mg/3mL GPC
SPE cartridges
• Fast auto clean-up on Gilson SPE robot
• Cartridges re-usable
c) Matrix effects - salts
• Use of Carbon SPE
• Levels of Na, Mg, K & Ca reduced by ~90-100%
• Na formate remains:
– some interference with C1 MRM
2D Plot –ve ion mode MS scan: P. oyster before & after carbon SPE clean-up
c) Matrix effects - salts
• Na formate MRM
452.7 > 113
• Check elution
with C1 routinely
c) Removal of matrix effects
• Recovery of
carbamates hugely
improved
• Peak shape
acceptable
• Improved sensitivity
Comparison of mean recoveries (n=8) and LOQ
(µg STX.2HCl eq/kg) for mussel extracts diluted
1/20 in 80:20:0.25 acetonitrile/water/acetic acid
and with carbon SPE cleanup.
Method sensitivity
• Excellent
comparison
with LC-FLD
methods
• Up to x50
improvement
in LOQ vs
current LCFLD method
HILIC-MS/MS vs LC-FLD
Comparison of PST results (µg STX di-HCl eq/kg) generated by HILIC-MS/MS and LC-FLD
methods on shellfish tissues from the UK between 2009-2014 (n=1,141)
PST HILIC-MS/MS summary
• Good method performance
– Key points are SPE, optimised HILIC and use
of sensitive TQ-S
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Fully validated for 12 species of interest
Good equivalence vs. LC-FLD methods
Accepted for publication
Collaborative trial planned
Aiming for AOAC and/or CEN method
Other findings
1) Assessment of UK shellfish revealed first
detection of novel PSP toxins “M toxins”, not
detected with LC-FLD
2) LCMSMS method incorporates Tetrodotoxin (TTX)
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Pufferfish; Not expected in bivalves; never seen in European
bivalves
Potential bacterial source e.g. Vibrio
Analysis of vibrio-positive oysters & mussels from south
coast found contain TTX
First evidence for TTX in UK waters (or N. Europe)
First proof of TTX in European bivalve molluscs
Ongoing work
Method development
•Further develop faster LC methods
•Other sample clean-up methods
•Further automation - where possible
•Assessment of biomolecular methods in parallel
•RBA, rapid tests, SPR, “molecular” techniques
Expanding toxin discovery
•Assessment of TTX impact within UK and EU
•Cyanotoxin contamination with food products
•Other new/emerging toxins for UK:
•Other AZA analogues
•Pinnatoxins and other cyclic imines
•Brevetoxins
•Other fish toxins – potential concern re: import
Conclusions
•Current chemical testing methods effective, but
complex & time-consuming
•UPLC-MS/MS has presented opportunities for
method simplification and more accurate
quantitation
•HILIC-MS/MS is a great tool for small polar PSTs
•Highly sensitive MS/MS systems are great – but
do not solve all problems
•Salt-removal is key for successful PST method –
may be of benefit to other methods for small polar
analytes?
Today’s event summary?
“This meeting aims to cover developments in
analytical instrumentation that make it possible
to simultaneously analyse numerous pollutants
in complex matrices using minimal sample
clean-up”
•With UPLC-MS/MS – yes, BUT
•Cannot envisage completely losing need for
sample clean-up
•Highly sensitive mass specs help but not
the solution to all problems
Acknowledgements
•FSA England and Wales
•FSA Scotland
•Cawthron Institute, New Zealand
•All in Biotoxin Team at Cefas, Weymouth
RSC for inviting me today