testing for marine toxins and dsp
TRANSCRIPT
© 2007 Her Majesty the Queen in right of Canada
(Canadian Food Inspection Agency), all rights reserved. Use without permission is prohibited.
Testing for Marine Toxins and DSP
Wade Rourke
Canadian Food Inspection Agency Dartmouth Laboratory
Dartmouth, NS
Canadian DSP Symposium
Vancouver, BC
November 27, 2012
2
Outline
•Information about toxins and toxin groups
•Information about different methods
•Canadian context and perspective
•Observations from BC samples
3
What are we talking about?
Okadaic acid and analogues (DSP toxins)
Pectenotoxins
Azaspiracids
Yessotoxins
Cyclic Imines
4
Chemical Structures
O
OH OH
O OO
OH
H
OH
OH
O
OHOOC
Okadaic Acid
O
OO
OOH
OHO
O O
O
OOH O
O
Pectenotoxin
O O
O
O
O
NH
O
O
HO
O
OH
H
H
H
HH
H
H
CH3
HOOC
Azaspiracid
O
O
O
O
O
OO
O
O
O
O
OH
OH
NaO3SO
NaO3SO
Yessotoxin
O
O
N
O
H O H
Gymnodimine
5
“DTX3”
Multiple compounds
• DTX3 refers to the fatty acid forms of OA, DTX1 and DTX2
• Could be fatty acid chain of 14-22 carbons
•Difficult to detect directly
• Large number of compounds
• Lack of standards for each compound
•Use enzymatic or alkaline hydrolysis procedure
O
OH
O
OH
O
OO
O
OH
H
OH
OH
O
O
HO
7
6
The problem starts…with hunger
Time
Detoxification
Toxic
Plankton People Digestive Gland
Toxic Incident
7
World Distribution
http://www.whoi.edu/redtide/page.do?pid=18103&tid=542&cid=65844&c=3
8
Mouse Bioassay
• Samples are extracted and then injected into 2-3 mice
• May need multiple extractions depending on toxin profile
• Mice are monitored for 24 hours
• Positive result: ≥2 of 3 mice die
• Negative result: ≥2 of 3 mice live
9
Methods of Analysis: Biological Methods I
Method Advantage Disadvantage
Biological
Methods –
General
•Produce a single
result
•Complex
instruments not
required
•Need several assays to monitor
all toxin groups
•No toxin profile data
Live
Animal
Bioassays
•Potential to detect
unknown toxins
•Poor reproducibility and
sensitivity
•Non-specific
•Use animals
•False positives and negatives
10
Methods of Analysis: Biological Methods II
Method Advantage Disadvantage
Immuno
Assays
•Senstive
•Fast
•Very specific
•Good only as screen
•React to antibody not
receptor
•Different reactivity of
analogues to anitbody
Functional
Assays
•Very sensitive
•Fast
•Test at receptor level
•Independent of toxin
profile
•Requires reliable enzymes
•Must know toxin mode of
action
11
Methods of Analysis
Chemical
Method Advantage Disadvantage
Chemical
Methods -
General
•Toxin profile data
•Complex instruments
required
•Require individual
standards and TEFs
LC-FLD •Good within a group,
not between groups
•Difficult to add new toxins
•Derivatization required
LC-MS/MS •Specific and sensitive
•Analyse toxins directly
•Multi-toxin analysis
•Easiest to add new
toxins
•Prone to matrix effects
•Most expensive
instruments
12
EFSA Recommendations - 2008
• Change action limits
• Decrease limits for OAs, PTXs, AZAs
• Increase limit for YTXs
• Group toxins by structure/mode of action not symptoms
• Change reference method
• Use TEFs to reflect toxicity of individual compounds
• Resulted in reference method changing from MBA to LC-MS/MS in 2011
13
Toxins in Atlantic Canada before 1987
Halifax NS
NB PE
NL PSP
N
NNH2
+ NH
NH
NH2
+
OH
OH
H
R4
R1
R2
R3
Paralytic Shellfish Poisoning
(Saxitoxin group)
14
Toxins in Atlantic Canada, 2005
Halifax NS
NB PE
NL ASP
DSP
SPX
PTX
Ile-de-la-
Madeleine PSP
YTX
AZA
15
Changing Times
• Adopting new technology
• HPLC-FLD with derivatization
• HPLC-MS
• UPLC-MS/MS
• Simplified, efficient extraction
• Ability to monitor toxins directly
• Ability to monitor multiple toxin classes
simultaneously
• Proactive monitoring vs Reactive testing
16
Dartmouth Laboratory – LSTs
# of Toxins Monitored
2 24
5
14
20 20 20 20 20
25 25
29
0
5
10
15
20
25
30
35
2000
/200
1
2001
/200
2
2002
/200
3
2003
/200
4
2004
/200
5
2005
/200
6
2006
/200
7
2007
/200
8
2008
/200
9
2009
/201
0
2010
/201
1
2011
/201
2
2012
/201
3
17
Changing Times
HPLC-FLD
DSP Toxins
Time (min)
10 15 20 25 30 35
m/z 312.5
m/z 508.3
m/z 692.5
m/z 706.5
m/z 805.5
m/z 819.5
m/z 946.5
m/z 1160.7
m/z 876.5
m/z 894.5
m/z 842.5
DAGYM
SPXYTX
PTX2sa
OA
DTX2
OA-D8:2
PTX2
DTX1
AZA1
MA Quilliam. 8th Int. Conf. Harmful Algae, Vigo,
Spain, June 1997.
MA Quilliam et al., In: Mycotoxins and Phycotoxins in
Perspective at the Turn of the Century, Wageningen,
The Netherlands, W.J. deKoe, pp. 383-391, (2001).
LC-MS
Multi-toxin
18
Standards and CRMs
• Needed for instrumental methods
• Difficult to get
• NRC CRMP only supplier of CRMs
• Need to rely on in-house standards and gather material during toxic episodes
• Collaboration within community
• Difficult to validate methods without reliable standards, especially for toxin regulations of different countries with different toxic profiles
19
CRM Availability
• National Research Council Canada
• Certified Reference Material Program
• Available LST CRMs
• OA, DTX1, DTX2, PTX2, AZA1, AZA2, AZA3, YTX, homoYTX,
GYM, SPX1, DSP Mus, AZA Mus
• New materials in planning or in progress
• Zero Mus, FDMT, PnTX-G
20
0
500
1000
1500
2000
2500
2000/2
001
2001/2
002
2002/2
003
2003/2
004
2004/2
005
2005/2
006
2006/2
007
2007/2
008
2008/2
009
2009/2
010
2010/2
011
2011/2
012
2012/2
013
Fiscal Year
# o
f S
am
ple
s
BC Samples Eastern Canada Samples
East vs West Sample Distribution
21
Method Transition
• Comparison and validation of new EU reference method began in April, 2011
• Saw performance and efficiency gains
• Implemented new method in May, 2012
• Average turn-around-time
• November, 2009 – April, 2012: 4.1 days (2900 samples)
• May, 2012 – November, 2012: 3.4 days (1300 samples)
22
Turn-Around-Time: Western Samples
Monday Tuesday Wednesday Thursday Friday
•Samples
arrive in
afternoon
•Day 1
•Samples are
extracted and
analysed
•Day 2
•Data are
analysed and
results
reported
•Day 3
•Samples are
extracted and
analysed
•Day 2
•Data are
analysed and
results
reported
•Day 3
•Samples
arrive in
afternoon
•Day 1
23
Turn-Around-Time
• Laboratory service standard is 5 business days
• Average turn-around-time is 3.4 days
• No difference between Eastern and Western samples
• Applies to regular samples
• Priority samples are analysed as soon as possible
24
LC-MS/MS Chromatogram
DSP Toxins
3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0Time, min
OA
DTX-1
DTX-2
25
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8Time, min
3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0Time, min
3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0Time, min
3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0Time, min
3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0Time, min
LC-MS/MS Chromatogram
Lipophilic Shellfish Toxins
CIs PTXs
AZAs
YTXs
DSPs
26
Yessotoxins - EU
• No human illnesses have been linked to YTXs
• Regulated in EU but not in Canada
• YTX, homoYTX, 45-OH YTX, 45-OH homoYTX
• EU maximum limit: 1.0 μg/g
27
Yessotoxins - Canada
• Maximum YTX levels found in Canada: 12 μg/g
• Combination of YTX and 45-OH YTX
• Results forwarded to HC for risk assessment
• These levels do not pose human health risk
28
Gorge Harbour - Monitoring
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.452011/0
6/2
1
2011/0
7/0
5
2011/0
7/1
9
2011/0
8/0
2
2011/0
8/1
6
2011/0
8/3
0
2011/0
9/1
3
µg
/g t
oxin
DTX1 esters
OA+DTX1
29
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
µg
/g t
oxin
DTX1 Esters
OA+DTX1
Manila Clams OystersMussels
July
31
August
04
August
02
August
02
August
02
August
02
July
26
July
26
August
02
August
04
August
02
August
02
Gorge Harbour - Investigation
30
Summary
• Lipophilic Shellfish Toxins (LSTs) include many different toxins, including the DSP toxins
• Many different methods, but LC-MS/MS offers most potential for multi-toxin analysis
• Taking advantage of newer technology provides potential to decrease turn-around-time
• CFIA looks for regulated and “emerging” toxins
• Monitoring program in place to prevent outbreaks
31 © 2007 Her Majesty the Queen in right of Canada
(Canadian Food Inspection Agency), all rights reserved. Use without permission is prohibited.