Testing for Marine Toxins and DSP

Size: px

Start display at page:

Download "Testing for Marine Toxins and DSP"

Shauna Wilkins
5 years ago
Views:

Laboratory Dartmouth, NS 2007 Her Majesty the Queen in right of Canada

1 Canadian DSP Symposium Vancouver, BC November 27, 2012 Testing for Marine Toxins and DSP Wade Rourke Canadian Food Inspection Agency Dartmouth Laboratory Dartmouth, NS 2007 Her Majesty the Queen in right of Canada (Canadian Food Inspection Agency), all rights reserved. Use without permission is prohibited.

2 utline Information about toxins and toxin groups Information about different methods Canadian context and perspective bservations from BC samples 2

3 What are we talking about? kadaic acid and analogues (DSP toxins) Pectenotoxins Azaspiracids Yessotoxins Cyclic Imines 3

4 Chemical Structures HC H H H H H kadaic Acid H H N H Gymnodimine H H HC Azaspiracid H H NH H H H H H CH H 3 H H Na 3 S Yessotoxin H H Pectenotoxin Na 3 S 4

5 DTX3 H H 7 H H H H Multiple compounds H DTX3 refers to the fatty acid forms of A, DTX1 and DTX2 Could be fatty acid chain of carbons Difficult to detect directly Large number of compounds Lack of standards for each compound Use enzymatic or alkaline hydrolysis procedure 5

6 The problem starts with hunger Toxic Plankton Digestive Gland People Time Toxic Incident Detoxification 6

7 World Distribution 7

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 8

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 9

10 Methods of Analysis: Biological Methods II Method Advantage Disadvantage Immuno Assays Functional Assays Senstive Fast Very specific Very sensitive Fast Test at receptor level Independent of toxin profile Good only as screen React to antibody not receptor Different reactivity of analogues to anitbody Requires reliable enzymes Must know toxin mode of action 10

11 Methods of Analysis Chemical Method Advantage Disadvantage Chemical Methods - General LC-FLD Toxin profile data Good within a group, not between groups LC-MS/MS Specific and sensitive Analyse toxins directly Multi-toxin analysis Easiest to add new toxins Complex instruments required Require individual standards and TEFs Difficult to add new toxins Derivatization required Prone to matrix effects Most expensive instruments 11

12 EFSA Recommendations Change action limits Decrease limits for As, 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

13 Toxins in Atlantic Canada before 1987 NL PSP NB PE Paralytic Shellfish Poisoning (Saxitoxin group) R 4 NS Halifax R 1 H NH N + H 2 N N NH H NH 2 + H R 2 R 3 13

14 Toxins in Atlantic Canada, 2005 Ile-de-la- Madeleine NL PSP ASP NB PE DSP SPX NS Halifax PTX YTX AZA 14

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 15

16 Dartmouth Laboratory LSTs # of Toxins Monitored / / / / / / / / / / / / /

17 Changing Times HPLC-FLD DSP Toxins DTX1 AZA1 A-D8:2 MA Quilliam. 8th Int. Conf. Harmful Algae, Vigo, Spain, June MA Quilliam et al., In: Mycotoxins and Phycotoxins in Perspective at the Turn of the Century, Wageningen, The Netherlands, W.J. dekoe, pp , (2001). DA GYM SPX DTX2 PTX2sa PTX2 YTX A LC-MS Multi-toxin m/z m/z m/z m/z m/z m/z m/z m/z m/z m/z m/z Time (min) 17

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 18

19 CRM Availability National Research Council Canada Certified Reference Material Program Available LST CRMs A, 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 19

20 2000/ / / / / / / / / / / / /2013 # of Samples East vs West Sample Distribution Fiscal Year BC Samples Eastern Canada Samples 20

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) 21

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 22

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 23

24 LC-MS/MS Chromatogram DSP Toxins A DTX-1 DTX Time, min 24

25 LC-MS/MS Chromatogram Lipophilic Shellfish Toxins YTXs Time, min Time, min DSPs AZAs Time, min CIs Time, min Time, min PTXs 25

26 Yessotoxins - EU No human illnesses have been linked to YTXs Regulated in EU but not in Canada YTX, homoytx, 45-H YTX, 45-H homoytx EU maximum limit: 1.0 μg/g 26

27 Yessotoxins - Canada Maximum YTX levels found in Canada: 12 μg/g Combination of YTX and 45-H YTX Results forwarded to HC for risk assessment These levels do not pose human health risk 27

28 2011/06/ /07/ /07/ /08/ /08/ /08/ /09/13 µg/g toxin Gorge Harbour - Monitoring DTX1 esters A+DTX

29 July 31 August 02 August 02 August 02 August 04 August 02 July 26 July 26 August 02 August 02 August 02 August 04 µg/g toxin Gorge Harbour - Investigation Mussels Manila Clams ysters DTX1 Esters A+DTX

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 30

2007 Her Majesty the Queen in right of Canada (Canadian Food

UPLC/MS/MS Method for the Routine Quantification of Regulated and Non-Regulated Lipophilic Marine Biotoxins in Shellfish

UPLC/MS/MS Method for the Routine Quantification of Regulated and n-regulated Lipophilic Marine Biotoxins in Shellfish Arjen Gerssen, 1 Mirjam D. Klijnstra, 1 Simon Cubbon, 2 and Antonietta Gledhill 2