MARINE BIOTOXINS – MONITORING AND RESEARCH ......Marine Institute - Shellfish Safety • National...
Transcript of MARINE BIOTOXINS – MONITORING AND RESEARCH ......Marine Institute - Shellfish Safety • National...
MARINE BIOTOXINS – MONITORING AND RESEARCH USING THE XEVO G2-S QTOF
Jane Kilcoyne
Marine Institute, Rinville, Oranmore, Co. Galway, Ireland. Email: [email protected]
Marine Institute - Shellfish Safety • National Reference Laboratory
for marine biotoxins – responsible for monitoring of toxins in shellfish using INAB accredited methods
• Research – methods, novel toxins, toxin purification and characterisation, production of RMs and CRMs
Shellfish industry • Rapidly growing industry,
worth ~€63 million annually. • Important industry for
Western/coastal Ireland. • Mussels, oysters, clams,
razorfish, etc. • Target of 78% increase in
production by 2020
Shellfish toxins are associated with algal blooms
• Shellfish are filter feeders
• At certain times of the year phytoplankton blooms can produce biotoxins which can accumulate in shellfish Loss to shellfish farmers
and Threat to human health
Many species – producing different toxins
Okadaic acid group
Domoic acid
Saxitoxins
Azaspiracids
Cyclic imines
EU regulated toxins (in shellfish) Classification Regulated toxin Causative
organism(s) Poisoning syndromes
Chemical detection methods
Biological detection methods
Hydrophilic Saxitoxins
Alexandrium spp., Gymnodinium catenatum, Pyrodinium bahamense, certain cyanobacteria
Paralytic shellfish poisoning – potent neurotoxin
LC-FD Microscopy
Domoic Acid
Pseudo-nitzschia spp., benthic diatoms
Amnesiac shellfish poisoning - neurotoxin
LC-PDA Microscopy
Lipophilic Azaspiracids
Azadinium spp. Amphidoma languida
Azaspiracid shellfish poisoning
LC-MS/MS Molecular probe
Okadaic acid group Dinophysis spp. and Prorocentrum spp.
Diarrhetic shellfish poisoning – neurotoxic, immunotoxic and embyrotoxic
LC-MS/MS Microscopy
Pectenotoxin 2
Dinophysis spp. and Protoperidinium spp.
Diarrhetic shellfish poisoning – toxic by i.p. but not orally
LC-MS/MS Microscopy
Yessotoxin Lingulodinium polyedrum, Protoceratium reticulatum
Diarrhetic shellfish poisoning – toxic by i.p. but not orally
LC-MS/MS Microscopy
Other toxins of interest Regulated toxin Causative
organism(s) Poisoning syndromes
Chemical detection method
Pinnatoxins
V. rugosum No human toxicity reported but toxic to mice by i.p.
LC-MS/MS
Spirolides
Alexandrium spp. No human toxicity reported but toxic to mice by i.p.
LC-MS/MS
Prorocentrolides
Prorocentrum spp. No human toxicity reported but toxic to mice by i.p.
LC-MS/MS
Prorocentin Prorocentrum spp. No human toxicity reported but toxic to mice by i.p.
LC-MS/MS
Palytoxins
Ostreopsis spp. Palytoxin poisoning – intense vasocontr ictor , potent neurotoxin
LC-MS/MS
Tetradotoxin
Bacterial producers Potent neurotoxin LC-MS/MS
2015 2014 2013
Harvesting site Site closure AZAs (≥ 160 µg/kg) Site closure OA group (≥ 160 µg/kg)
2012
2010
2011
2009 2008 2007 2006
2005 2003 2004 2002
Impact of HABs
Waters LC-MS instruments
Xevo TQ – Acquity UPLC Xevo G2-S Qtof – Acquity UPLC
Sample extraction procedure
• ~ 4,000 samples analysed by LC-MS per year
• Results reported within 24−48 hr of receipt of sample
2 g double extract with MeOH
Final volume 25 mL (SSR = 12.5)
Centrifuge and decant
Filter (0.2 µm)
Xevo G2S Qtof – method conditions Parameter YTX and OA group toxins Test method ID BCT-96 LC Method Time (min) A B
0 95 5 1 50 50 2 10 90 3 10 90 3.01 95 5 4 95 5
Flow rate 0.3 ml/min Injection vol 2 µl Column Acquity BEH C18 1.7 µm, 2.1 x 50 mm at 30 °C Mobile Phase A 95% H2O: 5% 2 mM ammonium formate and 50 mM formic acid Mobile Phase B 95% MeCN: 5% 2 mM ammonium formate and 50 mM formic Run Time 4 min MS method MSe 100−1200 m/z, negative mode, sensitivity
Parameter AZAs, PTX2 and DA Test method ID BCT-96 LC conditions Time (min) A B
0 70 30 5 10 90 5.5 10 90 5.51 70 30 6.5 70 30
Flow rate 0.3 ml/min Injection vol 2 µl Column Acquity BEH C18 1.7 µm, 2.1 x 50 mm at 30 °C Mobile Phase A 95% H2O: 5% 2 mM ammonium formate and 50 mM formic acid Mobile Phase B 95% MeCN: 5% 2 mM ammonium formate and 50 mM formic Run Time 6.5 min MS conditions MSe 100−1200 m/z, positive mode, sensitivity
Peak identification based on: • Mass ± 0.005 Da • Retention time (CRMs
available for majority of regulated toxins)
Xevo G2S Qtof – trueness and LOD/LOQ
FDMT FDMT
OA DTX1 DTX2 YTX DA AZA1 AZA2 AZA3 PTX2
Average 93.1 111.5 91.1 81.6 109.1 Average 84.4 78.1 74.3 94.7
Std dev 8.7 16.4 11.3 12.1 17.0 Std dev 6.2 5.8 5.8 14.8
%CV 10.6 13.1 12.5 14.8 15.2 %CV 7.4 7.5 7.8 15.8
Toxin LOD (s/n = 3)
(ng/mL)
LOQ (s/n = 10)
(ng/mL)
OA 0.20 1.2
DTX2 0.20 1.0
DTX1 0.12 1.1
YTX 0.34 1.7
DA 2.20 11.1
AZA1 0.18 0.9
AZA2 0.08 0.4
AZA3 0.06 0.3
PTX2 0.16 0.8
LOD/LOQ
Trueness
Xevo G2S Qtof – matrix effects
0
20
40
60
80
100
120
140
160
AZA1 % ME
0
20
40
60
80
100
120
OA % ME
0
20
40
60
80
100
120
140
160
YTX % ME
0.0
20.0
40.0
60.0
80.0
100.0
120.0
140.0
PTX2 % ME
• Can be difficult to assess and overcome
• Can be dependant on shellfish variety but also within the same varieties there can be differences due to sampling location and time of year
Xevo G2S Qtof – proficiency testing
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5Toxin z score z score z score z score z score
AZA1 0.21 -0.17 -0.27 0.11AZA2 0.36 -0.41 -1.49AZA3 -0.93 -0.79 -0.98AZA total 0.22 -0.01 -0.46 -0.43Free DTX1 *5.54 0.88Free DTX2 -0.36 -1.05 -0.31 -0.67Free OA -0.71 -1.93 -2.22 -0.33Total free OA group -0.71 0.04 -0.5 -1.14Total DTX1 2.05 0.63Total DTX2 -0.99 -0.69 -1.41Total OA -1.83 -1.6 -1.82Total OA group -0.99 -0.61 -1.62PTX1PTX2YTX -0.11 -1.36homo-YTX45-OH-homo-YTX45-OH-YTXTotal YTX -0.39 -1.32
• QUASIMEME - QUality ASsurance In Marine Environmental Monitoring in Europe, twice yearly
• EU-RL-Marine biotoxins, once yearly
Azaspiracid
Azadinium spp.
Azaspiracids shellfish poisoning
Biogeography of AZAs, Azadinium and AZP
>30 AZA analogues identified
Where do AZAs come from? AZA1, AZA2 and AZA3 are regulated in shellfish (160 µg/kg)
Accumulation
Production
Filter-feeding bivalves
Processing, cooking, etc.
160 µg/kg regulatory limit (AZA1, -2 and -3)
AZA1, AZA2 (and AZA33 and AZA34) AZA1, AZA2
metabolised producing other AZA analogues
Heat treatment catalyses toxin conversions (decarboxylation)
Severe vomiting, diarrhea
AZA2
AZA11 AZA19 AZA12
AZA23 AZA6 AZA45
AZA9 AZA27 AZA10
AZA49 AZA16 AZA28
AZA47 AZA15
AZA1
AZA7 AZA17 AZA8
AZA21 AZA3 AZA44
AZA4 AZA25 AZA5
AZA48 AZA14 AZA26
AZA46 AZA13
Hydroxylation at C-3
Hydroxylation at C-23
Oxidation of 22-Me
22-Decarboxylation
Dehydration
5 µm
A. spinosum A. obesum A. poporum A. polongum A. caudatumvar. margalefii
Amphidomalanguida
A. caudatumvar. caudatum
iAZA1, -2, -33 , -34 No known AZAsAZA2, -11, -36, -37, -40, -41 No known AZAs No known AZAs AZA38, -39
A. dalianense
No known AZAs
A. species # 1 A. species # 2 A. species # 3
No known AZAs No known AZAs No known AZAs
Ireland, Scotland, Shetland Is, Denmark
Scotland, Denmark, Korea, China
Shetland Is Scotland Ireland
China Greenland, Iceland Greenland, Iceland Greenland, Iceland
Currently no isolates
More species discovered…….
Why isolate?
• Calibration standards and reference materials
• Toxicology studies
• Further research – mitigation, biosensors, pharmacology, etc
Isolation of AZAs from shellfish Step 1
Extraction
Step 2Partitioning 1
Step 3 Partitioning 2
Step 4Silica gel
Step 5LH20
Step 6Flash-phenyl hexyl
Step 7Prep HPLC-C18/C8
Ethyl acetate1M NaCl
Aq. MeOHHexane
70% EtOAc/MeOH
Fr 8-15(AZA1,-2, -3 and -6)
Hexane100% EtOAc
90% EtOAc/MeOH50% EtOAc/MeOH
100% MeOH
AZA3: Fr 10-15AZA6: Fr 16-23 AZA1: Fr 24-34AZA2: Fr 35-45
Purified AZAs
Step 1Extraction
Step 2Partitioning 1
Step 3 Partitioning 2
Step 4Silica gel
Step 5LH20
Step 6Flash-phenyl hexyl
Step 7Prep HPLC-C18/C8
Step 1Extraction
Step 2Partitioning 1
Step 3 Partitioning 2
Step 4Silica gel
Step 1Extraction
Step 2Partitioning 1
Step 3 Partitioning 2
Step 4Silica gel
Step 5LH20
Step 6Flash-phenyl hexyl
Step 7Prep HPLC-C18/C8
Ethyl acetate1M NaCl
Aq. MeOHHexane
70% EtOAc/MeOH
Fr 8-15(AZA1,-2, -3 and -6)
Hexane100% EtOAc
90% EtOAc/MeOH50% EtOAc/MeOH
100% MeOH
AZA3: Fr 10-15AZA6: Fr 16-23 AZA1: Fr 24-34AZA2: Fr 35-45
Purified AZAs
• Lengthy and labour intensive.
• µg – mg amounts isolated (dependant on levels in shellfish).
*52% recoveries
*Kilcoyne, J., Keogh, A., Clancy, G., LeBlanc, P., Burton, I., Quilliam, M. A., Hess, P., and Miles, C. O. (2012) Improved isolation procedure for azaspiracids from shellfish, structural elucidation of azaspiracid-6, and stability studies, J. Agric. Food Chem. 60, 2447–2455.
Isolation of AZAs from A. spinosum
• Fewer steps • Greater recoveries • AZA1, -2, -33, -34, -36 and -37 isolated
Ethyl acetate
70% EtOAc/MeOH
AZA1: Fr 24-34AZA2: Fr 35-45
Step 4Flash-phenyl hexyl
1M NaCl
Hexane100% EtOAc
90% EtOAc/MeOH50% EtOAc/MeOH
100% MeOH
Purified AZAsStep 5Prep HPLC-C18/C8
Step 1Extraction
Step 2Partitioning 1
Step 3Silica gel
Ethyl acetate
70% EtOAc/MeOH
AZA1: Fr 24-34AZA2: Fr 35-45
Step 4Flash-phenyl hexyl
1M NaCl
Hexane100% EtOAc
90% EtOAc/MeOH50% EtOAc/MeOH
100% MeOH
Purified AZAsStep 5Prep HPLC-C18/C8
Step 1Extraction
Step 2Partitioning 1
Step 3Silica gel
Step 1Extraction
Step 2Partitioning 1
Step 3Silica gel
*70% recoveries
*Jauffrais, T., Kilcoyne, J., Séchet, V., Herrenknecht, C., Truquet, P., Hervé, F., Bérard, J. B., Nulty, C., Taylor, S., Tillmann, U., Miles, C. O., and Hess, P. (2012) Production and isolation of azaspiracid-1 and -2 from Azadinium spinosum culture in pilot scale photobioreactors, Mar. Drugs 10, 1360–1382.
List of purified AZA analogues isolated in ASTOX 2
Type§ R1 7,8 R2 R3 R4 R5 R6 [M+H]+ Origin Status Amount isolated (mg) AZA1 a1 H Δ H H CH3 H CH3 842.5 A. spinosum phycotoxin 39.0 37-epi-AZA1 a1 H Δ H H CH3 H CH3 842.5 A. spinosum artefact 0.3 AZA2 a1 H Δ CH3 H CH3 H CH3 856.5 A. spinosum phycotoxin 9.0 AZA3 a1 H Δ H H H H CH3 828.5 shellfish metabolite 7.6 AZA4 a1 OH Δ H H H H CH3 844.5 shellfish metabolite 0.3 AZA5 a1 H Δ H H H OH CH3 844.5 shellfish metabolite 0.2 AZA6 a1 H Δ CH3 H H H CH3 842.5 shellfish metabolite 0.6 AZA7 a1 OH Δ H H CH3 H CH3 858.5 shellfish metabolite 0.2 AZA8 a1 H Δ H H CH3 OH CH3 858.5 shellfish metabolite 0.2 AZA9 a1 OH Δ CH3 H H H CH3 858.5 shellfish metabolite 0.2 AZA10 a1 H Δ CH3 H H OH CH3 858.5 shellfish metabolite 0.2 AZA26 a2 H Δ H - - - - 824.5 shellfish metabolite 0.1 AZA33 b1 - Δ - H CH3 H CH3 716.5 A. spinosum phycotoxin 0.8 AZA34 c1 - Δ - H CH3 H CH3 816.5 A. spinosum phycotoxin 0.3 AZA36 a1 OH Δ CH3 H CH3 H H 858.5 A. poporum phycotoxin 0.9 AZA37 a1 OH - H H CH3 H H 846.5 A. poporum phycotoxin 1.3
§ The type refers to variations of the LHS and RHS parts of the molecule.
Left hand side (LHS) Right hand side (RHS)
a
b
c
1
2
1 3
7
19 2120
8
2223
30
39
AB
C D EF
GH
I37
• Structures determined by LC-MS and NMR
• Reference standards prepared for each analogue
• All analogues tested for toxicity in vitro
AZA2
AZA11 AZA19 AZA12
AZA23 AZA6
AZA9 AZA10
AZA16
AZA15
AZA1
AZA7 AZA17 AZA8
AZA21 AZA3
AZA4 AZA5
AZA14
AZA13
Hydroxylation at C-3
Hydroxylation at C-23
Oxidation of 22-Me
22-Decarboxylation
• Rehmann, N.; Hess, P.; Quilliam, M. A. Discovery of new analogs of the marine biotoxin azaspiracid in blue mussels (Mytilus edulis) by ultra-performance liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 2008, 22, 549–558.
• Jauffrais, T.; Marcaillou, C.; Herrenknecht, C.; Truquet, P.; Séchet, V.;
Nicolau, E.; Tillmann, U.; Hess, P. Azaspiracid accumulation, detoxification and biotransformation in blue mussels (Mytilus edulis) experimentally fed Azadinium spinosum. Toxicon 2012, 60, 582–595.
Identification of unknowns
Xevo QTof MSe, MS/MS
AZA2
AZA11 AZA19 AZA12
AZA23 AZA6 AZA45
AZA9 AZA27 AZA10
AZA49 AZA16 AZA28
AZA47 AZA15
AZA1
AZA7 AZA17 AZA8
AZA21 AZA3 AZA44
AZA4 AZA25 AZA5
AZA48 AZA14 AZA26
AZA46 AZA13
Hydroxylation at C-3
Hydroxylation at C-23
Oxidation of 22-Me
22-Decarboxylation
Dehydration
• Kilcoyne, J.; McCarron, P.; Hess, P.; Miles, C. O. Effects of heating on proportions of azaspiracids 1−10 in mussels (Mytilus edulis) and identification of carboxylated precursors for azaspiracids 5, 10, 13 and 15. J. Agric. Food Chem. 2015, 63, 10980–10987.
• Kilcoyne, J.; McCarron, P.; Twiner, M. J.; Rise, F.; Hess, P.; Wilkins, A.
L.; Miles, C. O. Structural elucidation and in vitro toxicity of a unique azaspiracid isolated from mussels (Mytilus edulis) and identification of dehydrated analogues. J. Nat. Prod. 2016, Submitted.
Xevo G2-S Qtof has multiple benefits: • Highly sensitive and specific
• Used for both regulatory monitoring and research
• MSe mode excellent tool for retrospective analysis
• Add-on software packages e.g. UNIFI, Progenesis allow for
faster and effective identification of unknowns
Conclusions
Thanks to my colleagues:
Prof. Christopher O. Miles, Norwegian Veterinary Institute Dr. Philipp Hess, Ifremer, France Dr. Urban Tillmann, Alfred Wegener Institute, Germany Dr. Bernd Krock, Alfred Wegener Institute, Germany Dr. Thierry Jauffrais, Ifremer, France Ms. Ciara Nulty, Marine Institute, Ireland Mr. Conor Duffy, Marine Institute, Ireland Dr. Michael Quilliam, National Research Council, Canada Dr. Pearse McCarron, National Research Council, Canada
Thanks to Clare O’Donnell