MARINE BIOTOXINS – MONITORING AND RESEARCH USING THE XEVO G2-S QTOF

Jane Kilcoyne

Marine Institute, Rinville, Oranmore, Co. Galway, Ireland. Email: jane.kilcoyne@marine.ie

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