Routine Quantification of Lipophilic Marine Biotoxins in Shellfish by LC/MS/MS - Waters Corporation...

©2015 Waters Corporation 1

Routine Quantification of Regulated and

Non-Regulated Lipophilic Marine Biotoxins in

Shellfish

Collaboration carried out with Arjen Gerssen and Mirjam Klijnstra: RIKILT


Marine Biotoxins Introduction

Occur naturally as a result of harmful algal

blooms (HABs) in saltwater environments

Bioaccumulate in filter-feeding bivalve

molluscs

When mussels, oysters etc. are ingested

by humans, toxins & metabolites can

cause serious illnesses

Blooms are very difficult to predict or

control (e.g. climate change), so careful

monitoring is paramount


Health Effects of Marine Biotoxins

Toxins can all accumulate in the digestive gland

(hepatopancreas) of filterfeeding molluscan shellfish

(e.g. mussels, oysters, cockles, clams and scallops)

Pose a health risk to humans if contaminated shellfish are

consumed

Marine biotoxin-related illnesses:

– Headaches, vomiting, diarrhea,…neurological problems, & …death


Background Classification

Toxins vary in hydrophilicity and are

classed by their effects:

– Amnesic Shellfish poisoning (ASP)

Domoic acid

– Paralytic Shellfish poisoning (PSP)

Saxitoxins

– Neurotoxic Shellfish poisoning (NSP)

Brevetoxins

– Diarrhetic Shellfish poisoning (DSP)

Okadaic acid (OA) group,

dinophysistoxin (DTX)

– Yessotoxins (YTX)

– Pectenotoxins (PTX)

– Azaspiracid Shellfish poisoning (AZA)

Azaspiracids

Hyd

ro

ph

ilic

Lip

op

hilic


Worldwide Regulations / Procedures

In order to prevent serious health effects from fish consumption there are regulations that support routine monitoring

The regulation of the lipophilic marine toxins is country dependent

– USA:

o FDA –via the FDA no routine monitoring programs for these toxins have been established yet, legislation exists for OA and DTX1

• Federal Government: NOAA Marine Biotoxins Programs – monitoring programmes existed since 1992 (and other local programmes also exist)

– Canada:

o CFIA Regions must have in place a program to adequately monitor marine biotoxins. As levels begin to rise, sampling frequency may be increased in accordance with the speed of the rise to ensure timely closure. The objective is to ensure that shellfish areas are closed when:

• PSP toxin levels reach 80 µg/100 g

• ASP toxin levels reach 20 µg/g

• DSP (okadaic acid and/or DTX-1, singly /in combination) toxin levels reach 0.2 µg/g

• Pectenotoxins (PTXs) levels reach 0.2 µg/g (whole tissue)


Worldwide Regulations / Procedures

Europe: – Most types of lipophilic marine toxins can be found in shellfish and as a

result EU legislation covers OA, DTXs, PTXs, YTXs and AZAs

– EU COMMISSION REGULATION No 15/2011

o …amending Regulation (EC) No 2074/2005 of 10 January 2011 as regards recognised testing methods for detecting marine biotoxins in live bivalve molluscs

o …Recognises testing of marine biotoxins in live bivalve molluscs the EU-RL LC-MS/MS methods as the reference method for the detection of these lipophilic toxins, and used as routine, both for the purposes of official controls at any stage of the food chain and own-checks by food business operators

Lipophilic biotoxins that should be monitored are: o Okadaic acid (OA)

o Dinophysistoxin-1, -2, -3 (DTX1,-2,-3), (DTX3 are the ester forms of respectively OA), DTX1 and -2

o Pectenotoxin-1, -2 (PTX1, -2)

o Yessotoxin (YTX), 45OH yessotoxin (45OH YTX), homo yessotoxin (homoYTX), 45OH homo yessotoxin (45OH homoYTX)

o Azaspiracid-1, -2 and -3 (AZA1, -2, -3)


Worldwide Regulations / Procedures European Limits


Lipophilic Toxins

Chemical structure of a) regulated toxins and b) non-regulated cyclic imines

Toxin R1 R2

Okadaic acid CH3 H

Dinophysistoxin-1 CH3 CH3

Dinophysistoxin-2 H CH3

Toxin R1 R2

Azaspiracid-1 H CH3

Azaspiracid-2 CH3 CH3

Azaspiracid-3 H H

Toxin R1

Pectenotoxin-1 OH

Pectenotoxin-2 H

Toxin R1 n

Yessotoxin H 1

Homo Yessotoxin H 2

45OH Yessotoxin OH 1

45OH Homo Yessotoxin OH 2

a) b)

Toxin R1 R2 R3 R4

Pinnatoxin-E H OH CH3

Pinnatoxin-F H OH CH3

Pinnatoxin-G O

H

H H

13-desmethyl spirolide C

Gymnodimine


EU: Accepted Testing Protocols

Before July 2011, official method = mouse bioassay

There were two main issues with this method

– Unethical

– Not scientifically robust to determine trace amounts of specific toxins

o The presence of cyclic imines would cause a physical reaction using the bioassay

– the result often fatal for the animal

Since July 2011, the official method for control of shellfish on the presence

of lipophilic marine biotoxins has been LC-MS/MS

The EU reference method is based on a

– Fixed extraction procedure

– Separation using LC – either an acidic mobile phase or alkaline mobile phase

– Detection by tandem quadrupole MS

…. Post-July 2011 …. Pre-July 2011


Acidic Method

Alkali Method

Analytical Trends using LC-MS/MS Lipophilic Marine Biotoxins

J. Chrom. A 1216 (2009) 1421–1430 Technology: HPLC-MS/MS


Project Aims

Produce a faster method using alkaline

conditions

– HPLC = 15 mins

– UPLC = 5 mins

Develop the method for regulated and

some non-regulated cyclic imines

compounds

Test method on different matrices

Obtain single day lab validation data


Single Day Validation Performance Requirements & Criteria

Single day validation study was performed to determine method performance

Characteristics and criteria assessed:

o Linearity

o Recovery

o Repeatability

o Within-laboratory reproducibility

o Selectivity and decision criteria (CCα)

Blank shellfish from the Dutch national monitoring program were used:

o Mussels (Mytilus edulis) (4 samples)

o Oysters (Crassosrea gigas) (1 sample)

o Cockles (Cerastoderma edule) (1 sample)

o Ensis (Ensis directus) (1 sample)

Seven different shellfish samples were extracted and spiked at:

o 0.0, 0.5, 1.0 and 1.5 times validation level


Sample Preparation & Extraction


Sample Extraction

Homogenized whole flesh shellfish tissue (1 g) was

extracted with methanol

Extract was vortex-mixed and centrifuged

Supernatant was transferred to a 10 mL volumetric

flask and made up to 10 mL with methanol

Filter crude shellfish extract prior to spiking / analysis

For DTX3 (ester forms of OA, DTX1 and -2)

– Extracts also subjected to alkaline hydrolysis using 2.5 M sodium

hydroxide

– Heat alkaline mixture for 40 min at 76˚C, cool to RT and neutralise

using 2.5M HCl


Standards Preparation

Certified standards OA, DTX1, -2, PTX2, YTX, AZA1, -2, -3,

gymnodimine (GYM) and 13-desmethyl spirolide C (SPX1):

NRC-CNRC, Canada

Semi-purified standards for pinnatoxin-E (PnTX-E), -F

(PnTX-F) and –G (PnTX-G): Cawthron Institute, New Zealand

For each toxin, standard stock solution was prepared (MeOH):

from these matrix matched standards (MMS) calibration

curves were prepared in blank mussel extract


Instrument Set-up

and

Method Optimisation


HPLC to UPLC Use Generic Gradient Conditions

UPLC system: ACQUITY UPLC

Runtime: 5.00 min

Column: ACQUITY UPLC BEH C18 1.7µm, 2.1 × 100 mm

Column temp.:

40 ˚C

Sample temp.: 10 ˚C

Mobile phase A: Water containing 6.7 mM ammonium hydroxide

Mobile phase B:

9:1 acetonitrile:water containing 6.7 mM ammonium hydroxide

Weak wash:

9:1 water:acetonitrile

Strong wash: 9:1 acetonitrile:water

Flow rate: 0.6 mL/min

Injection volume: 5 µL Time (min)

%age A %age B

0.00 70 30

0.50 70 3

3.50 10 90

4.00 10 90

4.10 70 30

5.00 70 30


MS conditions

MS system:

Xevo TQ-S

Ionization mode: ESI - / +

Capillary voltage: 3.0 kV

Source temp.: 150 ˚C

Desolvation temp.: 500 ˚C

Desolvation gas: 800 L/hr

550 444

396

125

396


MRM Transitions ESI Negative

Compound name Parent

(m/z)

Daughter

(m/z) Ionisation

Dwell

(s) Cone (V)

Collision

(eV)

Standard

available

trinor YTX 550.4 396.4 - 0.003 75 30

No 550.4 467.4 - 0.003 75 30

YTX 570.4 396.4 - 0.003 75 30

Yes 570.4 467.4 - 0.003 75 30

homoYTX 577.4 403.4 - 0.003 75 30

No 577.4 474.4 - 0.003 75 30

45OH YTX 578.4 396.4 - 0.003 75 30

No 578.4 467.4 - 0.003 75 30

45OH Homo YTX 585.4 403.4 - 0.003 75 30

No 585.4 474.4 - 0.003 75 30

COOH YTX 586.4 396.4 - 0.003 75 30

No 586.4 467.4 - 0.003 75 30

COOH OH YTX 593.4 396.4 - 0.003 75 30

No 593.4 403.4 - 0.003 75 30

COOH Homo YTX 593.4 467.4 - 0.003 75 30

No 593.4 474.4 - 0.003 75 30

OA/DTX2 803.5 113.1 - 0.003 80 60

Yes 803.5 255.2 - 0.003 80 45

DTX1 817.5 113.1 - 0.003 80 60

Yes 817.5 255.2 - 0.003 80 45


MRM Transitions ESI Positive

Compound name Parent

(m/z)

Daughter

(m/z) Ionisation

Dwell

(s) Cone (V)

Collision

(eV)

Standard

available

GYM 508.2 162.2 + 0.003 60 55

Yes 508.2 490.2 + 0.003 60 40

SPX1 692.5 164.3 + 0.003 60 55

Yes 692.5 444.2 + 0.003 60 40

PnTX-G 694.5 164.3 + 0.003 60 55

Yes 694.5 676.5 + 0.003 60 40

20-Me SPX G 706.5 164.3 + 0.003 60 55

No 706.5 346.2 + 0.003 60 40

PnTX-F 766.5 164.3 + 0.003 60 55

Yes 766.5 748.5 + 0.003 60 40

PnTX-E 784.5 164.3 + 0.003 60 55

Yes 784.5 766.5 + 0.003 60 40

AZA3 828.5 658.4 + 0.003 35 40

Yes 828.5 792.5 + 0.003 35 30

AZA6 842.5 658.4 + 0.003 35 40 Yes

AZA1 842.5 672.4 + 0.003 35 40 Yes

AZA1/6 842.5 824.5 + 0.003 35 30 Yes/No

AZA4 844.5 658.4 + 0.003 35 40 No

AZA5 844.5 674.4 + 0.003 35 40 No

AZA4/5 844.5 826.5 + 0.003 35 30 No

AZA2 856.5 672.4 + 0.003 35 40

Yes 856.5 838.5 + 0.003 35 30

PTX12 874.5 213.1 + 0.003 40 30

No 874.5 821.5 + 0.003 40 30

PTX2 876.5 213.1 + 0.003 40 30

Yes 876.5 823.5 + 0.003 40 30

PTX11 892.5 213.1 + 0.003 40 30

No 892.5 839.5 + 0.003 40 30

PTX2sa 894.5 213.1 + 0.003 40 30

No 894.5 805.2 + 0.003 40 30


MRMs of Matrix Matched Standard Mussel extract

= toxins currently regulated = toxins currently non-regulated


Single day validation results

Compound

Concentration

(µg/kg)

Recovery

(%)

RSDr

(%)

RSDrl

(%)

CCα

(µg/kg)

OA 160 99 2.7 4.1 171

DTX1 160 99 7.6 12.2 192

DTX2 160 102 2.6 4.1 171

YTX 1000 100 2.5 4.0 1070

AZA1 160 98 1.3 2.1 166

AZA2 160 98 1.9 3.0 168

AZA3 160 99 1.9 3.0 168

PTX2 160 103 8.7 13.9 197

GYM 200 99 3.9 6.3 221

SPX11 100 108 14.6 23.4* 141

SPX12 100 104 12.8 20.4 135

PinE 200 122 23.1 36.9* 347

PinF 200 91 5.1 8.1 224

PinG 50 102 3.9 4.8 54


Summary and Conclusions

A five minute ACQUITY UPLC method has been developed - with

good separation for all different toxin classes

Method (e.g. MRM transitions) has been developed to analyse and

report the regulated and non-regulated lipophilic toxins

– Can be used as a confirmatory method

– Experiments are integrated into Quanpedia database and allows any user

of this system to have access to this method protocol

High selectivity and sensitivity of the Xevo TQ-S suitable for this

application

– Peaks can be easily detected at the sensitivity levels required by

– Option to dilute the matrix and still obtain the required levels

Single-day validation, using the different method performance

parameters, provided excellent linearity for all toxins

Routine Quantification of Lipophilic Marine Biotoxins in Shellfish by LC/MS/MS - Waters Corporation...

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