routine quantification of lipophilic marine biotoxins in shellfish by lc/ms/ms - waters corporation...
TRANSCRIPT
©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
©2015 Waters Corporation 2
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
©2015 Waters Corporation 3
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
©2015 Waters Corporation 4
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
©2015 Waters Corporation 5
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)
©2015 Waters Corporation 6
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)
©2015 Waters Corporation 7
Worldwide Regulations / Procedures European Limits
©2015 Waters Corporation 8
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
©2015 Waters Corporation 9
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
©2015 Waters Corporation 10
Acidic Method
Alkali Method
Analytical Trends using LC-MS/MS Lipophilic Marine Biotoxins
J. Chrom. A 1216 (2009) 1421–1430 Technology: HPLC-MS/MS
©2015 Waters Corporation 11
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
©2015 Waters Corporation 12
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
©2015 Waters Corporation 13
Sample Preparation & Extraction
©2015 Waters Corporation 14
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
©2015 Waters Corporation 15
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
©2015 Waters Corporation 16
Instrument Set-up
and
Method Optimisation
©2015 Waters Corporation 17
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
©2015 Waters Corporation 18
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
©2015 Waters Corporation 19
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
©2015 Waters Corporation 20
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
©2015 Waters Corporation 21
MRMs of Matrix Matched Standard Mussel extract
= toxins currently regulated = toxins currently non-regulated
©2015 Waters Corporation 22
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
©2015 Waters Corporation 23
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
©2015 Waters Corporation 24