a generic method for regulated and unregulated phycotoxins in various matrices … ·...

A GENERIC METHOD FOR REGULATED

AND UNREGULATED PHYCOTOXINS IN

VARIOUS MATRICES WITH LC-HRMS Mirjam Klijnstra

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MSc Chemistry

Analytical sciences

Master Thesis

A generic method for regulated and unregulated

phycotoxins in various matrices with LC-hrMS

by

M. D. Klijnstra

April 2016

Supervisor:

dr W. Th. Kok

Daily Supervisor:

dr A. Gerssen

RIKILT Wageningen UR

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ABSTRACT

Phycotoxins such as marine biotoxins and cyanotoxins are produced by certain algae and cyanobacteria which

are naturally occurring in marine and fresh waters. Phycotoxins can accumulate in various marine and fresh

water species such as fish, crabs or shellfish (mussels, oysters, scallops and clams). By processing toxin

producing algae, contaminated fish or shellfish, phycotoxins can also end up in food or food supplements.

When these contaminated products are consumed or when there is contact with certain toxins, e.g., in

swimming water, they may cause severe intoxication symptoms, such as skin irritation, paralysis, diarrhoea and

even death may occur. Every year phycotoxins are held responsible for approximately 60,000 human

intoxications.

Legislation has been developed and monitoring programs have been established worldwide in order to prevent

intoxication of the consumer. Analytical methods are available to analyse regulated phycotoxins. However,

these methods are only suitable for a small specific group of toxins and/or a specific matrix (mainly shellfish).

Due to their different chemical properties it is difficult to analyse all phycotoxins in one single analytical

method. For instance, lipophilic compounds dissolve more easily in organic solvents and can be separated on

reversed phase liquid chromatography (LC) columns. Hydrophilic compounds dissolve best in water and can be

retained on hydrophilic interaction LC columns (HILIC).

For cases in which symptoms cannot be directly related to specific regulated phycotoxins, a screening method

is required. In this work such a screening method was developed and validated for all types of phycotoxins in

different matrices such as tissue, fresh and sea water and food supplements. Two LC methods were developed

to analyse sample extracts; one for hydrophilic and one for lipophilic phycotoxins. Sample extracts were

measured in full scan mode with an Orbitrap high resolution mass spectrometry (hrMS). Additionally, a

database was created to process the data. Unlike various other modes of MS/MS acquisition, LC-hrMS allows

untargeted measurements with the possibility to detect additional compounds, which were not foreseen to be

of interest at the time of the measurement, retrospectively.

The validation of the screening method for tissue samples was successful. Furthermore, it was shown that

regulated lipophilic phycotoxins, domoic acid and some paralytic shellfish poisoning (PSP) toxins can be

quantified in shellfish at 0.5 or 1 times the permitted level. However, some PSPs gave poor peak shapes which

caused difficulties during processing of the results. The validation of the screening method for water samples

was also successful, except for hydrophilic phycotoxins in sea water. During validation it appeared that the

method for sea water had to be modified slightly due to problems with the salt content. Recoveries of lipophilic

phycotoxins spiked to food supplements ranged from 9 to 102%, depending on matrix effects from sample to

sample. Therefore it was difficult to validate the method for lipophilic phycotoxins in food supplements.

When phycotoxins are found based on the library and confirmed with a fragment ion using the screening

method, it is still a tentative confirmation because fragment ions are analysed over a range of precursor ions

and retention times are often unknown. To confirm the presence of phycotoxins a standard is needed, or NMR

analysis needs to be done. For most phycotoxins for which standards are available confirmation methods

already exist. For the palytoxin-group toxins (PlTXs) an analytical method was not yet available and therefore

an LC-MS/MS method was established for the quantitation and confirmation of PlTXs. However, high quantities

of PlTXs were necessary to detect the compounds with the screening method, since the spectrum is

complicated (multiple precursor ions), and therefore a poor sensitivity was obtained. Within the confirmation

method cleavage fragments from an oxidative fragmentation reaction are included which solves any sensitivity

issues for PlTXs. However, the oxidation step was too specific to be included in the screening method.

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CONTENTS

1. Introduction ..................................................................................................................................... 9

2. Materials and methods ................................................................................................................. 13

2.1 Chemicals and standards ....................................................................................................... 13

2.2 Preparation of standard solutions ......................................................................................... 15

2.3 Preparation of extracts .......................................................................................................... 15

2.4 Instrumentation .................................................................................................................... 17

2.5 Validation .............................................................................................................................. 19

3. Results and discussion ................................................................................................................... 21

3.1 Optimization of extraction procedures ................................................................................. 21

3.2 Chromatography.................................................................................................................... 26

3.3 MS measurements................................................................................................................. 30

3.3 Validation .............................................................................................................................. 30

3.4 Confirmation of palytoxin-group toxins by LC-MS/MS ......................................................... 33

4. Conclusion ..................................................................................................................................... 37

5. Acknowledgement ......................................................................................................................... 38

6. References ..................................................................................................................................... 39

Appendices ............................................................................................................................................ 43

Appendix 1: Structures ...................................................................................................................... 44

Appendix 2: Database (part as example) .......................................................................................... 53

Appendix 3: RIKILT Standards operating procedures phycotoxins ................................................... 57

Appendix 4: Validation results .......................................................................................................... 63

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1. INTRODUCTION

Phycotoxins such as marine biotoxins and cyanotoxins are produced by certain algae and cyanobacteria that

are naturally occurring in marine and fresh waters. When a population of algae is rapidly increasing or

accumulating in a water system this is called an algal bloom. An algal bloom that has negative effects for other

organisms (e.g., via production of phycotoxins or by shading) is called a harmful algal blooms (HAB).

HABs are caused by the presence of high nutrient concentrations in the water by influx through river deltas and

rainfall and HAB formation can be enhanced by other weather factors such as wind and temperature [1].

When phycotoxins are present they can accumulate in various marine species such as fish, crabs or shellfish

(mussels, oysters, scallops and clams). In shellfish, toxins accumulate mainly in the digestive glands without

causing intoxication to the shellfish itself [2]. Various shellfish species can metabolize some phycotoxins into

fatty acid esters, which can cause a delayed onset of toxic symptoms. This esterification is known for

brevetoxins (PbTxs), cyclic imines (CIs), pectenotoxins (PTXs), okadaic acid (OA) and dinophysistoxins (DTXs) [3-

8]. Furthermore yessotoxins (YTXs) and PTXs can be metabolized by shellfish to analogues [9].

By processing toxin producing algae, contaminated fish or shellfish, phycotoxins can also end up in food or food

supplements. When these contaminated products are consumed or when there is contact with certain toxins,

e.g., in swimming water, they may cause severe intoxication symptoms, such as skin irritation, paralysis,

diarrhoea and even death may occur [10, 11]. In the past intoxications have been reported [12-14] and

throughout the world, phycotoxins produced by algae are held responsible for approximately 60,000 human

intoxications every year [15].

Some phycotoxins are classified by the syndromes they cause: paralytic shellfish poisoning (PSP) is caused by

saxitoxin (STX) and analogues, diarrhetic shellfish poisoning (DSP) is caused by OA and DTXs, amnesic shellfish

poisoning (ASP) is caused by domoic acid (DA), and neurotoxic shellfish poisoning (NSP) is caused by PbTxs [16].

However, there are many more phycotoxins known, including: azaspiracids (AZAs), YTXs, CIs, PTXs, microcystins

and nodularins (MCs), cylindrospermopsins (CYNs), anatoxins (ATXs), tetrodotoxins (TTXs), ciguatoxins (CTXs)

and palytoxins (PlTXs). AZAs cause diarrhoea; PTXs, CYNs and MCs are hepatotoxic; CIs, ATXs, and TTXs are

neurotoxic; CTXs show gastrointestinal and neurological symptoms and PlTXs cause gastrointestinal problems

or respiratory distress [17-22]. When human are exposed to high concentrations, some phycotoxins i.e. TTXs

and PbTxs can be lethal. YTXs are lethal to mice after intraperitoneal injection but not for human after

consumption of contaminated shellfish [23]. All mentioned toxin groups contain multiple analogues; the largest

group are the MCs with more than 160 different structures reported in literature and registered in Scifinder

[24, 25]. Some analogues are more toxic than others and therefore for some phycotoxin groups toxicity

equivalent factors (TEF) have been assigned.

Less than ten percent of all phycotoxins described in literature are available as a (certified) standard. Standards

are isolated from contaminated shellfish or algae and there are not more than a few producers worldwide that

produce certified phycotoxin standards. Furthermore the availability of contaminated shellfish and algae is

limited and it is time-consuming to produce such purified standards, and therefore standards are relatively

expensive [26].

Appendix 1 gives an overview of abbreviations and structures of all available standards. Molecular masses vary

from 118 Da to 3380 Da and phycotoxins can be divided into two classes based on their water solubility. In

general all phycotoxins with a molecular mass below 500 Da are considered to be hydrophilic phycotoxins and

all phycotoxins with a mass above 500 Da are considered to be lipophilic. The lipophilic phycotoxins include

compounds such as OA, DTXs, AZAs, PTXs, and YTXs. The hydrophilic phycotoxins include DA, STXs and CYNs.

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The analysis of lipophilic and hydrophilic compounds is very different. Lipophilic compounds tend to dissolve

better in organic solvents and are separated on reversed phase LC-columns. Hydrophilic compounds dissolve

better in water and are preferably separated with Hydrophilic Interaction Liquid Chromatography (HILIC)

columns.

Legislation and monitoring programs are established worldwide in order to prevent intoxication of shellfish

consumers [27, 28]. However there are some differences within legislation methods applied around the world.

Table 1 gives an overview of legislation in the European Union, CODEX guidelines and opinions of the European

Food Safety Authority (EFSA) on their maximum allowed concentrations. These levels are only applicable to fish

or shellfish and they are the sum of the concentrations of all analogues taking TEFs into account. Legislation in

the European Union is comparable with CODEX guidelines except for PTXs and YTXs, which are additional, and

PbTxs, as PbTxs do not occur in European waters. However, EFSA opinions are very different from the EU and

CODEX, where for most groups toxic levels are estimated lower by EFSA. This could be a concern as most of the

recommended levels by EFSA are allowed to be exceeded. For MCs there is a guideline for drinking water

issued by the World Health Organisation of 1 µg L-1

.

TABLE 1: LEGISLATION, GUIDELINES AND OPINIONS [29-40]

Toxin group EU (µg kg-1) CODEX (µg kg-1) EFSA opinions (µg kg-1)

OA and DTXs (DSP) YES – 160 YES – 160 45

PTXs YES – 160* NO 120

YTXs YES – 3750 NO 3750

AZAs YES – 160 YES – 160 30

DA (ASP) YES – 20000 YES – 20000 4500

STXs (PSP) YES – 800 YES – 800 75

CIs NO NO More research needed

PlTXs NO NO 30

CTXs YES YES – 0.01 More research needed

PbTxs (NSP) NO YES – 800 -

MCs NO NO -

TTXs NO NO -

ATXs NO NO -

CYNs NO NO -

* Included in the OA and DTX group

Besides variations in legislation there are also differences in the methods for analysis applied. Bioassays such as

mouse and rat bioassays were common methods for the determination of phycotoxins in the past. With the

official mouse bioassays for PSP and DSP toxins a mouse is injected intraperitoneally with a shellfish extract,

when the mouse dies within 10 minutes (PSP) or 24 hours (DSP) the test result is positive [30, 33]. For the

official rat bioassay for DPS toxins rats are starved for 24 hours and then fed with shellfish meat, the test result

is positive when the rat is suffering from diarrhoea after 16 hours [30]. The disadvantages are that the response

to possible phycotoxins might be specific for the animal and humans react different. Furthermore, the

administration route of the mouse bioassay is different from normal consumption which makes it difficult to

extrapolate results to human potency. Moreover, there is a growing resistance against the use of animals for

experiments [41]. The main advantage of animal testing is that the selectivity is low; therefore the assay may

also be sensitive towards possible unknown toxins. Until now, LC-MS/MS, HPLC-FLD and HPLC-UV have been

shown to be the techniques with the highest sensitivities and selectivities [42] and for all regulated phycotoxins

such analytical methods are available; however these methods are only suitable for a small specific group of

toxins and/or a specific matrix (mainly shellfish). Moreover, these analytical methods do not detect any

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phycotoxins that are not present during method development and included in the method. In general methods

are only available for phycotoxins for which regulatory limits are established. Furthermore, limits of detection

are for some methods above regulatory limits.

For PSP toxins the official inter-lab validated analytical methods are with pre- or post-column derivatisation of

the sample extracts and analysis with fluorescence detection [33]. Liquid chromatography coupled to (triple

quad) mass spectrometry (LC-MS/MS) methods for PSP toxins are described with HILIC separation, with various

types of columns as zwitterion or amide based columns [43-45] and ion-pair LC-MS [46]. Furthermore TTXs can

be separated with a HILIC column [47]. CYNs, further hydrophilic compounds, can be separated with a Zorbax

Sb–Aq or a Luna PFP(2) column [48, 49]. In general HILIC methods are more sensitive to matrix effects which

may cause shifts in retention time.

DSP toxins can be combined together with PTXs, YTXs, AZAs and CIs in one method for lipophilic toxins. Acidic,

neutral or alkaline reversed phase chromatographic conditions are used in combination with a C8 or C18 column

for separation of these lipophilic phycotoxins [42, 50]. When measured with MS, OA, DTXs and YTX are

preferably analysed in the negative ionisation mode to obtain better sensitivity, and AZAs and CIs in the

positive ionisation mode. The advantage of neutral and acidic conditions is that AZAs have better peak shapes

as AZAs are zwitterions at a high pH; the advantage of alkaline conditions is that no polarity switch is needed to

measure all compounds in the same run, because compounds analysed in the negative and positive mode are

separated. Under acidic or neutral conditions the charge of the CIs is different and CIs elute more rapidly.

When a conventional MS is used the disadvantage of polarity switching is that less data points can be

generated which is compromising peak shapes and sensitivity. DSP toxins tend to form esters when present in

shellfish. Many different esters, from C16 to C22 chains and with different saturations, are formed. They can be

analysed intact [7], however, it is impossible to have standards for all of them and therefore esters can be

transformed to the deconjugated form by alkaline hydrolysis [51].

Existing methods described for MCs are mostly suitable for water samples. MCs can be separated under acidic

conditions in combination with a C18 column and are analysed in the positive ionisation mode [52]. PlTXs, PbTxs

and CTXs are like MCs separated under acidic conditions in combination with a C18 column and are also

analysed in the positive ionisation mode [53-55]. DA is retained on reversed phase columns as well as on HILIC

columns; therefore DA is often included in multitoxin methods for DSP or PSP toxins and can be analysed in the

positive and the negative ionisation mode [56, 57].

PlTXs is the group with the largest molecules. Because of the many functional groups at the molecule multiple

adducts, charge states and losses of water are seen in the spectrum, which causes poor sensitivity. To obtain a

better sensitivity for PlTXs an oxidative fragmentation reaction is described for reduction of molecules to

specific cleavage fragments [58].

For all EU regulated phycotoxins or phycotoxins where an EFSA opinion is available, confirmative methods are

available within RIKILT, except for PlTXs. For PlTXs an LC-MS/MS method needed to be developed, this was part

of the research conducted within this thesis.

For untargeted analysis liquid chromatography combined with high resolution mass spectrometry (LC-hrMS) is

used. This technique allows untargeted measurements with the possibility to detect additional compounds,

which were not foreseen to be of interest at the time of the measurement, retrospectively, unlike triple

quadrupole instruments operating in multiple reaction monitoring (MRM) mode. It has been shown that LC-

hrMS is a useful technique to screen food samples for the presence of a high variety of analytes [59, 60]. For

phycotoxins, only a few methods are described using LC-hrMS, all suitable for only lipophilic toxins [61-63]. LC-

hrMS makes it possible to approach food toxicant analysis in a different way with other possibilities; however,

this brings also new challenges. The number of analytes that can be detected is too large to process and verify

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manually as it is typically done for quantitative MS/MS methods. For this, databases with target analytes and

sufficient information as retention or fragmentation information are needed to facilitate automated detection.

When such a database is available, extraction of the analytes of interest from the raw data can be done

automatically by software. There are several software packages to process data like MetAlign [64] which is

developed at RIKILT or Tracefinder from Thermo Scientific. However, at this stage MetAlign cannot handle

multiple charged fragments and Tracefinder is still under development because there are still some issues with

the software to be solved.

In this study we have investigated the possibilities of LC-hrMS as a generic technique to screen for all kinds of

phycotoxins in different matrices such as tissue, fresh and sea water and food supplements. This method will

be applied in case of an incident where symptoms cannot be directly related to the regulated phycotoxins. The

aim was to develop a method to extract different sample types with a generic extraction method and to

analyse sample extracts with an LC-method for hydrophilic and one for lipophilic phycotoxins. In order to

process the data a database needed to be created from literature and a protocol for data processing needed to

be established. Furthermore, as mentioned before, for the PlTXs an LC-MS/MS confirmatory method needed to

be developed.

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2. MATERIALS AND METHODS

2.1 CHEMICALS AND STANDARDS

Table 2 lists all chemicals used, abbreviations, concentration or purity and suppliers. Table 3 and Table 4 list all

standards used divided into hydrophilic and lipophilic phycotoxins.

TABLE 2: CHEMICALS

Chemicals Abbreviation Concentration or purity Supplier

Acetic acid HAc 100% Merck1

Acetonitrile ACN Ultra LC-MS grade Actu-All2

Ammonium hydroxide NH4OH 25% VWR3

Ammonium formate Amm.form >97% Sigma-Aldrich4

Formic acid FA 98-100% Merck1

n-Hexane HPLC grade Actu-All2

Methanol MeOH Ultra LC-MS grade Actu-All2

Periodic acid >99% Sigma-Aldrich4

Water H2O Ultra LC-MS grade Actu-All2

1 Merck, Darmstadt, Germany 2 Actu-All, Oss, The Netherlands 3 VWR International, Amsterdam, The Netherlands 4 Sigma-Aldrich, Zwijndrecht, The Netherlands

TABLE 3: HYDROPHILIC STANDARDS


L-2-Amino-3-methylaminopropionic acid BMAA >97% Sigma-Aldrich1

Anatoxin ATX 4.96 ± 0.18 μg mL-1 NRC2

Cylindrospermopsin CYN 12.6 ± 0.8 μg mL-1 NRC2

Decarbamoylgonyautoxin 2&3 dcGTX2 40.9 ± 1.8 μg mL-1

NRC2 dcGTX3 9.2 ± 0.3 μg mL-1

Decarbamoylsaxitoxin dcSTX 16.7 ± 0.5 μg mL-1 NRC2

Decarbamoylneosaxitoxin dcNEO 8.0 ± 0.3 μg mL-1 NRC2

L-2,4-Diaminobutyric acid DBA >95% Sigma-Aldrich1

Domoic acid DA >90% Sigma-Aldrich1

Gonyautoxin 1&4 GTX1 24.8 ± 1.3 μg mL-1

NRC2 GTX4 8.1 ± 0.7 μg mL-1

Gonyautoxin 2&3 GTX2 45.2 ± 2.3 μg mL-1

NRC2 GTX3 17.2 ± 0.9 μg mL-1

Gonyautoxin 5 GTX5 24.7 ± 1.1 μg mL-1 NRC2

Neosaxitoxin NEO 20.7 ± 1.1 μg mL-1 NRC2

Saxitoxin STX 19.8 ± 0.4 μg mL-1 NRC2

N-Sulfocarbamoylgonyautoxin-2&3 C1 53.9 ± 1.8 μg mL-1

NRC2 C2 16.1 ± 1.3 μg mL-1

Tetrodotoxin TTX 96% Latoxan3

Tetrodotoxin & 4,9-anhydro TTX TTX 25.6 ± 1.8 µg mL-1

CIFGA4 anhTTX 3.0 ± 0.2 µg mL-1

1 Sigma-Aldrich, Zwijndrecht, The Netherlands 2 National Research Council, Measurement science and standards, Halifax, Canada 3 Latoxan, Valence, France 4 CIFGA, Lugo, Spain

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TABLE 4: LIPOPHILIC STANDARDS


Azaspiracid-1 AZA1 1.24 ± 0.07 µg mL-1 NRC1



Azaspiracid-4 AZA4 1.19 ± 0.07 μg mL-1 CIFGA2

Azaspiracid-5 AZA5 1.20 ± 0.07 μg mL-1 CIFGA2

Brevetoxin 2 PbTx 2 95% Latoxan3



13-Desmethyl spirolide C SPX1 7.0 ± 0.4 µg mL-1 NRC1

13,19-Didesmethyl spirolide C 13,19-didesMeSPXC 10.24 ± 0.98 μg mL-1 CIFGA2

Dinophysistoxin-1 DTX1 15.1 ± 1.1 µg mL-1 NRC1

Dinophysistoxin-2 DTX2 7.8 ± 0.4 µg mL-1 NRC1

Gymnodimine GYM 5.0 ± 0.2 µg mL-1 NRC1

Homoyessotoxin hYTX 5.8 ± 0.3 µg mL-1 NRC1

20-Methyl spirolide G 20MeSPXG 7.01 ± 0.61 µg mL-1 CIFGA2

Microcystin-HilR MC-HilR >95% Enzo Life Sciences4

Microcystin-HtyR MC-HtyR >95% Enzo Life Sciences4

Microcystin-LA MC-LA >95% Enzo Life Sciences4

Microcystin-LF MC-LF >95% Enzo Life Sciences4

Microcystin-LR MC-LR >95% Enzo Life Sciences4

[D-Asp3]Microcystin-LR Asp MC-LR >95% Enzo Life Sciences4

Microcystin-LW MC-LW >95% Enzo Life Sciences4

Microcystin-LY MC-LY >95% Enzo Life Sciences4

Microcystin-RR MC-RR >95% Enzo Life Sciences4

Microcustin-YR MC-YR >95% Enzo Life Sciences4

Nodularin NOD >95% Enzo Life Sciences4

Okadaic acid OA 13.7 ± 0.6 µg mL-1 NRC1

Okadaic acid C8-diol ester OA C8-diol ester >90% Enzo Life Sciences4

Okadaic acid methyl ester OA methyl ester >90% Enzo Life Sciences4

Pacific ciguatoxin 1 pCTX1 No certified concentration University of Queensland5

Pacific ciguatoxin 2 pCTX2 No certified concentration University of Queensland5

Pacific ciguataxin 3 pCTX3 No certified concentration University of Queensland5

7-O-Palmitoylokadaic acid 16:0 OA ester, DTX3 90-94% MP Biomedicals6

Palytoxin PlTX >90% Wako7

Pectenotoxin-2 PTX2 8.6 ± 0.3 µg mL-1 NRC1

Pinnatoxin E PnTX E No certified concentration Cawthron Institute8

Pinnatoxin F PnTX F No certified concentration Cawthron Institute8

Pinnatoxin G PnTX G No certified concentration Cawthron Institute8

Yessotoxin YTX 5.6 ± 0.2 µg mL-1 NRC1

1 National Research Council, Measurement science and standards, Halifax, Canada 2 CIFGA, Lugo, Spain 3 Latoxan, Valence, France 4 Enzo Life Sciences, Antwerp, Belgium 5 Professor Lewis, Institute for molecular Bioscience, The University of Queensland, Australia 6 MP Biomedicals, Santa Ana, United states 7 Wako, Osaka, Japan 8 Cawthron Institute, Nelson, New Zealand

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2.2 PREPARATION OF STANDARD SOLUTIONS

For method development 5 mixtures were prepared. Standard mixture 1 contained all hydrophilic phycotoxins

except PSP toxins, at a concentration of 1 µg mL-1

in water: TTX, anhTTX (117 ng mL-1

), BMAA, DBA, DA, ATX

and CYN. Standard mixture 2 contained all PSP toxins at a concentration of 1 µg mL-1

in water containing 0.03

M acetic acid: STX, NEO, dcSTX, dcNEO, GTX1&4 (GTX4 325 ng mL-1

), GTX2&3 (GTX3 380 ng mL-1

), GTX5,

dcGTX2&3 (dcGTX3 224 ng mL-1

) and C1&2 (C2 299 ng mL-1

). Standard mixture 3 contained all microcystins at a

concentration of 1 µg mL-1

in methanol/water (80:20 v/v): MC-LA, MC-LF, MC-LR, MC-LW, MC-LY, MC-RR, MC-

WR, MC-HilR, MC-HtyR, MC-YR, Asp MC-LD and NOD. Standard mixture 4 contained lipophilic toxins at a

concentration of 100 ng mL-1

in methanol: OA, DTX1, DTX2, YTX, hYTX, PTX2, SPX1, GYM, 13,19-didesMeSPXC,

20MeSPXG, 16:0 OA ester, OA C8-diol ester, AZA1, AZA2, AZA3 and AZA4. Standard mixture 5 contained all

other lipophilic phycotoxins at a concentration of 100 ng mL-1

in methanol: PnTX E, PnTX F, PnTX G, pCTX1 (50

ng mL-1

), pCTX2 (50 ng mL-1

), pCTX3 (50 ng mL-1

), PbTx2, PbTx3, PbTx9, PlTX, AZA5, OA methyl ester and DA.

For the validation some phycotoxins were excluded due to lack of standards (pCTX1, 2 and 3), poor sensitivity

(PlTX) or poor peak shapes (DBA and BMAA) during method development. For the validation the standards of

mixtures 4 and 5 were combined in a single mixture.

2.3 PREPARATION OF EXTRACTS

TISSUE SAMPLES

The extraction method of phycotoxins from tissue samples like fish and shellfish was developed and optimised.

Multiple extraction solvents and volumes were tested with naturally contaminated samples. Additionally some

different extraction methods were tested. Recoveries were obtained with spiked samples and compared to

existing confirmatory methods for PSP toxins, DSP toxins, AZAs, and MCs.

The optimised extraction procedure was as follows: 1.0 ± 0.05 g tissue homogenate was weighed and extracted

with 4 mL methanol. The sample was vortex mixed for one minute using a multipulse vortex. The sample was

centrifuged at 2,000 x g for 5 minutes and the supernatant was decanted from the pellet to a graduated tube. 5

mL of H2O/ACN/Amm.form/FA (55:45 v/v, 2 mM, 0.5 mM) was added to the pellet. The sample was again

vortex mixed for one minute using a multipulse vortex. The sample was centrifuged at 2,000 x g for 5 minutes

and the supernatant was combined with the previous obtained methanol extract. The tube was filled to 10 mL

with acetonitrile. To avoid loss of compounds two different filters were used suitable for lipophilic and

hydrophilic phycotoxins. For the analysis of lipophilic phycotoxins an aliquot of the extract was filtered with a

0.2 µm HT Tuffryn filter (Sigma-Aldrich, Zwijndrecht, The Netherlands). For analysis of hydrophilic phycotoxins

an aliquot of the extract was filtered with a 0.45 µm PVDF filter (Sigma-Aldrich, Zwijndrecht, The Netherlands).

The filtered extracts were transferred into a glass vial and used for analysis with LC-hrMS.

WATER SAMPLES

For the analysis of the phycotoxins in water a clean-up procedure was developed and further optimised. Two

clean-up methods are needed, one specific for lipophilic phycotoxins and one for hydrophilic phycotoxins. For

both phycotoxin groups solid phase extraction (SPE) is used. Because water samples might contain algal cells

which can hold phycotoxins, a method to disrupt whole algal cells was optimised. It was assumed that the

disruption method with the maximal yield of lipophilic phycotoxins is also suitable for hydrophilic phycotoxins.

For the clean-up procedure of lipophilic phycotoxins, the SPE cartridge was selected which gave the best results

for regulated lipophilic phycotoxins in shellfish extracts [65]. The washing step of the SPE procedure was

further optimised for a wider range of compounds in water samples. For hydrophilic phycotoxins SPE cartridges

16

were tested in order to reduce matrix effects and to concentrate the hydrophilic toxins. Another option tested

to concentrate the phycotoxins was by evaporation. Recoveries were obtained with spiked samples and

compared to a standard solution for PSP toxins, regulated lipophilic phycotoxins, some CIs and MCs.

The optimised clean-up procedures were as follows: for the clean-up of water containing lipophilic phycotoxins

a 30 mg Strata-X polymeric reversed phase cartridge (Phenomenex, Utrecht, The Netherlands) was used. The

cartridge was activated and conditioned with 1 mL methanol followed by 1 mL water. A water sample of 1 mL

was loaded onto the cartridge and the cartridge was washed with 1 mL water. Subsequently, the lipophilic

phycotoxins were eluted with 1 mL methanol. The eluent was transferred into a glass vial and used for analysis

with LC-hrMS. To extract hydrophilic phycotoxins from water a 500 mg Chromabond HILIC cartridge (Macherey-

Nagel, Düren, Germany) was used. The cartridge was activated and conditioned with 1 mL water followed by 6

mL acetonitrile. A water sample of 1 mL diluted with 9 mL acetonitrile was loaded onto the cartridge and

subsequently washed with 2 mL acetonitrile. The hydrophilic phycotoxins were eluted with 2 mL water. The

eluent was diluted with 2 mL acetonitrile and transferred into a glass vial for analysis with LC-hrMS.

FOOD SUPPLEMENTS

The extraction and clean-up procedure for food supplements was based on the methods for tissue and water.

Pills were grinded or capsules were removed on forehand. 1.0 ± 0.05 g of food supplement was weighed and

extracted with 4 mL methanol. The sample was vortex mixed for one minute using a multipulse vortex.

Subsequently the sample was ultrasonic disrupted for 1 minute at 11 W to disrupt possible whole algal cells

present. The sample was centrifuged at 2,000 x g for 5 minutes and the supernatant was decanted from the

pellet to a graduated tube. 5 mL of H2O/ACN/Amm.form/FA (55:45 v/v, 2 mM, 0.5 mM) was added to the

pellet. The sample was vortex mixed for one minute using a multipulse vortex. The sample was centrifuged at

2,000 x g for 5 minutes and the supernatant was combined with the methanol extract. The tube was filled to 10

mL with acetonitrile. The extract was diluted with 67.5 mL water in order to obtain a 10% organic strength prior

to the SPE procedure for lipophilic phycotoxins. A 60 mg Strata-X polymeric reversed phase cartridge was

activated and conditioned with 3 mL methanol and 3 mL water. The diluted extract was loaded onto the

cartridge and was washed with 3 mL water. Respectively, the lipophilic phycotoxins were eluted with 2 mL

methanol and the eluate was transferred to a sample vial for analysis with LC-hrMS. The clean-up method for

hydrophilic phycotoxins in food supplements is still in development. When it is developed all quantities of the

extraction procedure can be doubled and then the extract can be split for both clean-up methods.

EXTRACTION OF PALYTOXIN-GROUP TOXINS FOR CONFIRMATION

Selwood et al. developed a method for fish and shellfish to analyse PlTXs like palytoxins, ovatoxins and

ostreocins using LC-MS after micro-scale oxidation [58]. The oxidative fragmentation reaction causes the PlTXs

to fragment to an amide aldehyde specific oxidation product for each compound and a common amino

aldehyde as shown in Figure 1. The method was further optimised. Multiple extraction solvents were tested; LC

and the MS conditions were optimised. Subsequently repeatability, extraction efficiency, matrix effects, limit of

detection (LOD), limit of quantitation (LOQ) and linearity were determined in shellfish extracts for the oxidation

products: amide aldehyde of palytoxin and the common amino aldehyde.

17

FIGURE 1: OXIDATIVE FRAGMENT PRODUCTS OF PALYTOXIN

The optimised extraction procedure was as follows: 1.0 ± 0.05 g tissue homogenate was weighed and extracted

with 3 mL methanol. The sample was vortex mixed for one minute using a multipulse vortex. The sample was

centrifuged at 2,000 x g for 5 minutes and the supernatant was decanted from the pellet to a graduated tube.

The extraction was repeated twice. After the third extraction 9 mL water was added to the 9 mL supernatant to

dilute the extract before solid phase extraction was applied. A 60 mg Strata-X polymeric reversed phase

cartridge was conditioned with 3 mL methanol followed by 3 mL water. Subsequently the entire diluted sample

extract was loaded onto the cartridge. The cartridge was washed with 2 mL methanol/water (40:60 v/v)

followed by 2 mL water and then the sample was oxidized with 2 mL of 50 mM periodic acid. The cartridge was

washed for a second time with 2 mL water. Thereafter the oxidation products were eluted with 3 mL

MeOH/H2O/HAc (60:40:0.1 v/v). The eluent was transferred into a glass vial and used for analysis with LC-

MS/MS.

2.4 INSTRUMENTATION

SCREENING OF PHYCOTOXINS BY LC-HRMS

For the screening of phycotoxins a Thermo Scientific UltiMate 3000 LC-system (Thermo Fisher Scientific,

Waltham, USA) coupled to a Thermo Scientific Q Exactive focus hybrid quadrupole-orbitrap mass spectrometer

was used. Hydrophilic phycotoxins are not well retained on a reversed phase column and the lipophilic

phycotoxins are not well retained on the HILIC column. Therefore it was decided to develop two different LC

methods based on reversed phase and HILIC conditions. The mobile phase compositions were kept identical for

both LC methods. Mobile phase A consisted of water and mobile phase B consisted of acetonitrile/water (9:1

v/v), both containing 2 mM ammonium formate and 0.5 mM formic acid. Chromatographic separation of

H2N O

OH

OH

OH

O

OH

OH

HO

OH OH

OH

OH

O

OH

OH

OH

OH

HO

OOH

OHHO

OH

OHOH

OOH

OH

OH

HOOH

OH

OH

OHHO

OHO

OH

OH

HO

NH

NH

HO

OH

OH

OH

OH

OO

OH

OH

HO

O

O

O

O

H

H

common amino aldehyde

specific amide aldehyde

18

lipophilic phycotoxins was achieved on a reversed phase ACQUITY BEH C18 1.7 µm, 100 · 2.1 mm UPLC column

(Waters, Milford, MA, USA). The column temperature was set at 35°C and the total run time was 28 minutes.

The gradient elution with a flow of 0.3 mL min-1

was as follows: 0.1 minute at 10% mobile phase B, then linearly

increased to 100% mobile phase B in 12.9 minutes and kept at 100% mobile phase B for 12 minutes.

Subsequently the gradient went back to 10% mobile phase B in 0.1 minute and kept at 10% mobile phase B for

2.9 minutes to equilibrate the column for the next run. Chromatographic separation of hydrophilic phycotoxins

was achieved on a TSKgel Amide-80 2 µm, 150 · 3 mm HPLC column (Tosoh Bioscience, Tokyo, Japan). The

column temperature was set at 35°C and the total run time was 20 minutes. The gradient elution with a flow of

0.5 mL min-1

was as follows: 0.1 minute at 90% mobile phase B, then linearly decreased to 45% mobile phase B

in 13.9 minutes and subsequently linearly decreased to 20% mobile phase B in 0.1 minute and kept at 20%

mobile phase B for 1.9 minutes. Subsequently the gradient went back to 90% mobile phase B in 0.1 minute and

kept at 90% mobile phase B for 3.9 minutes to equilibrate the column. For both chromatographic methods the

injection volume was set at 10 µL.

In order to detect the phycotoxins, electrospray ionisation (ESI) in both positive and negative mode was used.

The positive and negative ESI signals were acquired in two separate runs. The spray voltage in positive

ionisation mode was set at 3.5 kV and in negative ionisation mode at -2.5 kV. The capillary temperature was set

at 260°C. A full MS scan event of 100 to 1500 m/z with a resolution of 70,000 full width at half maximum

(FWHM) was acquired. In order to obtain additional information on the phycotoxins, fragmentation spectra

were also acquired. The so called MS2 scans were obtained by selecting all ions in respectively m/z mass range

windows of 100 to 500, 500 to 1000 and 1000 to 1500. As collision gas nitrogen was used. The normalized

collision energy (NCE) was set at 40 during fragmentation of all ion mass ranges, except for 100 to 500 m/z in

negative ionisation mode where the NCE was set at 30. Then after fragmentation the ions were scanned

respectively from 50 to 500, 50 to 1000 and 50 to 1500 m/z with a resolution set at 17,500 FWHM. The

automatic gain control representing the maximum capacity of the C-trap was set at a maximum of 106 ions or a

maximum injection time of 200 ms for both the full scan and MS2 scans were allowed.

To process the data Tracefinder (Thermo Fisher Scientific, Waltham, USA) was used. A database containing over

800 phycotoxins was constructed from literature. A part of the database is shown in Appendix 2 for illustration.

Relevant parts of the database for data processing were transferred to a Tracefinder compound database and

data was searched. There was a mass error allowed of 5 parts per million (ppm) for the m/z of the precursor

ions and for regulated phycotoxins at least one fragment ion should be present within a 5 ppm mass error.

Furthermore, if the retention time of a compound was known it should be within a ± 0.2 minutes retention

window.

CONFIRMATION OF PALYTOXIN-GROUP TOXINS BY LC-MS/MS

For PlTXs a Waters Acquity UPLC system coupled to a Waters Xevo TQ-S tandem mass spectrometer (Waters,

Milford, USA) was used. Chromatographic separation was achieved by a Waters Acquity BEH C18 100 · 2.1 mm,

1.7 µm UPLC column at 80°C, with a gradient of 5 minutes. Mobile phase A consisted of water and mobile

phase B consisted of acetonitrile/water (9:1 v/v), both containing 6.7 mM ammonium hydroxide. The gradient

with a flow of 0.6 mL min-1

was kept the first 0.5 minutes at 0% mobile phase B, and then it linearly increased

to 90% mobile phase B in 3 minutes and kept at 90% mobile phase B for 0.5 minutes. Thereafter the gradient

went back to 0% mobile phase B in 0.1 minutes and kept for 0.9 minutes at 0% mobile phase B to equilibrate

the column for the next run. The injection volume was set at 10 µL.

The MS system was operated in positive electrospray mode and data was recorded in MRM mode using two

transitions per PlTX oxidative fragmentation product. Table 5 shows the MS/MS conditions used to acquire the

19

MRM data. Furthermore, a capillary voltage of 3 kV, source temperature of 150 °C, desolvation temperature of

500 °C and desolvation gas flow of 800 L h-1

was set.

TABLE 5: MS/MS CONDITIONS FOR THE CONFIRMATION OF PALYTOXIN-GROUP TOXINS

Compound Precursor ion(m/z)

Product ion (m/z)

Cone (V)

Collision energy (eV)

Common amino aldehyde 300.2 107.0 30 25

300.2 151.0 30 25

Neo-palytoxin amide aldehyde 325.2 76.0 30 20

325.2 307.0 30 20

Ostreocin D amide aldehyde 329.2 76.0 30 13

329.2 149.0 30 13

Palytoxin, Tahitian palytoxin, Ovatoxin-a, 343.2 76.0 30 13

42-OH palytoxin amide aldehyde 343.2 123.0 30 13

Homopalytoxin amide aldehyde 357.2 76.0 30 20

357.2 339.0 30 20

Bishomopalytoxin amide aldehyde 371.2 76.0 30 20

371.2 353.0 30 20

2.5 VALIDATION

The following phycotoxins were validated: OA, DTX1, DTX2, YTX, hYTX, PTX2, SPX1, GYM, 13,19 didesm SPX C,

20 meth SPX G, OA methyl ester, 16:0 OA ester, OA C8 diol ester, AZA1, AZA2, AZA3, AZA4, AZA5, PnTX E, PnTX

F, PnTX G, CYN, DA, MC-LA, MC-LF, MC-LR, MC-LW, MC-LY, MC-RR, MC-WR, MC-HilR, MC-HtyR, MC-YR, Asp

MC-LR, NOD, STX, dcSTX, NEO, dcNEO, GTX1&4, GTX2&3, GTX5, dcGTX2&3, C1&2, TTX and ATX. The validation

of the screening method was based on the estimated screening detection limit (SDL). The SDL of the qualitative

screening method is the lowest level at which an analyte has been detected in at least 95% of the samples. 20

blank tissue samples, including 5 mussel-, 5 oyster-, 5 cockle-, 5 ensis- and 5 fish homogenates, were spiked

after extraction with 600 µg kg-1

hydrophilic phycotoxins, 150 µg kg-1

microcystins and 80 µg kg-1

of all other

lipophilic phycotoxins included in the validation. Five blanks, one of each matrix, were included to determine

false positives. 20 blank water samples, including 6 sea water, 6 brackish water, 6 fresh water and 2 tap water

were spiked before clean-up with 120 µg L-1

hydrophilic phycotoxins, 10 µg L-1

MCs and 5 µg L-1

of all other

lipophilic phycotoxins. DA was included in both clean-ups for hydrophilic phycotoxins and lipophilic

phycotoxins. Furthermore 4 blanks were included to determine false positives. 20 blank solid food supplements

were spiked before extraction with 30 µg kg-1

MCs and 15 µg kg-1

of all other lipophilic phycotoxins. 20 blank

liquid food supplements (mostly oils) were spiked before extraction with 50 µg kg-1

MCs and 15 µg kg-1

of all

other lipophilic phycotoxins. 9 blank food supplements were included to determine false positives. For each

procedure also an empty tube was included to determine if there were any interfering contaminants from the

procedures itself. Furthermore, the confirmation and quantitation of regulated lipophilic phycotoxins and some

CIs, PSP toxins and DA in shellfish were validated: OA, DTX1, DTX2, YTX, hYTX, SPX1, GYM, AZA1, AZA2, AZA3,

PnTX G, DA, STX, dcSTX, NEO, dcNEO, GTX1&4, GTX2&3, GTX5 and dcGTX2&3. 5 blank mussel homogenates

were spiked after extraction at 0.5 and 1 times the permitted level (PL) according to the European Union. One

blank mussel homogenate was spiked before extraction at 0.5 PL and reference materials were included to

determine the recovery. Matrix matched standards were spiked at 5 (ASP and PSP) or 6 (lipophilic phycotoxins)

levels. Concentrations are shown in Table 6.

20

TABLE 6: SPIKE LEVELS OF ASP, PSP AND DSP FOR QUANTITATIVE VALIDATION IN SHELLFISH [30-34]

Compound Toxin group 0.5 PL (ug kg-1)

1 PL (ug kg-1)

Matrix matched standards (ug kg-1)

DA ASP 10,000 20,000 0, 5, 10, 20, 50 mg kg-1

STX, dcSTX, NEO, dcNEO, GTX1&41, GTX2&31, GTX5, dcGTX2&31 PSP 400 800 0, 400, 600, 800, 1200

OA, DTX1, DTX2 DSP 80 160 0, 20, 40, 80, 160, 240

AZA1, AZA2, AZA3 80 160 0, 20, 40, 80, 160, 240

YTX, hYTX2 250 500 0, 62.5, 125, 250, 500, 750

SPX13 200 400 0, 50, 100, 200, 400, 600

GYM3 100 200 0, 25, 50, 100, 200, 300

PnTX G3 25 50 0, 6.25, 12.5, 25, 50, 75

1 Concentration of the highest isomer present. 2 The permitted level of YTX and hYTX is 3750 µg kg-1, values are target values. 3 There are no permitted levels for SPX, GYM and PnTX G. Values are target values.

21

3. RESULTS AND DISCUSSION

3.1 OPTIMIZATION OF EXTRACTION PROCEDURES

For development of extraction and clean-up procedures measurements were carried out with available RIKILT

standards operation procedures (SOPs). All methods were applied on LC-MS/MS; SOPs are described in

Appendix 3. Most recoveries obtained during method development were established by comparing results with

results from SOPs or by a mutual comparison between tested procedures. These were comparisons of

recoveries of the extraction and/or clean-up procedure including the influence of matrix effects in the purified

extracts. Therefore, unless stated otherwise, recoveries reported are apparent recoveries. SOP results were in

all cases considered as 100% recovery.

TISSUE SAMPLES

To develop an extraction method for both lipophilic and hydrophilic phycotoxins in tissue samples several

extraction methods, volumes and duration of the extraction were tested. Natural contaminated samples were

used containing OA, DTX2, AZAs, SPX1 and PSP toxins.

To test different extraction methods extractions were carried out with 4 mL methanol, followed by 4 mL

H2O/ACN/Amm.form/FA (55:45 v/v, 2 mM, 0.5 mM) or with 4 mL H2O/ACN/Amm.form/FA (55:45 v/v, 2 mM,

0.5 mM), followed by 4 mL methanol. Methanol was used to extract lipophilic phycotoxins and

H2O/ACN/Amm.form/FA was used to extract hydrophilic phycotoxins. After adding each extraction solvent

samples were vortex mixed for 1 minute, placed in an ultrasonic bath for 5 minutes, ultrasonic disrupted for 30

seconds at 11 W or heated for 5 minutes at 70 °C, followed by centrifugation and decanting of the extract. To

obtain a recovery results were compared to the SOP for lipophilic phycotoxins, which is a triplicate extraction

with 3 mL methanol, followed by vortex mixing for 1 minute, centrifugation and decanting of the extract after

each time adding methanol. The complete SOP extraction procedure is describes in Appendix 3. All extracts

were complemented to 10 mL with methanol and filtered. Recoveries (average of n=2) are shown in Table 7.

TABLE 7: AVERAGE RECOVERIES (%) OF LIPOPHILIC PHYCOTOXINS WITH VARIOUS EXTRACTION METHODS

Solvents* Extraction OA DTX2 DTX3

(OA 16:0) AZA1 AZA2 AZA3 SPX1 Average

MeOH, MP Vortex 95.2 118.5 89.1 87.8 90.2 91.2 148.6 102.9

100.1 MeOH, MP Ultrasonic bath 85.1 123.6 73.1 78.7 84.4 93.0 130.4 95.5

MeOH, MP Ultrasonic disruptor 89.7 116.4 79.9 83.5 90.1 94.5 136.8 98.7

MeOH, MP Heat 70 °C 76.5 112.3 54.6 89.5 90.4 171.4 128.2 103.3

MP, MeOH Vortex 87.6 121.0 49.9 84.5 81.3 98.6 123.4 92.3

93.0 MP, MeOH Ultrasonic bath 76.1 112.3 38.2 87.8 78.8 131.0 99.6 89.1

MP, MeOH Ultrasonic disruptor 99.2 115.4 52.9 81.3 79.6 119.7 151.0 99.9

MP, MeOH Heat 70 °C 74.2 101.4 37.2 86.9 78.5 157.7 98.1 90.6

*MP = Mobile phase A/B (50:50 v/v), which is H2O/ACN/Amm.form/FA (55:45 v/v, 2 mM, 0.5 mM)

All extraction methods showed good recoveries for the lipophilic phycotoxins present in the samples except for

DTX3 and AZA3. On average the recovery was higher and the RSD was lower when starting the extraction with

methanol. To obtain a higher recovery for DTX3 a multiple extractions with methanol are needed due to the

fatty acid ester chains in the molecules. The recovery of AZA3 in combination with heat was significantly higher

because AZA17 present in the sample was converted to AZA3 [66]. Heating was avoided in order to prevent

conversion within other samples. For further experiments there was chosen to continue with a combination of

vortex mixing and ultrasonic disruption.

22

To optimize the extraction procedure for lipophilic phycotoxins in tissue the duration of vortex mixing and

ultrasonic disruption was varied. 4 mL methanol was added to the samples, the sample was vortex mixed for 1

or 5 minutes, followed by centrifugation and decanting of the extract. Subsequently, 4 mL

H2O/ACN/Amm.form/FA (55:45 v/v, 2 mM, 0.5 mM) was added and ultrasonic disrupted for 30 seconds, 1

minute or 5 minutes at 11 W, followed by centrifugation and decanting of the extract. The results were

compared to the results of the SOP for lipophilic phycotoxins to obtain a recovery. Recoveries (average of n=2)

are shown in Table 8.

TABLE 8: AVERAGE RECOVERIES (%) OF LIPOPHILIC PHYCOTOXINS WITH VARIOUS DURATION OF EXTRACTION

Vortex Ultrasonic disruptor

OA DTX2 DTX3

(OA 16:0) AZA1 AZA2 AZA3 SPX1 Average

1 minute 0.5 minute 101.2 94.0 44.4 112.3 104.9 92.3 87.4 90.9

91.2 1 minute 1 minute 121.1 127.9 48.5 89.6 85.0 87.2 85.6 92.1

1 minute 5 minutes 108.0 119.2 43.7 96.3 95.3 91.7 80.2 90.6

5 minutes 0.5 minute 94.7 99.3 27.6 104.8 103.1 94.2 79.4 86.2

84.5 5 minutes 1 minute 80.0 67.0 14.2 102.8 103.3 93.1 69.4 75.7

5 minutes 5 minutes 103.8 109.7 37.7 105.2 106.6 97.2 80.4 91.5

A longer time of vortex mixing or ultrasonic disruption did not have an effect on the recovery. Because a longer

extraction time did not have a positive effect on the recovery there was continued with 1 minute of vortex

mixing and 1 minute of ultrasonic disrupting. After the lipophilic procedure, extraction optimization for the

hydrophilic phycotoxins was optimised. Different extraction volumes were tested. Natural contaminated

samples containing PSP toxins were used. 4 mL methanol was added to the samples, the sample was ultrasonic

disrupted for 1 minute at 11 W, followed by centrifugation and decanting of the extract. Subsequently, 4 or 5

mL H2O/ACN/Amm.form/FA (55:45 v/v, 2 mM, 0.5 mM) was added and vortex mixed for 1 minute, followed by

centrifugation and decanting of the extract. To obtain a recovery, results were compared to the results of a

triplicate extraction with 3 mL H2O/ACN/Amm.form/FA (55:45 v/v, 2 mM, 0.5 mM), followed by vortex mixing

for 1 minute, centrifugation and decanting of the extract after each time adding extraction solvent. All extracts

were complemented to 10 mL with acetonitrile and filtered. Recoveries (average of n=2) are shown in Table 9.

TABLE 9: AVERAGE RECOVERIES (%) OF HYDROPHILIC PHYCOTOXINS WITH VARIOUS VOLUMES OF EXTRACTION SOLVENT

MeOH MP* dcSTX NEO dcNEO GTX3 GTX4 GTX5 dcGTX3 C2 C1 GTX6 Average

4 mL 4 mL 99.1 56.3 99.6 82.7 96.8 94.2 94.3 91.9 55.8 94.9 86.6

4mL 5 mL 94.0 87.8 99.1 83.7 99.5 94.8 89.0 90.2 55.9 94.4 88.8

*MP = Mobile phase A/B (50:50 v/v), which is H2O/ACN/Amm.form/FA (55:45 v/v, 2 mM, 0.5 mM)

On average the recovery was slightly higher when 5 mL H2O/ACN/Amm.form/FA (55:45 v/v, 2 mM, 0.5 mM)

was used during the second extraction step. Especially the recovery for NEO is improved from 56% to 88%.

To verify the recovery of other phycotoxins blank shellfish samples were spiked with standards and to further

improve the efficiency of the method, ultrasonic disrupting was replaced by vortex mixing. 4 mL methanol was

added to the samples, the sample was ultrasonic disrupted or vortex mixed for 1 minute, followed by

centrifugation and decanting of the extract. Subsequently, 5 mL H2O/ACN/Amm.form/FA (55:45 v/v, 2 mM, 0.5

mM) was added and vortex mixed for 1 minute, followed by centrifugation and decanting of the extract. To

obtain a recovery the results were compared to the results of samples extracted with SOPs for lipophilic toxins,

PSP toxins and MCs. Extraction procedures of the SOPs are described in Appendix 3. Recoveries (average of

n=2) are shown in Table 10.

23

TABLE 10: AVERAGE RECOVERIES (%) OF SPIKED STANDARDS WITH AND WITHOUT ULTRASONIC DISRUPTION

First extraction OA DTX1 DTX2 AZA1 AZA2 AZA3 AZA4 YTX hYTX SPX1 GYM Average

Ultrasonic disruptor 119.5 123.9 120.3 91.7 90.8 98.1 91.2 96.9 103.9 100.9 101.5

Vortex 131.5 120.9 128.0 93.3 91.5 93.9 87.9 92.5 99.5 117.9 106.3

STX dcSTX NEO dcNEO GTX3 GTX4 GTX5 dcGTX3 TTX DA ATX CYN

Ultrasonic disruptor 79.9 87.8 78.9 111.0 67.2 157.6 55.1 106.8 111.1 101.9 200.6 109.1

Vortex 81.7 88.8 78.3 97.6 73.5 131.5 69.9 101.3 106.8 94.6 204.4 118.1

MC- WR HtyR YR LW HilR LY LR LF aspLR LA NOD RR

Ultrasonic disruptor 149.6 110.7 105.7 157.9 119.9 158.0 128.7 97.9 120.2 140.2 88.7 158.1 112.6

Vortex 167.8 129.5 119.7 146.1 129.8 162.2 133.9 88.4 132.0 141.4 104.7 152.9 114.8

All recoveries are above 80% except for some PSP toxins. On average the recovery for extraction with only

vortex mixing is higher than an extraction with ultrasonic disruption. The final procedure is described in the

materials and methods chapter.

WATER SAMPLES

Water samples can contain algal cells. Assumed was that phycotoxins can be present in algal cells and that

those cells might not break open during a clean-up procedure. When cells do not break open during clean-up,

the phycotoxins present in the whole cells are not measured and results give unreliable information about the

toxicity of the sample. To make sure cells were disrupted different disrupting methods were tested with a stain

of Alexandrium Ostenfeldii from the Ouwerkerkse kreek in The Netherlands producing SPX1 and GYM.

Parts of the sample were ultrasonic disrupted for 1 minute at 11W, placed in an ultrasonic bath for 5 minutes,

frozen, 20 seconds grinded twice with a Precellys (VWR, Amsterdam, The Netherlands) at 6500 rpm or used

without any treatment. Then all whole cells were removed by using a 0.2 µm HT Tuffryn filter (Sigma-Aldrich,

Zwijndrecht, The Netherlands). Furthermore, two filters used for samples without treatment were washed with

5 mL water or 1 mL methanol to determine any osmotic effects or effects of organic solvents on the algae.

Subsequently, 1 mL of the filtered samples was cleaned-up with solid phase extraction (SPE). Also a sample

without any treatment or filtration was cleaned-up. Methanol was diluted with 4 mL water before SPE to

ensure retention of SPX1 and GYM on the SPE cartridge. A 30 mg Strata-X polymeric reversed phase cartridge

(Phenomenex, Utrecht, The Netherlands) was activated and conditioned with 1 mL methanol followed by 1 mL

water. A water sample of 1 mL was loaded onto the cartridge and the cartridge was washed with 1 mL water.

Subsequently, the lipophilic phycotoxins were eluted with 1 mL methanol. Because the concentration of SPX1

and GYM in the water sample was unknown, the result with the highest response was set at a 100% recovery,

other result were compared to this. The recoveries (average of n=2) are shown in Table 11.

TABLE 11: AVERAGE RECOVERIES (%) AFTER ALGAL DISRUPTION

Sample treatment SPX1 GYM

Filter – SPE 14.4 12.8

Ultrasonic disruptor – filter – SPE 68.8 78.8

Ultrasonic bath – filter – SPE 15.6 14.8

Frozen – filter – SPE 58.9 66.7

Precellys – filter – SPE 53.7 67.0

Filter – water wash – SPE 12.6 14.8

Filter – MeOH wash – SPE 40.2 46.5

SPE 100.0 100.0

With filtration only phycotoxins present in the water were measured. It appears that only a small amount (14%)

of SPX1 and GYM was excreted by the algae into the water. Ultrasonic disruption, freezing, grinding with the

24

Precellys and treatment with methanol disrupted some of the algae and phycotoxins were released. There was

no osmotic effect because the concentration after washing with water was not increased compared to only

filtration. However, the best results were achieved with use of only the SPE clean-up. The experiment was

repeated without the filtration step; nevertheless, there was no increase in recovery when ultrasonic disrupted

samples were directly cleaned-up with SPE. This could mean that all toxins are released from the algal cells

during the SPE procedure. Most probably the cells were lysed during the addition of 1 mL methanol.

Brackish medium was used to cultivate the strain of Alexandrium Ostenfeldii. Therefore, to test the SPE clean-

up method for other lipophilic phycotoxins a blank brackish medium was spiked with lipophilic phycotoxins.

Different washing steps were tested to optimize the method. A 30 mg Strata-X polymeric reversed phase

cartridge was activated and conditioned with 1 mL methanol followed by 1 mL water. A water sample of 1 mL

was loaded onto the cartridge and the cartridge was washed with 1 mL water or water/methanol (80:20 v/v).

Subsequently, the lipophilic phycotoxins were eluted with 1 mL methanol. The results (average of n=2) were

compared to a standard solution to obtain a recovery. Results are shown in Table 12.

TABLE 12: AVERAGE RECOVERIES (%) OF LIPOPHILIC PHYCOTOXINS IN WATER AFTER SOLID PHASE EXTRACTION

Washing step OA DTX1 DTX2 AZA1 AZA2 AZA3 AZA4 YTX hYTX SPX1 GYM PnTX G Average

Water 148.0 86.8 141.1 41.4 33.5 49.7 57.6 120.9 98.6 79.1 85.2 70.1 84.3

20% MeOH 124.0 83.6 125.1 50.6 41.1 61.9 69.7 93.1 89.9 92.3 102.8 84.9 84.9


Water 29.5 63.8 61.9 10.4 40.2 42.8 54.6 18.7 35.1 47.7 75.8 97.2 48.1

20% MeOH 29.6 40.9 48.6 10.2 32.4 36.1 48.1 16.2 31.8 39.4 76.1 67.8 39.8

Besides the compounds in Table 12, DA was analysed as well but had no recovery. Therefore concluded was

that DA was not retained on the SPE cartridge, since DA is also not retained on the reversed phase LC column

with the conditions of this developed screening method. Recoveries of the MCs appear to be low (10 to 97%),

compared to a standard solution without clean-up, however compared to the results of the SOP for MCs in

water (data not shown, procedure in appendix 3) recoveries are 60 to 480% with an average of 196%.

Therefore the developed procedure is an improvement of existing SOP. Some of the compounds gave a

recovery >100%, all of these compounds were measured in negative ionisation mode. Although matrix effects

are generally less in negative ionisation mode, enhancement of these compounds could be due to matrix

effects. Because MCs had a better recovery when washed with water, this was included in the final procedure

for lipophilic phycotoxins in water samples. The final procedure is as described before in the materials and

methods chapter.

For the hydrophilic phycotoxins a different approach was needed, because hydrophilic phycotoxins do not

retain well on a reversed phase cartridge and are eluting simultaneously with salts present in the sample. At

first there was attempt to evaporate the sample and reconstitute in a high organic solvent. A high

concentration of acetonitrile was needed to trap the phycotoxins of interest on the HILIC LC-column. By

evaporating the sample in advance and thereafter dilute with a smaller volume acetonitrile the final

concentration would be higher as the initial concentration of the sample and there would be no loss of

sensitivity during LC-MS analysis. 100 mL of brackish water sample was evaporated to dryness and then

reconstituted in 10 mL of solvents ranging from 10 to 25% water with ACN/Amm.form/FA (90 to 75% v/v, 2

mM, 0.5 mM). Due to the high salt content in the sample two immiscible layers were formed. These extracts

cannot be used for LC-MS/MS analysis. Subsequently, different volumes of H2O/ACN/Amm.form/FA (55:45 v/v,

2 mM, 0.5 mM) were used to attempt to reconstitute the sample without formation of immiscible layers.

25

However, even after adding 60 mL there were still two layers present and it was decided that a procedure

including evaporation was not applicable for water samples with a relative high salt content. Then SPE with a

HILIC cartridge was attempted. Blank brackish water samples of 1 mL were spiked with 100 ng/mL hydrophilic

phycotoxins and diluted with 3 mL acetonitrile (75%) or 9 mL acetonitrile (90%) to test how much organic

strength was needed for the samples to obtain a good retention on the SPE cartridge. A 500 mg Chromabond

HILIC cartridge was activated and conditioned with 1 mL water followed by 6 mL acetonitrile. The diluted water

sample was loaded onto the cartridge and subsequently washed with 2 mL acetonitrile. The hydrophilic

phycotoxins were eluted with 2 mL water. The effluent of the sample, washing solvent and eluent were

collected. The effluent of the sample was evaporated and reconstituted in 75% acetonitrile, the washing

solvent was diluted with water and the eluent was diluted with acetonitrile. All fractions (n=2) were compared

to a standard solution to obtain a recovery. The results (average of n=2) are given in Table 13.

TABLE 13: AVERAGE RECOVERIES (%) OF HYDROPHILIC PHYCOTOXINS WITH CHROMABOND SOLID PHASE EXTRACTION

Organic strength

SPE fraction

STX

dcSTX

NEO

dcNEO GTX3 GTX4 GTX5 dcGTX3 C 1 TTX DA ATX CYN

75% Sample 10.4 10.8 9.7 7.7 15.7 0.8 16.1 15.0 0.1 16.4 0.2 52.0 36.2

Wash 21.3 24.2 38.8 26.0 20.3 6.3 22.4 18.4 0.2 24.7 0.1 26.1 19.7

Elution 51.6 55.6 84.8 67.8 46.0 57.9 56.7 56.1 3.0 42.1 7.6 1.7 18.7

Total 83.3 90.6 133.3 101.4 82.0 65.0 95.2 89.5 3.4 83.1 7.8 79.8 74.6

90% Sample 0.1 0.3 0.1 5.4 0.1 0.0 0.1 0.1 0.1 0.1 0.7 55.5 0.2

Wash 0.1 0.2 0.4 1.9 0.1 0.0 0.1 0.1 0.1 0.1 0.2 22.4 1.0

Elution 74.4 66.7 114.7 101.0 68.3 58.9 77.5 75.3 2.7 78.9 7.2 3.2 69.4

When the sample was diluted to only 75% acetonitrile most hydrophilic phycotoxins were not well retained on

the cartridge as most compounds eluted with loading of the sample and during the washing step. However,

when the sample was diluted to 90% acetonitrile most hydrophilic phycotoxins were well retained during solid

phase extraction, except for ATX which was still eluting during loading of the sample and the washing step. C1

and DA were showing bad recoveries; although there is no clear explanation it might that these phycotoxins

precipitated during dilution of the sample together with the salts, were not eluted from the cartridge or were

suppressed due to matrix effects during LC-MS analysis. The final procedure is described in the materials and

methods chapter.

FOOD SUPPLEMENTS

There is a large variety of food supplements as shown in Figure 2. Samples can be oils, pills or powders. It can

be expected that extraction behaviour for these matrices are difficult and it might be impossible to obtain good

recoveries for all supplements that are available. Still the procedure which is a combination of the tissue and

water procedure was tested. A true recovery of the extraction and SPE was determined by spiking food

supplements before extraction and spiking another extract of the same product after SPE. Results (average of

n=2) are shown in Table 14.

26

FIGURE 2: VARIATY OF FOOD SUPPLEMENTS USED FOR RECOVERY EXPERIMENTS

TABLE 14: AVERAGE RECOVERIES (%) OF LIPOPHILIC PHYCOTOXINS IN FOOD SUPPLEMENTS

Sample OA DTX1 DTX2 AZA1 AZA2 AZA3 AZA4 YTX hYTX SPX1 GYM PTX2 Average

Oil 79.8 80.1 81.8 59.3 46.1 70.3 89.3 62.5 55.9 93.5 92.3 90.7 75.1

Oil capsules 56.2 67.4 66.2 27.4 21.6 39.2 83.8 13.1 9.3 90.8 84.0 64.4 52.0

Powder 91.3 60.8 79.2 49.1 48.2 53.2 57.4 25.0 26.9 62.2 73.5 53.6 56.7

Tablets 77.5 82.5 79.1 34.8 24.1 46.0 69.4 19.5 17.6 79.0 80.1 80.1 75.5

Average 76.2 72.7 76.6 42.7 35.0 52.2 75.0 30.0 27.4 81.4 82.5 72.2


Oil 84.0 78.8 74.8 80.7 83.5 84.5 86.4 89.5 82.0 72.8 83.6 76.0 81.4

Oil capsules 66.4 56.0 55.2 19.5 53.7 14.9 46.0 17.5 49.8 10.0 41.4 102.5 44.4

Powder 52.6 57.3 53.3 69.3 62.5 53.5 56.8 56.0 57.9 52.8 67.0 84.0 60.2

Tablets 54.4 62.2 64.9 63.3 64.6 44.7 59.6 50.2 64.7 44.1 63.0 50.3 57.2

Average 64.3 63.6 62.0 58.2 66.1 49.4 62.2 53.3 63.6 44.9 63.8 78.2

As expected a lot of different results are obtained for different matrices but also between the phycotoxins.

Lowest recoveries were obtained for YTX and hYTX. On average the recovery is 61%, however the recoveries

range from 9 to 102%. Because matrix effects are very different from each other it is difficult to develop a

method that suits for all types of food supplements with acceptable recoveries for all compounds. Since the

clean-up method worked properly for water samples it was assumed that when changing the SPE clean-up

method to remove more matrix also some phycotoxins will disappear. Therefore it is not an option to change

or add more clean-up steps. When samples are measured in the future, it is recommended that standard

addition is applied to determine recoveries of phycotoxins from individual samples.

3.2 CHROMATOGRAPHY

The separation of lipophilic and hydrophilic compounds is very different. Lipophilic compounds are retained on

reversed phase LC-columns. Hydrophilic compounds are retained with HILIC. To increase the efficiency of the

method there was attempt to use one generic mobile phase combination of water and organic solvent with

additives for both separations.

It was important that peaks were broad enough to create enough data points, as the resolution of the hrMS is

related to the scan speed. When a higher resolution is set the scan time is longer. MS measurements were

divided in four scan events. One full scan at a resolution of 70,000 FWHM and three MS2 scans at a resolution

of 17,500 FWHM with elevated NCE (30% or 40%) as earlier described in the materials and methods chapter. All

these events caused a relatively long total cycle time of 0.85 seconds. Therefore, when a minimum of 10 data

points per peak are desired, peaks needs to be at least 9 seconds broad.

27

REVERSED PHASE LIQUID CHROMATOGRAPHY

For separation of lipophilic phycotoxins an ACQUITY BEH C18 1.7 µm, 100 · 2.1 mm column was used, the same

type as used in the SOPs for lipophilic phycotoxins (Appendix 3). Mobile phases and other LC settings were

similar to a screening method for pesticides [67], except for mobile phase B, where methanol was replaced by

acetonitrile. To elute all compounds of interest methanol/water/amm.form/FA (95:5 v/v, 2 mM, 0.5 mM)

needed to be replaced by acetonitrile/water/amm.form/FA (90:10 v/v, 2 mM, 0.5 mM). The following gradient

was tested: 0.1 minute at 10% mobile phase B, then linearly increased to 100% mobile phase B in 12.9 minutes

and kept at 100% mobile phase B for 4 minutes. Subsequently the gradient went back to 10% mobile phase B in

0.1 minute and kept at 10% mobile phase B for 2.9 minutes to equilibrate the column. A good separation was

obtained, except that the 16:0 OA ester was still retained on the column. To elute the esters the gradient was

extended and was kept at 100% mobile phase B for 12 minutes. Although most standards were eluted after 7

minutes the percentage of mobile phase B was slowly increased to obtain a good retention for any unknown

phycotoxins eluting in the first 7 minutes. Chromatograms are shown in Figure 3. The final gradient and other

LC settings are described in the materials and methods chapter.

HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY

For HILIC methods in general it is known that matrix effect have a major influence on sensitivity and retention

times, especially when samples are treated with a non-selective sample treatment [68]. To obtain a sufficient

separation of hydrophilic phycotoxins two different HILIC columns were tested. The Nucleoshell HILIC 3 µm,

100 · 2.7 mm HPLC column (Macherey-Nagel, Düren, Germany) and the TOSOH Bioscience TSKgel Amide-80 2

µm, 150 · 3 mm HPLC column (Tosoh Bioscience, Tokyo, Japan) were tested and compared. The best results

were obtained with the TSKgel Amide column, as the Nucleoshell HILIC column, which is a zwitterionic column,

gave no peaks at all under the tested conditions. However, peaks were still relatively broad with the TSK Amide

column and there was no baseline separation for GTX1 and GTX4, which are isomers. Peak shapes of DBA and

BMAA were poor and therefore these compounds were excluded from the validation. Chromatograms are

shown in Figure 4. The gradient and other LC settings are described in the materials and methods chapter.

28

FIGURE 3: REVERSED PHASE LIQUID CHROMATOGRAPHY OF A) AZASPIRACIDS, SPIROLIDES, PINNATOXINS, GYMNODIMINE AND

OKADAIC ACID ESTERS MEASURED IN POSITIVE IONISATION MODE; B) DOMOIC ACID, MICROCYSTINS, CIGAUTOXINS, BREVETOXINS,

PECTENOTOXIN AND PALYTOXIN MEASURED IN POSITIVE IONISATION MODE; C) YESSOTOXINS, OKADAIC ACID AND DINOPHYSIS TOXINS

MEASURED IN NEGATIVE IONISATION MODE

0%

20%

40%

60%

80%

100%

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Re

lati

ve a

bu

nd

ance

tR (min)

Gym

PnTX E

13,19didesmSPXC

SPX1

20meSPXG

PnTXF

PnTXG

AZA4

AZA5

OAme-ester

OAdiolester

AZA3 AZA1

AZA2

A

0%

20%

40%

60%

80%

100%

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Re

lati

ve a

bu

nd

ance

tR (min)

DA

NOD

MCRR MCHtyR, MCLR, AspMCLR, MCLY

MCHilR

MCWR, PlTX

MCLA MCLY

MCLW

MCLF

pCTX1

PTX2

PbTx3

PbTx9

pCTX2

PbTx2

pCTX3

B

0%

20%

40%

60%

80%

100%

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Re

lati

ve a

bu

nd

ance

tR (min)

OA, YTX, hYTX

DTX2

DTX1

16:0 OA ester

C

29

FIGURE 4: HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY OF A) ANATOXIN, CYLINDROSPERMOPSIN, DOMOIC ACID,

TETRODOTOXIN, SAXITOXIN AND NEOSAXITOXIN MEASURED IN POSITIVE IONISATION MODE; B) C1&2, GONYAUTOXIN 2, 3 AND 5

MEASURED IN NOEGATIVE IONISATION MODE; C) DECARBAMOYLGONYAUTOXIN 2 AND 3 MEASURED IN NEGATIVE IONISATION MODE;

D) GONYAUTOXIN 1 AND 4 MEASURED IN NEGATIVE IONISATION MODE

0%

20%

40%

60%

80%

100%

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Re

lati

ve a

bu

nd

ance

tR (min)

ATX

CYN

DA

TTX

STX, dc STX NEO, dcNEO

A

0%

20%

40%

60%

80%

100%

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Re

lati

ve a

bu

nd

ance

tR (min)

C1

C2

GTX2

GTX3

GTX5

B

0%

20%

40%

60%

80%

100%

7 8 9 10 11

Re

lati

ve a

bu

nd

ance

tR (min)

dcGTX2

dcGTX3

C

7 8 9 10 11

tR (min)

GTX1

GTX4

D

30

3.3 MS MEASUREMENTS

Analyses were conducted in full scan mode from to 100 to 1500 m/z. Although there are phycotoxins with

higher masses stated is the database, literature shows that all compounds with a mass above 1500 Da in the

database are multiple charged, for example some DTX4 analogues and PlTX-group toxins [69-72]. Furthermore

MS2 scans were obtained from all precursor ions in the range of 100 to 1500 m/z divided into three separate

scan events in order to increase the selectivity. Fragmentation spectra were more selective this way, because

fragments are originating from a relatively small range of precursor ions. MS2

settings were optimised using

different NCEs. The chosen NCE gave for most of the phycotoxins tested at least one fragment ion in the MS2.

Still with unknowns it might that the NCE is not sufficient to get fragmentation information.

Palytoxin showed a low sensitivity. When looking closer to the spectrum it appeared that palytoxin had a

complex spectrum with multiple adducts, charges and water losses as showed in Figure 5. Because many

different adducts were formed with multiple charges and multiple losses of water occurred, sensitivity of

palytoxin was low. Furthermore, because of the size of the molecule also the isotopic pattern contributed to a

low sensitivity. The same applies for ovatoxins [71]. When palytoxin was ionised in negative mode only a peak

at m/z 1338.2325 ([M-2H]2-

) appeared and had an intensity of about half of the peak height of the [M+H+Na]2+

adduct. Because availability of palytoxin standard was limited palytoxin was excluded from the validation study.

FIGURE 5: IONISATION SPECTRUM OF PALYTOXIN STANDARD

3.3 VALIDATION

A validation study was performed as described in the materials and methods chapter. For each extraction or

clean-up method 20 blank samples were spiked with standards. In 95% percent of the samples the precursor

ion and at least one fragment ion of the phycotoxins should be discernible. Also some non-spiked samples were

included to determine false positives: the presence of signals at m/z traces representative for the precursor

ions and one of its fragment ions. All results and requirements of the quantitative validation are shown in

Appendix 4.

SCREENING TISSUE

Overall the validation results of the screening method for tissue samples were satisfactory. 45 out of 49

phycotoxins were successfully validated. Out of the 20 spiked samples for all compounds the precursor ion was

0%

20%

40%

60%

80%

100%

500 600 700 800 900 1000 1100 1200 1300 1400 1500

Re

lati

ve a

bu

nd

ance

m/z

1351.2353 [M+H+Na]2+

1359.2212 [M+H+K]2+

1331.2419 [M+2H-H2O]2+

1322.2337 [M+2H-2H2O]2+

1313.2326 [M+2H-3H2O]2+

875.8228 [M+3H-3H2O]3+

908.4884 [M+H+2Na]3+

895.1576 [M+2H+Na-H2O]3+

31

found except for YTX in 3 samples. In none of the samples was a fragment ion for 16:0 OA ester found due to

low sensitivity. C2 was lower in concentration compared to most other hydrophilic phycotoxins and a fragment

ion was therefore not found in 6 samples. A fragment ion of CYN was not found in 3 samples, TTX in 2 samples

(only ensis), C1 in 2 samples (only mussel) and AZA5 in 2 samples (only fish). Out of the 6 samples selected for

the determination of the false positives one of the samples (a cockle) did show some results. For this sample

traces were obtained for SPX1, 20MeSPXG and PnTX G.

SCREENING WATER

43 out of the 49 tested phycotoxins were successfully validated. As the number of brackish water samples were

lacking, artificial brackish water samples were created by mixing of sea and fresh water samples (50:50 v/v). For

all lipophilic phycotoxins the precursor and fragment ions were found except for YTX, hYTX, OA C8-diol ester

and 16:0 OA ester. Most hydrophilic phycotoxins were not found in all sea water samples. This was supposedly

due to the salt content in the samples. Prior to the solid phase extraction samples were diluted with

acetonitrile. Due to the high salt content a precipitate was formed during the dilution step presumably

containing some of the hydrophilic phycotoxins. This can be resolved by diluting the sea water samples first

with water (50:50 v/v) and subsequently diluting with acetonitrile, which was already tested during the

validation due to the lack of brackish water. This procedure is identical to the procedure to produce brackish

water samples. When the protocol is adapted for sea water samples it should be successful for all hydrophilic

phycotoxins, except DA due to lack of retention on the SPE cartridge and C2 due to the lower concentration.

One brackish water sample was not blank, which contained SPX1 and GYM.

SCREENING FOOD SUPPLEMENTS

The validation of the screening method for lipophilic phycotoxins in solid food supplements was unsuccessful.

Only for some CIs the precursor ion and a fragment ion were found in a sufficient number of samples.

Therefore the spike level of MCs was increased for the validation of the screening method for lipophilic

phycotoxins in liquid food supplements. For spiked liquid food supplements result were better. In some

samples multiple phycotoxin groups had a low recovery. It seems that it was depending on the sample matrix

whether the method performed properly or not. Only for CIs and MC-RR the precursor ion and a fragment ion

were found in a sufficient number of samples, which are the most sensitive compounds in the MS detector due

to their amino containing functional groups.

TARGET SCREENING WITH DATABASE

Blanks included in the validation were screened with the complete database. When tissue samples were

measured with the method for lipophilic phycotoxins, all blanks including blank chemicals used during clean-up

contained a peak with a mass equal to the mass of PnTX E amide. All samples with matrix contained multiple

peaks with masses equal to esterification products of GYM. Blank tissue samples measured with the method

for hydrophilic toxins contained mainly compounds with masses equal to ATX- and TTX derivatives, although

the retention times were not corresponding with the expected retention times of the compounds and no

fragment ions were found.

When water samples measured with the method for lipophilic phycotoxins all blanks including blank chemicals

used during clean-up, contained a peak with an equal mass as PnTX E amide, similar to the tissue samples. No

phycotoxins were found in blank water extracts measured with the method for hydrophilic phycotoxins.

32

For food supplements again all blanks including blank chemicals used during clean-up contained a peak with an

equal mass as PnTX E amide. All blank liquid samples and some blank solid samples contained multiple peaks

with masses equal to esterification products of GYM, OA or DTXs.

Besides the masses equal to masses of ATX- and TTX derivatives in tissue samples and esterification products in

tissue and food supplements none to a maximum of twenty other peaks, which were confirmed based on exact

mass and isotopic pattern. Most matches could be ruled out based on retention time or peak shape. For

remaining peaks fragments should be confirmed when known, and when fragments are present subsequently

be confirmed with a confirmation method. This is an acceptable low number of false positives.

QUANTITATION REGULATED TOXINS IN TISSUE

The calibration standards were injected before and after the samples. From the calibration standards a

calibration curve is constituted. The correlation coefficient should be greater than 0.99 and the back-calculated

concentration of the calibration standards should not exceed ± 20% of the theoretical concentration. The drift

between two bracketing calibration curves should not exceed 30% difference in the slopes. The correlation

coefficient of DSP toxins and DA complies with the requirements. Not all PSP toxins have calibration curves

with a correlation > 0.99. STX, NEO and dcNEO are late eluting compounds with poor peak shapes. Due to the

poor peak shapes a maximum smoothing level was required to integrate the peaks properly, for this reason

intensities of those peaks became unreliable. Furthermore linearity of GTX1&4 and C2 is below <0.99. This is

caused due to poor separation of GTX1&4 in combination with the low concentration of GTX4 and the low

concentration of C2. The non-complying correlation of dcGTX2 is inexplicable. For some compounds one level

of the calibration curve exceeds the deviation of 20% of their theoretical concentration. For DSP toxins this is

DTX2 and YTX at the lowest level, hYTX at 250 µg kg-1

, for DA at the lowest level, and for PSP toxins these are

NEO, dcNEO, GTX1&4, dcGTX2 and C2 at the lowest level. The maximum drift obtained was 25.3% for NEO,

which is below the criteria of 30%. The calibration standards are analysed every series of analyses. Thus, when

the criteria for the calibration curve comply within a series of analysis, quantitation of DSP toxins in shellfish is

allowed since all other parameters determined during the validation meet the requirements.

Results of the blank samples should be smaller than 30% of the LOQ. In some blanks the molecular ion of a

phycotoxin was found. However, all blanks measured for the validation of the screening method did not show a

fragment ion and were therefore not confirmed, except for one cockle sample which was in hindsight not a

blank, as described at the validation results of the screening in tissue samples.

The accuracy of the measurement was assessed by the recovery estimation with the extracts used to calculate

the repeatability. For each target analyte the recovery should be ranged in each matrix under the scope of this

method between 70 - 120%. The recovery of DSP toxins and DA were good in general. For PSP toxins STX,

dcSTX, NEO, dcNEO, GTX1&4, dcGTX2 and C2 did have a recovery below 70%. These are the same compounds

that did not meet the requirements for the calibration curve.

The repeatability (RSDr) was assessed in 5-fold at two different concentrations: 0.5 PL and 1 PL. In each matrix

the RSDr should be less than or equal to 20% for all compounds and only one outlier (Grubb’s test) was

allowed. C2, YTX and hYTX at 0.5 PL had one outlier and OA at 1 PL had one outlier.

For DSP toxins and DA all RSDr were below 20%. The RSDr did not comply for STX, dcSTX, NEO and dcNEO at

both spiking levels, dcGTX2 and C1 at 0.5 PL and C2 at 1 PL.

Regulated lipophilic phycotoxins, DA and some PSP toxins can be quantified in shellfish at 0.5 or 1 times the

permitted level. However, late eluting PSP toxins had poor peak shapes which gave difficulties during

processing of the results.

33

3.4 CONFIRMATION OF PALYTOXIN-GROUP TOXINS BY LC-MS/MS

When phycotoxins are found with the screening method including fragments it is still a tentative confirmation,

because fragment ions are analysed over a range of precursor ions and retention time are often unknown. For

most phycotoxins for which standards are available confirmation methods already existed. For the palytoxin-

group toxins (PlTXs) an analytical method was not yet available and therefore an LC-MS/MS method was

established for the quantitation and confirmation of PlTXs.

The extraction procedure for the extraction of PlTXs developed by Selwood et al. [58] was optimised by testing

six extraction solvent compositions. Natural contaminated samples were used containing PlTX and ostreocin-D.

The total amount of extraction solvent was added and then samples were grinded with an Ultraturrax. In Table

15 recoveries are given of the palytoxin amide aldehyde and the common amino aldehyde for the different

extraction solvents (average of n=2). The recovery calculation is based on comparison to extraction with

methanol/water (50:50 v/v). For the last experiment hexane was removed before SPE.

TABLE 15: PALYTOXIN RECOVERIES AND RELATIVE STANDARD DEVATIONS (RSD) OF SIX DIFFERENT EXTRACTION SOLVENTS

Palytoxin amide aldehyde Common amino aldehyde Extraction solvent (9 mL) Rec (%) RSD (%) Rec (%) RSD (%)

MeOH/H2O (50:50 v/v) 100.0 1.2 100.0 8.4

MeOH/H2O/FA (50:50:1 v/v) 127.8 11.7 130.9 5.7

MeOH/H2O/HN4OH (50:50:1 v/v) 74.5 14.1 75.6 4.3

MeOH 189.8 4.0 320.8 6.0

MeOH/H2O/ACN/Amm.form/FA (80:55:45 v/v, 1.1 mM, 0.28mM) 191.0 9.0 279.0 4.0

MeOH/H2O/ACN/Amm.form/FA (80:55:45 v/v, 1.1 mM, 0.28mM) + 5 mL hexane 135.8 11.6 178.9 10.5

Extraction with methanol and MeOH/H2O/ACN/Amm.form/FA (80:55:45 v/v, 1.1 mM, 0.28mM)gave the

highest recoveries compared to extraction with methanol/water (50:50 v/v), which is the same extraction

solvent as used for screening method (4 mL MeOH and 5 mL H2O/ACN/Amm.form/FA (55:45 v/v, 2 mM, 0.5

mM), but combined together in one single extraction step. Higher recoveries for the common amino aldehyde

can be due to the presence of ostreocin-D.

The reproducibility and extraction efficiency was tested for extraction with methanol. Samples were grinded on

forehand and the extraction was carried out with 3 times 3 mL methanol as described in the materials and

method chapter. To determine the reproducibility eight different blank mussel homogenates were spiked at 60

ng g-1

with PlTX. The relative standard deviation was 5.8% for the palytoxin amide aldehyde and 4.5% for the

common amino aldehyde.

For the extraction efficiency two natural contaminated samples containing approximately 35 and 350 ng g-1

PlTX and ostreocin-D and a blank mussel homogenate spiked with 60 ng g-1

PlTX were used. Three extra

extraction steps with water were added, as the initial method was extraction with MeOH/H2O (50:50 v/v). Each

extraction step was analysed separately. Figure 6 shows the average extraction efficiency of the three samples.

After extraction with 3 times 3 mL methanol more than 90% of the total amount of PlTXs is extracted for all

samples.

34

FIGURE 6: EXTRACTION EFFICIENCY PALYTOXINS (N=3)

In order to determine if there were any matrix effects, samples were diluted after sample preparation to 75,

50, and 25% of the extract with methanol. Figure 7 shows the calculated concentration in relation to the

dilution factor. The common amino aldehyde is not affected by matrix effects because deviations in calculated

concentrations are less than 10% in all dilutions. However, dilution of the palytoxin amide aldehyde shows an

effect. When the sample is less diluted (more matrix), calculated concentrations are lower. Samples diluted to

25% give a higher concentration from 14 to 28% compared to the undiluted samples. Furthermore, the

increase of the concentration after dilution is related to the amount of palytoxin present in the sample (14%

increment for the low contaminated sample; 18% increment for the spiked blank; 28% increment for the high

contaminated sample).

FIGURE 7: MATRIX EFFECTS AFTER SAMPLE PREPARATION OF THREE SAMPLES

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

3 mLMeOH

3 mLMeOH

3 mLMeOH

3 mLwater

3 mLwater

3 mLwater

Extr

acti

on

eff

icie

ncy

(%

)

Extraction step

Palytoxin amide aldehyde

Common amino aldehyde

240

260

280

300

320

340

360

con

cen

trat

ion

(n

g g-1

)

Sample 1 Palytoxin amide aldehyde

Sample 1 Common amino aldehyde

0

20

40

60

80

0 0.25 0.5 0.75 1

Cal

cula

ted

Dilution factor

Blank + 60 ng g-1 Palytoxin amide aldehyde

Blank + 60 ng g-1 Common amide aldehyde

Sample 2 Palytoxin amide aldehyde

Sample 2 Common amino aldehyde

35

Matrix effects are depended on the analyte concentration in the sample or on the matrix itself; therefore a

matrix matched standard should be spiked at comparable levels to quantify contaminated samples, which is a

standard spiked to a similar matrix before extraction to correct for any matrix effects.

Furthermore, the correlation is calculated from the constructed calibration curve of matrix matched standards

from 0 to 100 ng g-1

. The correlation should be ≥ 0.990. Figure 8 shows the linear matrix matched calibration

curves. The palytoxin amide aldehyde has a correlation (r) of 0.997 and the common amino aldehyde has a

correlation of 0.998. Both matrix matched calibration curves meet the requirements.

The LOD and the LOQ of the method are obtained from signal to noise ratios of the transition with the lowest

signal at 30 ng g-1

. The signal to noise ratio of the palytoxin amide aldehyde is 18 at 30 ng g-1

. Consequently the

LOD for the palytoxin amide aldehyde is 5 ng g-1

(S/N = 3) and the LOQ is 10 ng g-1

(S/N =6). The common amino

aldehyde has a signal to noise ratio of 6 at 30 ng g-1

which is therefor the LOQ. Consequently the LOD for the

common amino aldehyde is 15 ng g-1

.

A confirmation method for PlTXs by LC-MS/MS was developed. The next step would be validation.

Unfortunately there is a lack of PlTX standard which makes validation difficult. Now, for all the classes

mentioned in the EU legislation or mentioned by EFSA confirmatory methods for one or more toxins are

available within RIKILT.

FIGURE 8: LINEARITY OF THE PALYTOXIN AMYDE ALDEHYDE AND THE COMMON AMINO ALDEHYDE OF MATRIX MATCHED STANDARDS

0

10000

20000

30000

40000

50000

60000

0 20 40 60 80 100 120

Pe

ak a

rea

Concentration (ng g-1)

Palytoxin amide aldehyde

Common amino aldehyde

36

FIGURE 9: TWO TRANSITIONS OF PALYTOXIN AMIDE ALDEHYDE (LEFT) AND COMMON AMINO ALDEHYDE (RIGHT) WITH SIGNAL TO

NOISE RATIO OF THE LOWEST SIGNAL AT 30 NG G-1

Time 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100 MRM of 15 Channels ES+

300.2 > 107 4.81e5 1.63

MRM of 15 Channels ES+ 300.2 > 151

5.36e4 S/N:PtP=6.01

30 ng g-1

Time 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100 MRM of 15 Channels ES+

343.2 > 76 1.18e6 1.72

MRM of 15 Channels ES+ 343.2 > 123

3.23e5 S/N:PtP=18.30

37

4. CONCLUSION

A method was developed for the screening of a wide variety of phycotoxins in tissue, water and food

supplement samples. One extraction method was used for all types of phycotoxins. Because of the different

chemical properties of hydrophilic and lipophilic phycotoxins a separate clean-up had to be developed for

water and food supplements. A clean-up method for hydrophilic phycotoxins in food supplements is still in

development. Also chromatography had to be developed separately for hydrophilic and lipophilic toxins. A

validation study was performed for all methods developed as a screening method and/or for the quantitation

of regulated phycotoxins. The validation for the screening of tissue and water samples was successful, except

for hydrophilic phycotoxins in sea water. During validation it appeared that the method for sea water had to be

adjusted slightly due to problems with the high salt content. Recoveries of some lipophilic phycotoxins spiked

to food supplements ranged from 9 to 102%, depending on matrix effects, from sample to sample. Therefore it

was difficult to validate the method for lipophilic phycotoxins in solid and liquid food supplements. To discern

the phycotoxins in 95% of the food supplements higher spiking levels were needed, which was impracticable

due to availability and cost of the standards. However, normally food supplements are not taken in large

amounts and high levels of phycotoxins should be present to be harmful. Taking this into consideration the

method is probably sensitive enough to measure toxic levels of phycotoxins in food supplements. In all

matrices an acceptable low number of false positives were found when a target screening using the database

was performed.

Regulated lipophilic phycotoxins, DA and some PSP toxins can be quantified in shellfish at 0.5 or 1 times the

permitted level. However, late eluting PSP toxins had poor peak shapes which gave difficulties during

processing of the results.

A method is developed for the quantitation and confirmation of palytoxin-group toxins. When there appears to

be a palytoxin-group toxin present in a sample during the screening it can be identified and quantified by an LC-

MS/MS method. However, high quantities of palytoxin group toxins are necessary to pick up the compounds

with the screening method since the ionisation pattern is complicated and therefore the sensitivity is low. An

oxidative fragmentation step which reduces the molecules in the confirmation method solves this problem.

When a phycotoxin is found with the screening method including a fragment it is still a tentative confirmation,

because fragment ions are analysed over a range of precursor ions and retention time are often unknown. To

confirm the compound a standard is needed, or NMR analysis needs to be done.

For future experiments the LC method for hydrophilic phycotoxins could be further optimised. Furthermore the

clean-up of food supplement extracts still needs to be further developed and validated.

38

5. ACKNOWLEDGEMENT

My first acknowledgement goes to my supervisor Arjen Gerssen for his guidance during this project. He has

given a lot helpful advice on the continuation of the project. And for support during the presentation of this

subject during the Fifth Joint Symposium on Marine & Freshwater Toxins Analysis in Baiona, Spain.

Sincere thanks to Marlène Gaillot for conducting the first experiments for this project during her internship.

Next, I would like to thank my colleagues from the Natural toxins and pesticides group, and especially Susannah

de Witte for her moral support and Paul Zomer for his help with the Q-Exactive.

Furthermore, I would like to acknowledge Wim Kok from the University of Amsterdam and RIKILT for giving me

the opportunity to continue my education with this master.

39

6. REFERENCES

1. Manivasagan, P.; Kim, S.-K., Chapter 34 - An Overview of Harmful Algal Blooms on Marine Organisms. In Handbook of Marine Microalgae, Academic Press: Boston, 2015; pp 517-526.

2. Gerssen, A.; Pol-Hofstad, I. E.; Poelman, M.; Mulder, P. P. J.; van den Top, H. J.; de Boer, J., Marine Toxins: Chemistry, Toxicity, Occurrence and Detection, with Special Reference to the Dutch Situation. Toxins (Basel) 2010, 2, (4), 878-904.

3. de la Iglesia, P.; McCarron, P.; Diogène, J.; Quilliam, M. A., Discovery of gymnodimine fatty acid ester metabolites in shellfish using liquid chromatography/mass spectrometry. Rapid Communications in Mass Spectrometry 2013, 27, (5), 643-653.

4. Aasen, J. A. B.; Hardstaff, W.; Aune, T.; Quilliam, M. A., Discovery of fatty acid ester metabolites of spirolide toxins in mussels from Norway using liquid chromatography/tandem mass spectrometry. Rapid Communications in Mass Spectrometry 2006, 20, (10), 1531-1537.

5. McCarron, P.; Rourke, W. A.; Hardstaff, W.; Pooley, B.; Quilliam, M. A., Identification of Pinnatoxins and Discovery of Their Fatty Acid Ester Metabolites in Mussels (Mytilus edulis) from Eastern Canada. Journal of Agricultural and Food Chemistry 2012, 60, (6), 1437-1446.

6. Wilkins, A. L.; Rehmann, N.; Torgersen, T.; Rundberget, T.; Keogh, M.; Petersen, D.; Hess, P.; Rise, F.; Miles, C. O., Identification of Fatty Acid Esters of Pectenotoxin-2 Seco Acid in Blue Mussels (Mytilus edulis) from Ireland. Journal of Agricultural and Food Chemistry 2006, 54, (15), 5672-5678.

7. Torgersen, T.; Wilkins, A. L.; Rundberget, T.; Miles, C. O., Characterization of fatty acid esters of okadaic acid and related toxins in blue mussels (Mytilus edulis) from Norway. Rapid Communications in Mass Spectrometry 2008, 22, (8), 1127-1136.

8. Morohashi, A.; Satake, M.; Murata, K.; Naoki, H.; Kaspar, H. F.; Yasumoto, T., Brevetoxin B3, a new brevetoxin analog isolated from the greenshell mussel perna canaliculus involved in neurotoxic shellfish poisoning in new zealand. Tetrahedron Letters 1995, 36, (49), 8995-8998.

9. MacKenzie, L.; Holland, P.; McNabb, P.; Beuzenberg, V.; Selwood, A.; Suzuki, T., Complex toxin profiles in phytoplankton and Greenshell mussels (Perna canaliculus), revealed by LC–MS/MS analysis. Toxicon 2002, 40, (9), 1321-1330.

10. Nicolas, J.; Hendriksen, P. J. M.; Gerssen, A.; Bovee, T. F. H.; Rietjens, I. M. C. M., Marine neurotoxins: State of the art, bottlenecks, and perspectives for mode of action based methods of detection in seafood. Molecular Nutrition & Food Research 2014, 58, (1), 87-100.

11. Balmer-Hanchey, E. L.; Jaykus, L.-A.; Jaykus, L.-A.; McClellan-Green, P., Marine Biotoxins of Algal Origin and Seafood Safety. Journal of Aquatic Food Product Technology 2003, 12, (1), 29-53.

12. Lipp, E. K.; Rose, J. B., The role of seafood in foodborne diseases in the United States of America. Rev Sci Tech 1997, 16, (2), 620-40.

13. Mos, L., Domoic acid: a fascinating marine toxin. Environmental Toxicology and Pharmacology 2001, 9, (3), 79-85.

14. Sanders, W. E., Jr., Intoxications from the seas: ciguatera, scombroid, and paralytic shellfish poisoning. Infect Dis Clin North Am 1987, 1, (3), 665-76.

15. Van Dolah, F. M.; Ramsdell, J. S., Review and assessment of in vitro detection methods for algal toxins. J AOAC Int 2001, 84, (5), 1617-25.

16. Munday, R.; Reeve, J., Risk assessment of shellfish toxins. Toxins (Basel) 2013, 5, (11), 2109-37. 17. Ito, E.; Satake, M.; Ofuji, K.; Kurita, N.; McMahon, T.; James, K.; Yasumoto, T., Multiple organ damage

caused by a new toxin azaspiracid, isolated from mussels produced in Ireland. Toxicon 2000, 38, (7), 917-930.

18. Terao, K.; Ito, E.; Yanagi, T.; Yasumoto, T., Histopathological studies on experimental marine toxin poisoning. I. Ultrastructural changes in the small intestine and liver of suckling mice induced by dinophysistoxin-1 and pectenotoxin-1. Toxicon 1986, 24, (11–12), 1141-1151.

19. Carmichael, W. W., Health Effects of Toxin-Producing Cyanobacteria: “The CyanoHABs”. Human and Ecological Risk Assessment: An International Journal 2001, 7, (5), 1393-1407.

20. Bane, V.; Lehane, M.; Dikshit, M.; O’Riordan, A.; Furey, A., Tetrodotoxin: Chemistry, Toxicity, Source, Distribution and Detection. Toxins (Basel) 2014, 6, (2), 693-755.

21. Dickey, R. W.; Plakas, S. M., Ciguatera: A public health perspective. Toxicon 2010, 56, (2), 123-136. 22. Tubaro, A.; Durando, P.; Del Favero, G.; Ansaldi, F.; Icardi, G.; Deeds, J. R.; Sosa, S., Case definitions for

human poisonings postulated to palytoxins exposure. Toxicon 2011, 57, (3), 478-495.

40

23. Tubaro, A.; Dell'Ovo, V.; Sosa, S.; Florio, C., Yessotoxins: A toxicological overview. Toxicon 2010, 56, (2), 163-172.

24. Rastogi, R.; Sinha, R.; Incharoensakdi, A., The cyanotoxin-microcystins: current overview. Reviews in Environmental Science and Bio/Technology 2014, 13, (2), 215-249.

25. Zurawell, R. W.; Chen, H.; Burke, J. M.; Prepas, E. E., Hepatotoxic Cyanobacteria: A Review of the Biological Importance of Microcystins in Freshwater Environments. Journal of Toxicology and Environmental Health, Part B 2005, 8, (1), 1-37.

26. Hess, P.; McCarron, P.; Quilliam, M., Fit-for-purpose shellfish reference materials for internal and external quality control in the analysis of phycotoxins. Analytical and Bioanalytical Chemistry 2007, 387, (7), 2463-2474.

27. Paredes, I.; Rietjens, I. M. C. M.; Vieites, J. M.; Cabado, A. G., Update of risk assessments of main marine biotoxins in the European Union. Toxicon 2011, 58, (4), 336-354.

28. Wekell, J. C.; Hurst, J.; Lefebvre, K. A., The origin of the regulatory limits for PSP and ASP toxins in shellfish. Journal of Shellfish Research 2004, 23, (3), 927-930.

29. CODEX, Standard for live and raw bivalve molluscs CODEX STAN 292-2008. 2008, (292). 30. EFSA, Marine biotoxins in shellfish - Okadaic acid and analogues. The EFSA Journal 2008, 589, 1-62. 31. EFSA, Marine biotoxins in shellfish – Azaspiracid group. The EFSA Journal 2008, 723, 1-52. 32. EFSA, Marine biotoxins in shellfish – Domoic acid. The EFSA Journal 2009, 1181, 1-61. 33. EFSA, Marine biotoxins in shellfish – Saxitoxin group. The EFSA Journal 2009, 1019, 1-76. 34. EFSA, Marine biotoxins in shellfish – Yessotoxin group. The EFSA Journal 2009, 907, 1-62. 35. EFSA, Marine biotoxins in shellfish – Pectenotoxin group. The EFSA Journal 2009, 1109, 1-47. 36. EFSA, Scientific Opinion on marine biotoxins in shellfish – Palytoxin group. The EFSA Journal 2009,

1393, 1-40. 37. EFSA, Scientific Opinion on marine biotoxins in shellfish – Emerging toxins: Brevetoxin group. The EFSA

Journal 2010, 1677, 1-29. 38. EFSA, Scientific Opinion on marine biotoxins in shellfish – Emerging toxins: Ciguatoxin group. The EFSA

Journal 2010, 1627, 1-38. 39. EFSA, Scientific Opinion on marine biotoxins in shellfish – Cyclic imines (spirolides, gymnodimines,

pinnatoxins and pteriatoxins). The EFSA Journal 2010, 1628, 1-39. 40. EU, Regulation (EC) No 853/2004. 2004. 41. Christian, B.; Luckas, B., Determination of marine biotoxins relevant for regulations: from the mouse

bioassay to coupled LC-MS methods. Analytical and Bioanalytical Chemistry 2008, 391, (1), 117-134. 42. Gerssen, A.; Mulder, P. P. J.; McElhinney, M. A.; de Boer, J., Liquid chromatography–tandem mass

spectrometry method for the detection of marine lipophilic toxins under alkaline conditions. Journal of Chromatography A 2009, 1216, (9), 1421-1430.

43. Diener, M.; Erler, K.; Christian, B.; Luckas, B., Application of a new zwitterionic hydrophilic interaction chromatography column for determination of paralytic shellfish poisoning toxins. Journal of Separation Science 2007, 30, (12), 1821-1826.

44. Turner, A. D.; McNabb, P. S.; Harwood, D. T.; Selwood, A. I.; Boundy, M. J., Single-Laboratory Validation of a Multitoxin Ultra-Performance LC-Hydrophilic Interaction LC-MS/MS Method for Quantitation of Paralytic Shellfish Toxins in Bivalve Shellfish. J AOAC Int 2015, 98, (3), 609-621.

45. Dell’Aversano, C.; Hess, P.; Quilliam, M. A., Hydrophilic interaction liquid chromatography–mass spectrometry for the analysis of paralytic shellfish poisoning (PSP) toxins. Journal of Chromatography A 2005, 1081, (2), 190-201.

46. Quilliam, M. A.; Janeček, M.; Lawrence, J. F., Characterization of the oxidation products of paralytic shellfish poisoning toxins by liquid chromatography/mass spectrometry. Rapid Communications in Mass Spectrometry 1993, 7, (6), 482-487.

47. Turner, A. D.; Powell, A.; Schofield, A.; Lees, D. N.; Baker-Austin, C., Detection of the pufferfish toxin tetrodotoxin in European bivalves, England, 2013 to 2014. Eurosurveillance 2015, 20, (2).

48. Guzmán-Guillén, R.; Moreno, I.; Prieto Ortega, A. I.; Eugenia Soria-Díaz, M.; Vasconcelos, V.; Cameán, A. M., CYN determination in tissues from freshwater fish by LC–MS/MS: Validation and application in tissues from subchronically exposed tilapia (Oreochromis niloticus). Talanta 2015, 131, 452-459.

49. Gallo, P.; Fabbrocino, S.; Cerulo, M. G.; Ferranti, P.; Bruno, M.; Serpe, L., Determination of cylindrospermopsin in freshwaters and fish tissue by liquid chromatography coupled to electrospray ion trap mass spectrometry. Rapid Communications in Mass Spectrometry 2009, 23, (20), 3279-3284.

41

50. McCarron, P.; Giddings, S.; Reeves, K.; Hess, P.; Quilliam, M., A mussel (Mytilus edulis) tissue certified reference material for the marine biotoxins azaspiracids. Analytical and Bioanalytical Chemistry 2015, 407, (11), 2985-2996.

51. Villar-González, A.; Rodríguez-Velasco, M. L.; Ben-Gigirey, B.; Yasumoto, T.; Botana, L. M., Assessment of the hydrolysis process for the determination of okadaic acid-group toxin ester: Presence of okadaic acid 7-O-acyl-ester derivates in Spanish shellfish. Toxicon 2008, 51, (5), 765-773.

52. Kaloudis, T.; Zervou, S.-K.; Tsimeli, K.; Triantis, T. M.; Fotiou, T.; Hiskia, A., Determination of microcystins and nodularin (cyanobacterial toxins) in water by LC–MS/MS. Monitoring of Lake Marathonas, a water reservoir of Athens, Greece. Journal of Hazardous Materials 2013, 263, Part 1, 105-115.

53. Caillaud, A.; de la Iglesia, P.; Darius, H. T.; Pauillac, S.; Aligizaki, K.; Fraga, S.; Chinain, M.; Diogène, J., Update on Methodologies Available for Ciguatoxin Determination: Perspectives to Confront the Onset of Ciguatera Fish Poisoning in Europe. Marine Drugs 2010, 8, (6), 1838-1907.

54. Ishida, H.; Nozawa, A.; Nukaya, H.; Tsuji, K., Comparative concentrations of brevetoxins PbTx-2, PbTx-3, BTX-B1 and BTX-B5 in cockle, Austrovenus stutchburyi, greenshell mussel, Perna canaliculus, and Pacific oyster, Crassostrea gigas, involved neurotoxic shellfish poisoning in New Zealand. Toxicon 2004, 43, (7), 779-789.

55. Ciminiello, P.; Dell’Aversano, C.; Iacovo, E. D.; Fattorusso, E.; Forino, M.; Tartaglione, L., LC-MS of palytoxin and its analogues: State of the art and future perspectives. Toxicon 2011, 57, (3), 376-389.

56. McNabb, P.; Selwood, A. I.; Holland, P. T., Multiresidue Method for Determination of Algal Toxins in Shellfish: Single-Laboratory Validation and Interlaboratory Study. J AOAC Int 2005, 88, (3), 761-772.

57. Ciminiello, P.; Dell'Aversano, C.; Fattorusso, E.; Forino, M.; Magno, G. S.; Tartaglione, L.; Quilliam, M. A.; Tubaro, A.; Poletti, R., Hydrophilic interaction liquid chromatography/mass spectrometry for determination of domoic acid in Adriatic shellfish. Rapid Communications in Mass Spectrometry 2005, 19, (14), 2030-2038.

58. Selwood, A. I.; van Ginkel, R.; Harwood, D. T.; McNabb, P. S.; Rhodes, L. R.; Holland, P. T., A sensitive assay for palytoxins, ovatoxins and ostreocins using LC-MS/MS analysis of cleavage fragments from micro-scale oxidation. Toxicon 2012, 60, (5), 810-820.

59. Kaufmann, A., The current role of high-resolution mass spectrometry in food analysis. Analytical and Bioanalytical Chemistry 2012, 403, (5), 1233-1249.

60. Peters, R. J. B.; Stolker, A. A. M.; Mol, J. G. J.; Lommen, A.; Lyris, E.; Angelis, Y.; Vonaparti, A.; Stamou, M.; Georgakopoulos, C.; Nielen, M. W. F., Screening in veterinary drug analysis and sports doping control based on full-scan, accurate-mass spectrometry. TrAC Trends in Analytical Chemistry 2010, 29, (11), 1250-1268.

61. Gerssen, A.; Mulder, P. P. J.; de Boer, J., Screening of lipophilic marine toxins in shellfish and algae: Development of a library using liquid chromatography coupled to orbitrap mass spectrometry. Analytica Chimica Acta 2011, 685, (2), 176-185.

62. Blay, P.; Hui, J. P. M.; Chang, J.; Melanson, J. E., Screening for multiple classes of marine biotoxins by liquid chromatography-high-resolution mass spectrometry. Analytical and Bioanalytical Chemistry 2011, 400, (2), 577-585.

63. Škrabáková, Z.; O'Halloran, J.; van Pelt, F. N. A. M.; James, K. J., Food contaminant analysis at ultra-high mass resolution: Application of hybrid linear ion trap - orbitrap mass spectrometry for the determination of the polyether toxins, azaspiracids, in shellfish. Rapid Communications in Mass Spectrometry 2010, 24, (20), 2966-2974.

64. Lommen, A.; Kools, H. J., MetAlign 3.0: Performance enhancement by efficient use of advances in computer hardware. Metabolomics 2012, 8, (4), 719-726.

65. Gerssen, A.; McElhinney, M. A.; Mulder, P. P. J.; Bire, R.; Hess, P.; Boer, J., Solid phase extraction for removal of matrix effects in lipophilic marine toxin analysis by liquid chromatography-tandem mass spectrometry. Analytical and Bioanalytical Chemistry 2009, 394, (4), 1213-1226.

66. McCarron, P.; Kilcoyne, J.; Miles, C. O.; Hess, P., Formation of Azaspiracids-3, -4, -6, and -9 via Decarboxylation of Carboxyazaspiracid Metabolites from Shellfish. Journal of Agricultural and Food Chemistry 2008, 57, (1), 160-169.

67. Mol, H. J.; Zomer, P.; de Koning, M., Qualitative aspects and validation of a screening method for pesticides in vegetables and fruits based on liquid chromatography coupled to full scan high resolution (Orbitrap) mass spectrometry. Analytical and Bioanalytical Chemistry 2012, 403, (10), 2891-2908.

68. Periat, A.; Kohler, I.; Thomas, A.; Nicoli, R.; Boccard, J.; Veuthey, J.-L.; Schappler, J.; Guillarme, D., Systematic evaluation of matrix effects in hydrophilic interaction chromatography versus reversed

42

phase liquid chromatography coupled to mass spectrometry. Journal of Chromatography A 2016, 1439, 42-53.

69. Nascimento, S. M.; Purdie, D. A.; Morris, S., Morphology, toxin composition and pigment content of Prorocentrum lima strains isolated from a coastal lagoon in southern UK. Toxicon 2005, 45, (5), 633-649.

70. Lenoir, S.; Ten-Hage, L.; Turquet, J.; Quod, J.-P.; Bernard, C.; Hennion, M.-C., First evidence of palytoxin analogues from an Ostreopsis Mascarenensis (Dinophyceae) benthic bloom in Southwestern Indian ocean. Journal of Phycology 2004, 40, (6), 1042-1051.

71. García-Altares, M.; Tartaglione, L.; Dell’Aversano, C.; Carnicer, O.; de la Iglesia, P.; Forino, M.; Diogène, J.; Ciminiello, P., The novel ovatoxin-g and isobaric palytoxin (so far referred to as putative palytoxin) from Ostreopsis cf. ovata (NW Mediterranean Sea): structural insights by LC-high resolution MSn. Analytical and Bioanalytical Chemistry 2015, 407, (4), 1191-1204.

72. Miles, C. O.; Samdal, I. A.; Aasen, J. A. G.; Jensen, D. J.; Quilliam, M. A.; Petersen, D.; Briggs, L. M.; Wilkins, A. L.; Rise, F.; Cooney, J. M.; Lincoln MacKenzie, A., Evidence for numerous analogs of yessotoxin in Protoceratium reticulatum. Harmful Algae 2005, 4, (6), 1075-1091.

43

APPENDICES

APPENDIX 1: STRUCTURES

APPENDIX 2: DATABASE (PART AS EXAMPLE)

APPENDIX 3: RIKILT STANDARDS OPERATING PROCEDURES PHYCOTOXINS

APPENDIX 4: VALIDATION RESULTS

44

APPENDIX 1: STRUCTURES

Β-METHYLAMINO-L-ALANINE

Abbreviation: BMAA

Cas number: 15920-93-1

Molecular formula: C4H10N2O2

Exact mass: 118.074228

Group: Hydrophilic

D-2,4-DIAMINOBUTYRIC ACID

Abbreviation: DBA




Group: Hydrophilic

ANATOXIN-A

Abbreviation: ATX


Molecular formula: C10H15NO


Group: Hydrophilic

DOMOIC ACID

Abbreviation: DA


Molecular formula: C15H21NO6


Group: ASP, Hydrophilic

TETRODOTOXIN

Abbreviation: TTX




Group: Hydrophilic

OH

O

HN

H2N

H2N

O

OH

NH2

H2N O

OH

OH

HO

O

HO

O

HO

O

OH

HN

O

OHN

NH

HN

OH

HOOH

OHHO

OH

45

PSP TOXINS, HYDROPHILIC

Name Abbr. Cas number Molecular formula

Exact mass R1 R2 R3 R4

Saxitoxin STX 35523-89-8 C10H17N7O4 299.134203 H H H

Neosaxitoxin NEO 64296-20-4 C10H17N7O5 315.129118 OH H H

Gonyautoxin 1 GTX1 60748-39-2 C10H17N7O9S 411.080850 OH H OSO3-

Gonyautoxin 2 GTX2 60508-89-6 C10H17N7O8S 395.085935 H H OSO3-

Gonyautoxin 3 GTX3 60537-65-7 C10H17N7O8S 395.085935 H OSO3- H

Gonyautoxin 4 GTX4 64296-26-0 C10H17N7O9S 411.080850 OH OSO3- H

Gonyautoxin 5 GTX5 64296-25-9 C10H17N7O7S 379.091020 H H H

N-sulfocarbamoyl gonyautoxin 2 C1 80173-30-4 C10H17N7O11S2 475.042752 H H OSO3-

N-sulfocarbamoyl gonyautoxin 3 C2 80226-62-6 C10H17N7O11S2 475.042752 H OSO3- H

Decarbamoylsaxitoxin dcSTX 58911-04-9 C9H16N6O3 256.128389 H H H OH

Decarbamoylneosaxitoxin dcNEO 68683-58-9 C9H16N6O4 272.123304 OH H H OH

Decarbamoylgonyautoxin 2 dcGTX2 86996-87-4 C9H16N6O7S 352.080121 H H OSO3- OH

Decarbamoylgonyautoxin 3 dcGTX3 87038-53-7 C9H16N6O7S 352.080121 H OSO3- H OH

CYLINDROSPERMOPSIN

Abbreviation: CYN

Cas number: 143545-90-8

Molecular formula: C15H21N5O7S


Group: Hydrophilic

GYMNODIMINE A

Abbreviation: GYM A

Cas number: 173792-58-0



Group: Cyclic imines, Lipophilic

O

NH2

O

O

NH

O

S O-

O

O

S

OOH

OH

OO

OO

N NH

N

NH

NH

O

O

OH

O

N

H

H

OH

OH

R4

N NH

HN

N

HN

NH

R1

R2R3

46

SPIROLIDE C, CYCLIC IMINES, LIPOPHILIC


Exact mass R1

13-Desmethyl spirolide C SPX1 334974-07-1 C42H61NO7 691.444804 CH3

13,19-Didesmethyl spirolide C 13,19-didesMeSPXC 908118-02-5 C41H59NO7 677.429154 H

20-METHYL SPIROLIDE G

Abbreviation: 20MeSPXG

Cas number: 849215-95-8



Group: Cyclic imines, Lipophilic

PINNATOXINS, CYCLIC IMINES, LIPOPHILIC



Pinnatoxin E PnTX E 1227167-69-2 C45H69NO10 783.492149

H OH CH3

Pinnatoxin F PnTX F 1227167-70-5 C45H67NO9 765.481584

H OH CH3

Pinnatoxin G PnTX G 1312711-74-2 C42H63NO7 693.460454 OH H H

OH

CO2H

O O

O

N

O

HO

O

OO

HO R1

O

N

OO

OOOH

HO

N

OO

O

OHO

O

H

R1

R3R4

R2

47

AZASPIRACIDS, LIPOPHILIC



Azaspiracid 1 AZA1 214899-21-5 C47H71NO12 841.497629 H H CH3 H

Azaspiracid 2 AZA2 265996-92-7 C48H73NO12 855.513279 H CH3 CH3 H

Azaspiracid 3 AZA3 265996-93-8 C46H69NO12 827.481979 H H H H

Azaspiracid 4 AZA4 344422-49-7 C46H69NO13 843.476894 OH H H H

Azaspiracid 5 AZA5 344422-51-1 C46H69NO13 843.476894 H H H OH

PECTENOTOXIN 2

Abbreviation: PTX2


Molecular formula: C47H70O14


Group: Lipophilic

O

OO

O

O

O

O

HO

HO

O

OH

O

HNR3

H

H

R1

R2

R4

O

O

O

O

O

O

HO

O

O

O

O

OH

HO

O

H

H

H

48

O

OO OH

O OH

OO

O

O

NH

HN

HN

NNH

Y X

NODULARIN

– D-MeAsp – L-Arg – Adda – D-Glu – Mdhb –

Abbreviation: NOD

Cas number: 118399-22-7



Group: Lipophilic

MICROCYSTINS, LIPOPHILIC

– D-Ala – X –D- MeAsp – Y – Adda – D-Glu – Mdha –


Exact mass X Y

Microcystin HilR MC-HilR 169789-55-3 C50H76N10O12 1008.56442 Hil Arg

Microcystin HtyR MC-HtyR 478001-08-0 C53H74N10O13 1058.543685 Hty Arg

Microcystin LA MC-LA 96180-79-9 C46H67N7O12 909.484773 Leu Ala

Microcystin LF MC-LF 154037-70-4 C52H71N7O12 985.516073 Leu Phe

Microcystin LR MC-LR 101043-37-2 C49H74N10O12 994.548770 Leu Arg

Microcystin LW MC-LW 157622-02-1 C54H72N8O12 1024.526972 Leu Trp

Microcystin LY MC-LY 123304-10-9 C52H71N7O13 1001.510988 Leu Tyr

Microcystin RR MC-RR 111755-37-4 C49H75N13O12 1037.565817 Arg Arg

Microcystin WR MC-WR 138234-58-9 C54H73N11O12 1067.544019 Trp Arg

Microcystin YR MC-YR 101064-48-6 C52H72N10O13 1044.528035 Tyr Arg

Asp3 microcystin LR AspMC-LR 120011-66-7 C48H72N10O12 980.533120 Leu Arg

O

NH

O

HNN

O

OHO

NHO

HN

HN NH2

O

OHN

OH

O

49

Adda Ala Arg Glu

Hil Hty Leu MeAsp

Mdha Mdhb Phe Trp

Tyr

H2N O

OH

O

H2NO

OH

H2N

NH

O

OH

NHH2N

H2NO

OH

HO O

H2NO

OH

H2NO

OH

OH

H2NO

OH

H2NO

OH

O

OH

NH

O

OH

NH

O

OH

H2NO

OH

H2NO

OH

NH

H2NO

OH

OH

50

O

O O

O

O

O

O

O

O

O

O

HO

O

R1

DSP, LIPOPHILIC


Exact mass R1 R2 R3 R4 R5

Okadaic acid OA 78111-17-8 C44H68O13 804.465995 CH3 H H H H

Dinophysistoxin 1 DTX1 81720-10-7 C45H70O13 818.481645 CH3 CH3 H H H

Dinophysistoxin 2 DTX2 139933-46-3 C44H68O13 804.465995 H H CH3 H H

16:0 7-O-Acyl

okadaic acid DTX3 118745-19-0 C60H98O14 1042.695660 CH3 H H (CH2)14CH3 H

Okadaic acid

methyl ester

OA methyl

ester 78111-14-5 C45H70O13 818.481645 CH3 H H H CH3

Okadaic acid-D8a OA C8-diol

ester 318536-96-8 C52H80O14 928.554810 CH3 H H H

BREVETOXINS, LIPOPHILIC


Exact mass R1

Brevetoxin 2 PbTx 2 79580-28-2 C50H70O14 894.476560



OH

O

OH

OH

O

O

CH3

O

O

O

CH2

O

OR4CH3

H

HOH

OH CH3

H

R1

O

R2

R5O

O

H3C OH

R3

51

HO

HO

O

O

O

O

O

O

HH

OHH H

HH

OH H H H

HH

O

O

O

OR1

OH HH

H

OH

HH

HOH H

H

H

YESSOTOXINS, LIPOPHILIC


Exact mass R1

Yessotoxin YTX 112514-54-2 C55H82O21S2 1142.479009 (CH2)2OSO3-

1a-Homoyessotoxin hYTX 196309-94-1 C56H84O21S2 1156.494659 (CH2)3OSO3-

PACIFIC CIGUATOXINS, LIPOPHILIC


Exact mass R1

Pacific ciguatoxin 1 P-CTX-1 11050-21-8 C60H86O19 1110.576335



R1

O

OH

R1

O

R1

O

R1

O

O

O

O

O

O

O

O

O

O

OHO

H

H

H

H

HHHHHH

H H H

H

H

H

H

H

H

OH

O S

O

O

OH

52

PALYTOXIN

Abbreviation: PlTX



Exact mass: 2678.479607

Group: Lipophilic

H2N O

OH

OH

OH

O

OH

OH

HO

OH OH

OH

OH

O

OH

OH

OH

OH

HO

OOH

OHHO

OH

OHOH

OOH

OH

OH

HOOH

OH

OH

OHHO

OHO

OH

OH

HO

NH

NH

HO

OH

OH

OH

OH

OO

OH

OH

HO

O

O

O

O

H

H

53

APPENDIX 2: DATABASE (PART AS EXAMPLE)

Name Synonyms Standard

available Supplier Cas number LC method

Expected RT

(min) Molecular formula Exact mass

saxitoxin STX YES NRC-CNRC, Canada 35523-89-8 HILIC 15.34 C10H17N7O4 299.134203

neosaxitoxin NEO YES Cifga, Spain; NRC-CNRC, Canada 64296-20-4 HILIC 15.16 C10H17N7O5 315.129118

gonyautoxin 1 GTX1 YES NRC-CNRC, Canada 60748-39-2 HILIC 8.37 C10H17N7O9S 411.080850




gonyautoxin 5 GTX5; B1 YES Cifga, Spain; NRC-CNRC, Canada 64296-25-9 HILIC 10.06 C10H17N7O7S 379.091020

gonyautoxin 6 GTX6; B2 NO 82810-44-4 HILIC C10H17N7O8S 395.085935

N-sulfocarbamoyl gonyautoxin 1 C3; PX3 NO 89614-45-9 HILIC C10H17N7O12S2 491.037667

N-sulfocarbamoyl gonyautoxin 2 C1; PX1; epi-GTX8 YES Cifga, Spain; NRC-CNRC, Canada 80173-30-4 HILIC 6.25 C10H17N7O11S2 475.042752

N-sulfocarbamoyl gonyautoxin 3 C2; PX2; GTX8 YES Cifga, Spain; NRC-CNRC, Canada 80226-62-6 HILIC 6.87 C10H17N7O11S2 475.042752

N-sulfocarbamoyl gonyautoxin 4 C4; PX4 NO 89674-98-6 HILIC C10H17N7O12S2 491.037667

decarbamoylsaxitoxin dcSTX YES Cifga, Spain; NRC-CNRC, Canada 58911-04-9 HILIC 15.34 C9H16N6O3 256.128389

decarbamoylneosaxitoxin dcNEO; GTX7 YES NRC-CNRC, Canada 68683-58-9 HILIC 15.34 C9H16N6O4 272.123304

decarbamoylgonyautoxin 1 dcGTX1 NO 122075-86-9 HILIC C9H16N6O8S 368.075036

decarbamoylgonyautoxin 2 dcGTX2 YES Cifga, Spain; NRC-CNRC, Canada 86996-87-4 HILIC 8.61 C9H16N6O7S 352.080121

decarbamoylgonyautoxin 3 dcGTX3 YES Cifga, Spain; NRC-CNRC, Canada 87038-53-7 HILIC 9.26 C9H16N6O7S 352.080121

decarbamoylgonyautoxin 4 dcGTX4 NO 122169-51-1 HILIC C9H16N6O8S 368.075036

11α-hydroxy-N21-sulfocarbamoylsaxitoxin M1α NO 1055346-90-1 HILIC C10H17N7O8S 395.085935

11β-hydroxy-N21-sulfocarbamoylsaxitoxin M1β NO HILIC C10H17N7O8S 395.085935

11α-hydroxysaxitoxin M2α NO 99685-70-8 HILIC C10H17N7O5 315.129118

11β-hydroxysaxitoxin M2β NO 68107-89-1 HILIC C10H17N7O5 315.129118

11,11-dihydroxy-N21-sulfocarbamoylsaxitoxin M3 NO 1055346-91-2 HILIC C10H17N7O9S 411.080850

11,11-dihydroxysaxitoxin M4 NO 1055346-92-3 HILIC C10H17N7O6 331.124033

M5 (structure unknown) M5 NO HILIC C10H17N7O8S 395.085935

54

Name [M+H]+ [M+NH4]+ [M+Na]+ Other adduct /

charge +

Fragment 1+

elemental

composition

Fragment 1+

exact mass

Fragment 2+

elemental

composition

Fragment 2+

exact mass

saxitoxin 300.141479 317.168028 322.123424 C10H15N7O3 282.130914 C9H9N5O 204.087986

neosaxitoxin 316.136394 333.162943 338.118339 C10H15N7O4 298.125829 C9H9N5O2 220.082901

gonyautoxin 1 412.088126 429.114675 434.070071 C10H17N7O6 332.131309 C10H15N7O5 314.120744 C7H6N2O5 199.034949


gonyautoxin 3 396.093211 413.119760 418.075156 C10H17N7O5 316.136394 C10H15N7O4 298.125829




N-sulfocarbamoyl gonyautoxin 1 492.044943 509.071492 514.026888 C10H17N7O6 332.131309 C10H15N7O5 314.120744

N-sulfocarbamoyl gonyautoxin 2 476.050028 493.076577 498.031973 C10H17N7O8S 396.093211 C10H17N7O5 316.136394 C10H15N7O4 298.125829

N-sulfocarbamoyl gonyautoxin 3 476.050028 493.076577 498.031973 C10H17N7O8S 396.093211 C10H17N7O5 316.136394 C10H15N7O4 298.125829

N-sulfocarbamoyl gonyautoxin 4 492.044943 509.071492 514.026888 C10H17N7O6 332.131309 C10H15N7O5 314.120744

decarbamoylsaxitoxin 257.135665 274.162214 279.117610 C9H14N6O2 239.125100 C5H7N3O 126.066188

decarbamoylneosaxitoxin 273.130580 290.157129 295.112525 C9H14N6O3 255.120015 C7H9N5O 180.087986

decarbamoylgonyautoxin 1 369.082312 386.108861 391.064257 C9H16N6O5 289.125495 C9H14N6O4 271.114930

decarbamoylgonyautoxin 2 353.087397 370.113946 375.069342 C9H16N6O4 273.130580 C9H16N6O4 273.130580 C9H14N6O3 255.120015



11α-hydroxy-N21-sulfocarbamoylsaxitoxin 396.093211 413.119760 418.075156 C10H17N7O5 316.136394 C10H15N7O4 298.125829

11β-hydroxy-N21-sulfocarbamoylsaxitoxin 396.093211 413.119760 418.075156 C10H17N7O5 316.136394 C10H15N7O4 298.125829

11α-hydroxysaxitoxin 316.136394 333.162943 338.118339 C10H15N7O4 298.125829 C9H9N5O2 220.082901

11β-hydroxysaxitoxin 316.136394 333.162943 338.118339 C10H15N7O4 298.125829 C9H9N5O2 220.082901

11,11-dihydroxy-N21-sulfocarbamoylsaxitoxin 412.088126 429.114675 434.070071 C10H17N7O6 332.131309 C10H15N7O5 314.120744

11,11-dihydroxysaxitoxin 332.131309 349.157858 354.113254 C10H15N7O5 314.120744 C10H13N7O4 296.110179

M5 (structure unknown) 396.093211 413.119760 418.075156

55

Name [M-H]- Other adduct /

charge -

Fragment 1-

elemental

composition

Fragment 1-

exact mass

Fragment 2-

elemental

composition

Fragment 2-

exact mass

Toxin

group

Sym

pto

ms

sho

rt-t

erm

mem

ory

loss

neu

roto

xic

dia

rrh

oea

liver

dam

age

der

mat

itis

tum

or

pro

mo

ters

saxitoxin - STX PSP x

neosaxitoxin - STX PSP x

gonyautoxin 1 410.073574 C9H16O8N6S 367.067760 C9H14O7N6S 349.057195 STX PSP x

gonyautoxin 2 394.078659 C9H16O7N6S 351.072845 C9H14N6O6S 333.062280 STX PSP x

gonyautoxin 3 394.078659 C9H16O7N6S 351.072845 C9H14N6O6S 333.062280 STX PSP x

gonyautoxin 4 410.073574 C9H16O8N6S 367.067760 C9H14O7N6S 349.057195 STX PSP x

gonyautoxin 5 378.083744 C10H15N7O6S 360.073179 CO4HNS 121.955355 STX PSP x

gonyautoxin 6 394.078659 STX PSP x

N-sulfocarbamoyl gonyautoxin 1 490.030391 STX PSP

N-sulfocarbamoyl gonyautoxin 2 474.035476 C10H17N7O8S 394.078659 CO4HNS 121.955355 STX PSP

N-sulfocarbamoyl gonyautoxin 3 474.035476 CO4HNS 121.955355 STX PSP x

N-sulfocarbamoyl gonyautoxin 4 490.030391 STX PSP x

decarbamoylsaxitoxin - STX PSP x

decarbamoylneosaxitoxin - STX PSP x

decarbamoylgonyautoxin 1 367.067760 STX PSP

decarbamoylgonyautoxin 2 351.072845 C9H16O7N6S 351.072845 C9H14N6O6S 333.062280 STX PSP

decarbamoylgonyautoxin 3 351.072845 C9H16O7N6S 351.072845 C9H14N6O6S 333.062280 STX PSP

decarbamoylgonyautoxin 4 367.067760 STX PSP

11α-hydroxy-N21-sulfocarbamoylsaxitoxin 394.078659 STX PSP

11β-hydroxy-N21-sulfocarbamoylsaxitoxin 394.078659 STX PSP

11α-hydroxysaxitoxin STX PSP x

11β-hydroxysaxitoxin STX PSP x

11,11-dihydroxy-N21-sulfocarbamoylsaxitoxin 410.073574 STX PSP

11,11-dihydroxysaxitoxin STX PSP

M5 (structure unknown) 394.078659 STX PSP

56

Name Regulatory

limit Unit

Toxicity

equivalency

factor

Unit Produced by References

saxitoxin 800 µg STX-eq/kg 1 STX eq.* Alexandrium andersoni; A. catenella; A. circinalis; A.

excavatum; A. fundyense; A. lusitanicum; A. minutum; A.

monilatum; A. ostenfeldii; A. peruvianum; A. tamarense;

Anabaena lemmermannii; Aphanizomenon flos-aquae; Aph.

gracile; Aph. issatschenkoi; Cylindrospermopsis raciborskii;

Gymnodinium catenatum; Planktothrix sp.; Pyrodinium

bahamense

*The EFSA Journal (2009) 1019, 1-76

neosaxitoxin 800 µg STX-eq/kg 1 STX eq.* *The EFSA Journal (2009) 1019, 1-76

gonyautoxin 1 800 µg STX-eq/kg 1 STX eq.* *The EFSA Journal (2009) 1019, 1-76

gonyautoxin 2 800 µg STX-eq/kg 0.4 STX eq.* *The EFSA Journal (2009) 1019, 1-76





N-sulfocarbamoyl gonyautoxin 1 The EFSA Journal (2009) 1019, 1-76

N-sulfocarbamoyl gonyautoxin 2 The EFSA Journal (2009) 1019, 1-76

N-sulfocarbamoyl gonyautoxin 3 800 µg STX-eq/kg 0.1 STX eq.* *The EFSA Journal (2009) 1019, 1-76

N-sulfocarbamoyl gonyautoxin 4 800 µg STX-eq/kg 0.1 STX eq.* *The EFSA Journal (2009) 1019, 1-76

decarbamoylsaxitoxin 800 µg STX-eq/kg 1 STX eq.* *The EFSA Journal (2009) 1019, 1-76

decarbamoylneosaxitoxin 800 µg STX-eq/kg 0.4 STX eq.* *The EFSA Journal (2009) 1019, 1-76

decarbamoylgonyautoxin 1 The EFSA Journal (2009) 1019, 1-76




11α-hydroxy-N21-sulfocarbamoylsaxitoxin metabolites formed in shellfish The EFSA Journal (2009) 1019, 1-75

11β-hydroxy-N21-sulfocarbamoylsaxitoxin metabolites formed in shellfish The EFSA Journal (2009) 1019, 1-76

11α-hydroxysaxitoxin 800 µg STX-eq/kg 0.3 STX eq.* metabolites formed in shellfish *The EFSA Journal (2009) 1019, 1-76

11β-hydroxysaxitoxin 800 µg STX-eq/kg 0.3 STX eq.* metabolites formed in shellfish *The EFSA Journal (2009) 1019, 1-76

11,11-dihydroxy-N21-sulfocarbamoylsaxitoxin metabolites formed in shellfish The EFSA Journal (2009) 1019, 1-76

11,11-dihydroxysaxitoxin metabolites formed in shellfish The EFSA Journal (2009) 1019, 1-76

M5 (structure unknown) metabolites formed in shellfish Mar. Drugs 2010, 8, 2185-2211

57

APPENDIX 3: RIKILT STANDARDS OPERATING PROCEDURES PHYCOTOXINS

Note: Not the complete SOPs are described, only the parts used for this research project.

SOP A0983 Shellfish – The determination of PSP toxins (paralytic shellfish poisoning) – LC-MS/MS

Validated compounds: STX, dcSTX, NEO, dcNEO, GTX1&4, GX2&3, GTX5, dcGTX2&3

Extraction:

Weigh of every sample 1.0 g ± 0.05 g in a plastic tube of 12 mL

Add 2 mL H2O/MeOH/HAc (50:50 v/v, 15 mM) and vortex mix 20 seconds

Place the tubes in an overhead shaker for 15 minutes

Centrifuge 10 minutes at 3600 g

Transfer supernatant to a graduated tube of 15 mL

Add 1.5 mL H2O/MeOH/HAc (50:50 v/v, 15 mM) and vortex mix 20 seconds


Transfer supernatant to the same tube of 15 mL and make up to 4 mL mark with H2O/MeOH/HAc (50:50 v/v, 15

mM)

Mix and filter circa 2 mL extract using a 0.45 µm PVDF filter (Sigma-Aldrich, Zwijndrecht, The Netherlands)

Mix 100 µL filtered extract with 900 µL H2O/ACN/Hac (20:70 v/v, 6.7 mM)

Filter the diluted extract using a 0.45 µm PVDF filter

Transfer extracts to vial and cap

LC settings:

Column: TSKgel Amide-80 2 µm, 250 · 2 mm (TOSOH Bioscience, Tokyo, Japan)

Mobile phase A: water containing 50 mM FA

Mobile phase B: acetonitrile containing 50 mM FA

Flow: 0.2 mL min-1

Injection volume: 10 µL

Column temperature: 40 °C

Runtime: 20 minutes

Gradient: see Table 16

TABLE 16: GRADIENT PSP

Time (min) %A %B

0 30 70

1 30 70

8.5 95 5

13.5 95 5

14 30 70

MS settings:

Ionisation mode: ESI+

Capillary voltage: 2.7 kV

Cone voltage: 25 V

Source temperature: 120 °C

58

Desolvation temperature: 350 °C

Desolvation gas flow: 600 L h-1

Cone gas flow: 60 L h-1

CID gas: argon

Fragmentation: see Table 17

TABLE 17: FRAGMENTATION CONDITIONS PSP

Compound Precursor ion (m/z)

Product ion (m/z)


STX 300.2 204.1

282.1

25

19

dcSTX 257.2 126.1

239.1

25

25

NEO 316.1 298.1

220.1

20

20

dcNEO 273.2 255.1

180.1

19

19

GTX1&4 380.1 300.1

282.1

22

22

GTX2&3 412.1 314.1

332.1

20

20

GTX5 396.1 298.1

316.1

20

20

dcGTX2&3 353.1 255.1

273.1

18

18

59

SOP A1127 Shellfish – the quantitation and confirmation of marine lipophilic phycotoxins – LC-MS/MS

Validated compounds: OA, DTX1, DTX2, DTX3, PTX2, YTX, hYTX, AZA1, AZA2, AZA3, SPX1, GYM, PnTX E, PnTXF,

PnTX G

Extraction:


Add 3 mL methanol and vortex mix 1 minute


Transfer supernatant to a graduated tube of 15 mL



Transfer supernatant to the same tube of 15mL



Transfer supernatant to the same tube of 15 mL and make up to 10 mL mark with methanol

Mix and filter extract using a 0.2 µm HT Tuffryn filter (Sigma-Aldrich, Zwijndrecht, The Netherlands)

Transfer extracts to vial and cap

LC settings:

Column: ACQUITY UPLC BEH C18 1.7µm, 2.1 · 100 mm (Waters, Milford, MA, USA)

Mobile phase A: water containing 6.7 mM NH4OH

Mobile phase B: acetonitrile/water (9:1 v/v) containing 6.7 mM NH4OH

Flow: 0.6 mL min-1



Runtime: 5 minutes


TABLE 18: GRADIENT DSP

Time (min) %A %B

0 70 30

0.5 70 30

3.5 10 90

4 10 90

4.1 70 30

5 70 30

MS settings:

Ionisation mode: ESI- and ESI+





CID gas: argon


60

TABLE 19: FRAGMENTATION CONDIOTIONS DSP


Product ion (m/z)

Ionisation Cone (V) Collision energy (eV)

OA/DTX2 803.5 113.1

255.2

Negative

Negative

80

80

60

45

DTX1 817.5 113.1

255.2

Negative

Negative

80

80

60

45

YTX 570.4 396.4

467.4

Negative

Negative

75

75

30

30

hYTX 577.4 403.4

474.4

Negative

Negative

75

75

30

30

AZA1 842.5 672.4

824.5

Positive

Positive

35

35

40

30

AZA2 856.5 672.4

838.5

Positive

Positive

35

35

40

30

AZA3 828.5 658.4

792.5

Positive

Positive

35

35

40

30

SPX1 692.5 164.3

444.2

Positive

Positive

60

60

55

40

GYM 508.2 162.2

490.2

Positive

Positive

60

60

55

40

PnTX G 694.5 164.3

676.5

Positive

Positive

60

60

55

40

PTX2 876.5 213.1

823.5

Positive

Positive

40

40

30

30

61

SOP A1167 Microalgae, shellfish and water – detection of mycrocystins and nodularin – LC-MS/MS

Validated compounds: MC-RR, MC-YR, MC-LR, MC-LA, MC-LY, MC-LW, MC-LF, NOD

Extraction shellfish:


Add 15 mL of H2O/ACN/FA (25:75:1 v/v)

Place the tubes in an overhead shaker for 30 minutes


Pipette with a positive displacement pipette an aliquot of 2 ml of the clear supernatant in a 12 ml plastic tube

Add 2 mL of hexane saturated with acetonitrile

Shake vigorously for 30 seconds


Remove the hexane layer with the vacuum pump

Transfer an aliquot of 0.5 mL extract to a Mini-Uniprep filter (Sigma-Aldrich, Zwijndrecht, The Netherlands)

Apply the PFTE filter, 0.45 µm pore size and filter the extract with help of a compressor

Clean-up water:

An aliquot of 50 ml water is taken and ultrasonicated using a sonifier during 30 seconds at amplitude 75.

Activate an Oasis HLB cartridge (Waters, Etten-Leur, The Netherlands) with 6 ml MeOH followed by 6 ml water

Apply 25 ml of water resulting from onto the cartridge

Wash the cartridge with 6 ml MeOH/water (40:60; v/v)

Dry the cartridge under vacuum

Elute the compounds with 6 ml MeOH/water (80:20; v/v) in a 12 ml tube

Evaporate the eluate at 50 °C ± 5°C with nitrogen gas until the extract is dry

Dissolve the residue in 400 µl MeOH/water (80:20; v/v) and mix using a vortex

Transfer the extract to a Mini-Uniprep filter

Apply the PFTE filter, 0.45 µm pore size and filter the extract with help of a compressor

LC settings:

Column: ACQUITY UPLC BEH C18 1.7µm, 2.1 · 100 mm (Waters, Milford, MA, USA)

Mobile phase A: water containing 26.5 mM FA

Mobile phase B: acetonitrile containing 26.5 mM FA

Flow: 0.6 mL min-1



Runtime: 5 minutes


62

TABLE 20: GRADIENT MC

Time (min) %A %B

0 90 10

3.5 35 65

3.51 1 99

4 1 99

4.01 90 10

5 90 10

MS settings:

Ionisation mode: ESI+


Cone voltage: 50 V




Cone gas flow: 150 L h-1

CID gas: argon


TABLE 21: FRAGMENTATION CONDITIONS MC


Product ion (m/z)


NOD 825.5 135.1

163.1

55

55

MC-LA 910.5 213.2

163.1

50

50

MC-LF 986.5 213.2

375.3

40

40

MC-LR 995.7 135.1

213.2

60

60

MC-LY 1002.5 213.2

163.1

50

50

MC-LW 1025.6 213.2

375.3

70

70

MC-RR 1038.5 135.1

213.2

70

70

MC-YR 1045.5 135.1

213.2

60

60

63

APPENDIX 4: VALIDATION RESULTS

Screening of hydrophilic and lipophilic phycotoxins in tissue

Spiking levels: Hydrophilic phycotoxins 600 µg kg-1

*

Microcystins and nodularin 150 µg kg-1

Other lipophilic phycotoxins 80 µg kg-1

*Exceptions: dcGTX3 (134 µg kg-1

), GTX1&4 (795 µg kg-1

), GTX3 (228 µg kg-1

), C2 (180 µg kg-1

)

TABLE 22: VALIDATION RESULTS OF HYDROPHILIC PHYCOTOXINS IN TISSUE

HILIC POS S blanks mussel cockle oyster ensis fish S

ATX 3 1 0 1 0 0 1 2 3 2 2 2 3 3 3 3 2 2 3 2 2 3 3 3 3 3 3 3

dcNEO 3 0 0 0 0 0 0 3 2 1 3 3 3 3 2 3 3 3 3 2 2 3 3 3 3 2 3 3

dcSTX 3 0 0 0 0 0 0 3 3 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

NEO 3 0 0 0 0 0 0 3 3 1 3 3 3 3 2 3 3 3 3 3 2 3 3 3 3 2 3 3

STX 3 0 0 0 0 0 0 3 3 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

TTX 3 0 0 0 0 0 0 3 3 2 2 2 3 2 3 3 2 2 3 1 1 2 3 3 2 2 3 3

HILIC NEG

CYN 2 0 0 0 0 0 0 2 2 2 2 2 2 1 2 2 2 2 2 1 2 2 1 2 2 2 2 2

dcGTX2 3 0 1 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

dcGTX3 3 1 0 0 0 0 0 3 3 2 2 2 3 3 3 3 3 3 3 3 2 3 3 3 3 3 3 3

DA 2 0 1 1 0 0 0 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

GTX1&4 3 0 0 0 0 0 1 2 3 3 3 3 3 3 3 2 3 2 2 3 2 2 3 3 3 3 3 3

GTX2 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 3 3 3 3 3

GTX3 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 2 2 3 3 3 3 2 3 3

GTX5 3 0 0 0 0 1 0 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 3 3 3 3 3

C1 3 0 0 0 0 0 0 1 1 3 2 2 2 2 2 2 2 2 3 2 2 2 3 3 2 3 3 3

C2 2 0 0 1 0 0 0 2 2 2 2 2 1 2 2 1 1 1 2 1 1 2 2 2 2 2 2 2

LEGEND

0 No precursor ion found

1 Precursor ion found

2 Precursor ion + 1 fragment ion found

3 Precursor ion + 2 fragment ions found

S Standard solution

RP POS Measurements with reversed phase LC and positive electrospray ionisation

RP NEG Measurements with reversed phase LC and negative electrospray ionisation

HILIC POS Measurements with HILIC and positive electrospray ionisation

HILIC NEG Measurements with HILIC and negative electrospray ionisation

64

TABLE 23: VALIDATION RESULTS OF LIPOPHILIC PHYCOTOXINS IN TISSUE

RP POS S blanks mussel cockle oyster ensis fish S

MC-HilR 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

MC-HtyR 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

MC-LA 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

MC-LF 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

MC-LR 3 0 0 0 0 1 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

MC-LW 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

MC-LY 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

MC-RR 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

MC-WR 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

MC-YR 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

NOD 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

13,19-didesMeSPXC 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

SPX1 3 0 0 3 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

20MeSPXG 3 0 0 3 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

Asp MC-LR 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

AZA1 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 3 3

AZA2 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

AZA3 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

AZA4 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

AZA5 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 1 1 3 3

GYM 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

OA methyl ester 3 0 0 0 0 0 0 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

OA C8-diol ester 3 0 0 0 0 0 1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 3 3 2 3 3

PTX2 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 2 3 2 3 2 3 2 3 2 2 2 3

PnTX E 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

PnTX F 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

PnTX G 3 0 0 3 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

RP NEG

16:0 OA ester 1 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

hYTX 3 0 0 0 0 0 0 2 3 3 2 2 3 2 3 3 2 2 2 2 2 3 3 3 3 3 2 3

DTX1 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

DTX2 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

OA 3 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

YTX 3 0 0 0 0 0 0 0 2 2 3 3 0 2 3 2 2 2 0 2 2 2 2 3 2 2 3 3

LEGEND





S Standard solution





65

Screening of hydrophilic and lipophilic phycotoxins in water

Spiking levels: Hydrophilic phycotoxins 120 µg L-1

*

Microcystins 10 µg L-1

Other lipophilic phycotoxins 5 µg L-1

*Exceptions: dcGTX3 (27 µg L-1

), GTX1&4 (159 µg L-1

), GTX3 (46 µg L-1

), C2 (36 µg L-1

)

TABLE 24: VALIDATION RESULTS OF HYDROPHILIC PHYCOTOXINS IN WATER

HILIC POS S blanks sea water brackish water fresh water tap S

ATX 3 0 0 0 0 0 2 2 2 2 3 2 2 2 2 3 2 2 3 2 3 3 2 2 3 3 3

dcNEO 3 0 0 0 0 0 3 2 3 2 2 3 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3

dcSTX 3 0 0 0 0 0 3 3 3 1 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

NEO 3 0 0 0 0 0 3 1 3 1 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

STX 3 1 0 0 0 0 3 3 3 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

TTX 3 0 0 0 0 0 2 2 2 3 2 3 3 2 2 3 3 3 3 3 2 2 3 3 3 2 3

HILIC NEG

CYN 2 0 0 0 0 0 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

dcGTX2 3 0 0 0 0 0 3 3 3 3 1 3 3 3 3 3 3 3 3 2 3 3 3 3 3 3 3

dcGTX3 3 0 0 0 0 0 2 3 1 0 0 3 3 3 2 3 3 2 3 1 3 3 3 3 3 3 3

DA 2 0 0 0 0 0 0 2 0 2 0 0 1 0 0 0 0 1 0 0 1 2 1 1 0 0 2

GTX1&4 3 0 0 0 0 0 1 3 1 1 0 2 3 3 2 3 2 3 3 1 3 3 3 3 3 3 3

GTX2 3 0 0 0 0 0 3 3 3 1 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

GTX3 3 0 0 0 0 0 1 2 2 0 1 1 2 3 3 2 2 2 2 1 2 3 3 3 3 3 3

GTX5 3 0 0 0 0 0 0 3 0 3 0 0 3 2 2 2 2 2 3 2 3 3 3 3 3 3 3

C1 3 0 0 0 0 0 2 3 2 3 1 3 3 3 2 3 3 3 3 3 3 3 3 3 3 3 3

C2 2 0 0 0 0 0 1 1 2 2 0 1 2 1 1 1 2 1 2 1 2 2 2 2 2 2 2

LEGEND





S Standard solution





66

TABLE 25: VALIDATION RESULTS OF LIPOPHILIC PHYCOTOXINS IN WATER

RP POS S blanks sea water brackish water fresh water tap S

MC-HilR 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

MC-HtyR 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

MC-LA 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

MC-LF 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 3 3 3 3 3

MC-LR 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

MC-LW 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

MC-LY 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

MC-RR 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

MC-WR 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

MC-YR 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

NOD 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

13,19-didesMeSPXC 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

SPX1 3 0 0 3 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

20MeSPXG 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

Asp MC-LR 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

AZA1 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

AZA2 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

AZA3 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

AZA4 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

AZA5 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

GYM 3 0 0 2 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

OA methyl ester 2 0 0 0 0 0 2 2 2 2 2 2 2 2 3 3 2 2 2 3 3 2 2 3 3 3 3

OA C8-diol ester 3 0 0 0 0 0 3 1 3 3 3 3 3 2 2 3 3 3 1 3 2 1 1 2 3 2 3

PTX2 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

PnTX E 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

PnTX F 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

PnTX G 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

RP NEG

16:0 OA ester 1 0 0 0 0 0 0 0 1 1 1 0 0 0 1 0 1 1 0 1 0 0 1 1 0 1 1

hYTX 2 0 0 0 0 0 1 0 0 1 1 0 1 0 0 0 0 1 0 1 0 0 0 0 0 0 3

DTX1 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

DTX2 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

OA 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

YTX 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 2

DA 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 1 2

LEGEND





S Standard solution





67

Screening of lipophilic phycotoxins in food supplements

Spiking levels: Microcystins 30 µg L-1

for solids and 50 µg L-1

for liquids

Other lipophilic phycotoxins 15 µg L-1

TABLE 26: VALIDATION RESULTS OF LIPOPHILIC PHYCOTOXINS IN SOLID FOOD SUPPLEMENTS

RP POS S blanks solid food supplements S

MC-HilR 3 0 0 0 0 2 2 2 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 3 3

MC-HtyR 3 0 0 0 0 3 0 2 3 0 3 2 0 3 0 3 0 0 0 0 0 0 0 0 0 3 3

MC-LA 3 0 0 0 0 0 0 0 0 0 1 0 0 2 0 0 0 0 0 0 0 0 0 0 2 0 3

MC-LF 3 0 0 2 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 2 3

MC-LR 3 0 0 0 0 0 1 1 0 0 0 0 0 1 2 1 0 0 0 0 0 0 0 0 0 2 3

MC-LW 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 2 0 0 0 0 0 0 0 0 3 3

MC-LY 3 0 0 0 0 0 0 0 0 0 0 0 0 3 0 2 0 0 0 0 0 0 0 0 0 3 3

MC-RR 3 0 0 0 0 0 2 2 3 2 1 1 1 2 2 3 2 1 1 2 1 2 1 3 1 3 3

MC-WR 3 0 0 0 0 0 0 1 0 0 1 0 0 0 0 2 2 0 0 0 0 0 1 0 2 3 3

MC-YR 3 0 0 0 0 0 1 0 3 0 0 0 0 3 0 3 0 0 0 0 0 0 0 0 3 3 3

NOD 3 0 0 0 0 0 1 1 3 2 1 2 1 2 1 3 3 1 1 0 1 1 1 0 1 3 3

13,19-didesMeSPXC 3 0 0 0 0 0 3 3 3 3 3 3 2 2 2 2 2 3 3 3 2 3 3 2 3 3 3

SPX1 3 0 0 0 2 0 2 2 2 2 2 3 3 2 2 2 3 3 2 2 2 2 2 2 3 3 3

20MeSPXG 3 0 0 0 0 0 2 3 3 2 2 3 3 3 3 2 3 3 2 2 2 1 2 3 3 3 3

Asp MC-LR 3 0 0 0 0 0 1 1 2 2 0 1 1 3 0 1 0 0 0 0 0 0 1 0 1 1 3

AZA1 3 0 0 0 0 0 2 1 2 1 0 2 3 1 3 1 2 1 0 1 0 1 1 1 1 3 3

AZA2 3 0 0 0 0 0 1 1 3 1 0 1 3 1 3 1 2 1 1 1 0 2 1 1 2 1 3

AZA3 3 0 1 0 0 0 1 1 2 1 0 3 3 2 3 1 2 0 0 1 0 1 0 1 1 2 3

AZA4 3 0 0 0 0 0 1 1 1 1 0 1 1 1 3 1 2 1 0 1 0 1 0 1 1 3 3

AZA5 3 0 0 0 0 0 2 1 1 1 1 1 2 1 2 1 1 1 0 1 0 1 0 1 1 1 3

GYM 3 0 3 0 2 0 2 2 3 1 3 3 3 3 1 2 1 3 2 1 1 2 2 1 3 3 3

OA methyl ester 3 0 0 0 0 0 1 1 1 2 0 1 1 1 2 1 2 0 0 0 1 1 0 0 0 2 2

OA C8-diol ester 3 0 0 0 1 0 0 1 1 0 0 0 1 2 1 2 1 0 1 0 0 0 0 0 0 1 3

PTX2 3 0 0 0 0 0 1 1 2 1 2 0 2 2 2 2 2 2 0 1 0 2 1 0 2 1 3

PnTX E 3 0 0 0 0 0 2 2 2 2 2 2 2 2 0 2 2 2 2 2 2 2 2 2 2 3 3

PnTX F 3 0 0 0 0 0 2 2 3 2 2 2 2 2 2 2 3 2 2 2 1 1 2 2 2 3 3

PnTX G 3 0 0 0 0 0 3 2 3 3 2 3 3 3 3 3 3 3 2 2 1 2 2 3 3 3 3

RP NEG

16:0 OA ester 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1

hYTX 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3

DTX1 3 0 0 0 0 0 2 2 3 2 2 0 0 2 2 3 0 1 2 0 0 0 2 0 2 3 3

DTX2 3 0 0 0 0 0 2 3 2 2 0 0 2 3 2 3 3 0 0 0 0 0 0 1 1 2 3

OA 3 0 0 0 0 0 3 3 3 3 1 2 3 3 2 3 3 2 1 0 0 0 1 1 2 3 3

YTX 3 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3

LEGEND





S Standard solution





68

TABLE 27: VALIDATION RESULTS OF LIPOPHILIC PHYCOTOXINS IN LIQUID FOOD SUPPLEMENTS

RP POS S blanks liquid food supplements S

MC-HilR 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 0 0 3 3 3 3 3 3 3 0 3 3

MC-HtyR 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 0 3 3 3 3 3 3 3 3 0 3 3

MC-LA 3 0 0 0 0 0 3 3 3 3 3 0 3 2 3 0 0 0 2 3 0 3 3 3 0 3 3

MC-LF 3 0 0 0 0 0 3 3 3 3 3 0 3 3 3 0 0 0 3 3 3 3 3 3 2 3 3

MC-LR 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 0 0 1 2 3 3 3 3 3 2 3 3

MC-LW 3 0 0 0 0 0 3 3 3 3 3 0 3 3 3 0 0 0 3 3 3 3 0 3 2 3 3

MC-LY 3 0 0 0 0 0 3 3 3 3 3 0 3 2 3 0 0 0 3 3 0 3 0 3 2 3 3

MC-RR 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 0 3 3

MC-WR 3 0 0 0 0 0 3 3 3 3 3 3 3 2 3 0 3 3 3 3 3 3 3 3 0 3 3

MC-YR 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 0 0 3 3 3 3 3 3 3 0 3 3

NOD 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 0 2 3 2 3 3 3 3 3 2 3 3

13,19-didesMeSPXC 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

SPX1 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

20MeSPXG 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 2 3 3 3 3 3 3 3 3 3 3 3

Asp MC-LR 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 0 3 1 1 3 3 3 3 3 1 3 3

AZA1 3 0 0 0 0 0 3 3 3 3 3 1 3 2 3 0 1 1 3 3 3 1 3 3 2 3 3

AZA2 3 0 0 0 0 0 3 3 3 3 3 2 3 2 3 1 1 1 3 3 1 3 3 3 1 3 3

AZA3 3 0 0 0 0 0 3 3 3 3 3 2 3 3 3 1 1 1 3 3 2 2 3 3 3 3 3

AZA4 3 0 0 0 0 0 3 3 3 3 3 3 1 3 3 0 0 1 3 3 3 3 3 3 1 3 3

AZA5 3 0 0 0 0 0 3 3 3 3 3 3 1 1 3 1 1 1 3 3 3 3 3 3 1 3 3

GYM 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 3 3 3 1 3 3 3 3 3 3 3 3

OA methyl ester 2 0 0 0 0 0 2 2 3 2 2 1 1 1 2 0 0 1 2 2 0 0 0 2 1 2 2

OA C8-diol ester 3 0 0 0 0 0 2 1 2 2 3 1 3 3 2 0 1 1 2 2 0 0 0 2 1 2 3

PTX2 3 0 0 0 0 0 3 3 3 3 3 2 1 2 3 0 0 1 3 3 2 2 2 3 1 3 3

PnTX E 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 0 2 2 2 3 3 3 3 3 2 3 3

PnTX F 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 2 2 2 3 3 3 3 3 3 2 3 3

PnTX G 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 2 3 3 3 3 3 3 3 3 3 3 3

RP NEG

16:0 OA ester 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0

hYTX 3 0 0 0 0 0 3 3 3 2 2 0 0 0 3 0 0 0 1 2 1 1 1 2 0 1 2

DTX1 3 0 0 0 0 0 3 3 3 3 3 0 3 3 0 0 0 0 3 3 3 3 3 3 0 3 3

DTX2 3 0 0 0 0 0 3 3 3 3 3 2 3 3 3 0 2 3 3 3 3 3 3 3 1 3 3

OA 3 0 0 0 0 0 3 3 3 3 3 3 3 3 3 0 0 3 3 3 3 3 3 3 0 3 3

YTX 3 0 0 0 0 0 2 2 2 1 1 0 0 0 2 0 0 0 1 2 1 0 1 1 0 0 2

LEGEND





S Standard solution





69

TABLE 28: VALIDATED LEVELS SCREENING

Compound (Shell) fish (µg kg-1)

Water (µg L-1)

Food supplements solids (µg kg-1)

Food supplements liquids (µg kg-1)

HILIC POS

ATX 600 120

dcNEO 600 120

dcSTX 600 120

NEO 600 120

STX 600 120

TTX 600 (except ensis) 120

HILIC NEG

CYN * 120

dcGTX2 600 120

dcGTX3 134 27

DA 600 *

GTX1&4 795 159

GTX2 600 120

GTX3 228 46

GTX5 600 120

C1 600 (except mussel) 120

C2 * *

RP POS

MC-HilR 150 10 * *

MC-HtyR 150 10 * *

MC-LA 150 10 * *

MC-LF 150 10 * *

MC-LR 150 10 * *

MC-LW 150 10 * *

MC-LY 150 10 * *

MC-RR 150 10 * 50

MC-WR 150 10 * *

MC-YR 150 10 * *

NOD 150 10 * 50

13,19-didesMeSPXC 80 5 15 15

SPX1 80 5 15 15

20MeSPXG 80 5 15 15

Asp MC-LR 150 10 * *

AZA1 80 5 * *

AZA2 80 5 * *

AZA3 80 5 * *

AZA4 80 5 * *

AZA5 80 (except fish) 5 * *

GYM 80 5 * 15

OA methyl ester 80 5 * *

OA C8-diol ester 80 * * *

PTX2 80 5 * *

PnTX E 80 5 15 15

PnTX F 80 5 15 15

PnTX G 80 5 15 15

RP NEG

16:0 OA ester * * * *

hYTX 80 * * *

DTX1 80 5 * *

DTX2 80 5 * *

OA 80 5 * *

YTX * * * *

* Validated; without satisfactory results

70

Quantitation of ASP, PSP and DSP in tissue

TABLE 29: SPIKE LEVELS OF ASP, PSP AND DSP FOR QUANTITATIVE VALIDATION IN SHELLFISH

Compound Toxin group 0.5 PL (ug kg-1)

1 PL (ug kg-1)

Matrix matched standards (ug kg-1)

DA ASP 10,000 20,000 0, 5, 10, 20, 50 mg kg-1

STX, dcSTX, NEO, dcNEO, GTX1&41, GTX2&31, GTX5, dcGTX2&31 PSP 400 800 0, 400, 600, 800, 1200

OA, DTX1, DTX2 DSP 80 160 0, 20, 40, 80, 160, 240

AZA1, AZA2, AZA3 80 160 0, 20, 40, 80, 160, 240

YTX, hYTX2 250 500 0, 62.5, 125, 250, 500, 750

SPX13 200 400 0, 50, 100, 200, 400, 600

GYM3 100 200 0, 25, 50, 100, 200, 300

PnTX G3 25 50 0, 6.25, 12.5, 25, 50, 75

1 Concentrations of the highest isomer present given. 2 The permitted level of YTX and hYTX is 3750 µg kg-1, values are target values. 3 There are no permitted levels for SPX, GYM and PnTX G. Values are target values.

TABLE 30: VALIDATION RESULTS OF LIPOPHILIC PHYCOTOXINS IN TISSUE, MMS BEFORE SAMPLES, RECOVERY AND REPEATABLITLITY

Compound Linearity % Deviation from the back-calculated concentration

Recovery RSDr

0.5PL RSDr

1PL Level 1 Level 2 Level 3 Level 4 Level 5

Requirements >0.9900 <20% <20% <20% <20% <20% 70-120% <20% <20%

OA 0.999 3.3% -4.0% -7.0% 3.1% -0.5% 120.0% 17.1% *7.7%

DTX1 0.999 -9.6% 8.3% -4.1% -3.8% 2.0% 109.8% 4.6% 8.5%

DTX2 0.999 -20.8% -3.9% 6.2% 1.3% -1.0% 111.4% 15.0% 12.0%

YTX 0.995 -20.6% -10.5% -14.6% 4.5% 0.1% 90.1% *10.7% 18.1%

hYTX 0.994 6.6% -9.1% -21.0% -1.2% 3.1% 75.2% *7.1% 5.5%

AZA1 0.998 10.3% 7.9% -2.3% -7.2% 3.1% 95.0% 5.1% 3.0%

AZA2 0.999 12.1% -1.6% -2.7% 3.5% -1.3% 88.7% 5.5% 3.4%

AZA3 1.000 3.7% 5.8% -1.4% -0.3% 0.1% 108.1% 6.9% 4.8%

SPX1 0.999 -1.1% 1.1% -0.7% 4.5% -1.9% 109.9% 5.0% 7.9%

GYM 1.000 2.6% 2.5% -0.8% -1.5% 0.7% 102.3% 5.6% 6.5%

PnTX G 0.999 -4.2% -1.3% -1.8% 3.9% -1.5% 102.2% 7.1% 9.8%

* One outlier, tested with Grubbs test, removed from dataset

TABLE 31: VALIDATION RESULTS OF LIPOPHILIC PHYCOTOXINS IN TISSUE, MMS AFTER SAMPLES

Compound Linearity % Deviation from the back-calculated concentration Drift in

sensitivity Level 1 Level 2 Level 3 Level 4 Level 5

Requirements >0.9900 <20% <20% <20% <20% <20% <30%

OA 0.994 -1.4% -13.0% 0.1% 11.6% -4.8% -24.2%

DTX1 0.997 -13.3% 3.3% -10.1% 8.1% -2.5% -15.7%

DTX2 0.999 -12.8% -5.3% 4.6% 2.6% -1.4% 6.4%

YTX 0.993 -0.8% -5.8% -22.4% -1.0% 3.1% 11.4%

hYTX 0.995 3.4% -6.5% -15.1% -4.9% 4.0% 3.4%

AZA1 0.995 -3.0% -10.2% 4.5% 10.0% -4.6% -15.5%

AZA2 0.998 3.0% -4.0% -1.9% 6.9% -2.8% -4.3%

AZA3 0.997 -9.1% 4.8% 3.9% 6.6% -3.4% -4.3%

SPX1 1.000 -0.2% 0.0% -3.0% -0.4% 0.5% 8.8%

GYM 0.998 15.0% 3.9% -6.7% -5.0% 2.8% 9.1%

PnTX G 0.999 -6.9% -1.7% 5.9% -4.9% 1.6% 1.4%

71

TABLE 32: VALIDATION RESULTS OF HYDROPHILIC PHYCOTOXINS IN TISSUE, MMS BEFORE SAMPLES, RECOVERY AND REPEATABLITLITY

Compound Linearity % Deviation from the back-calculated concentration Recovery RSDr

0.5PL RSDr

1PL Level 1 Level 2 Level 3 Level 4

Requirements >0.9900 <20% <20% <20% <20% 70-120% <20% <20%

DA 0.994 -25.0% 9.6% 13.3% -3.4% 93.0% 3.4% 1.4%

STX 0.994 -16.9% 1.9% 8.5% -2.4% 39.2% 59.9% 42.7%

dcSTX 0.999 0.6% 3.2% 2.1% -1.8% 33.2% 53.8% 37.4%

NEO 0.966 -8.9% -31.3% 3.7% 7.2% 18.4% 69.1% 43.8%

dcNEO 0.992 -12.4% -12.0% 5.2% 2.1% 9.2% 50.0% 35.2%

GTX1&4 0.981 -23.7% -15.0% 3.2% 5.0% 85.2% 8.4% 12.5%

GTX2 0.995 -7.2% -3.9% -5.3% 4.1% 95.9% 7.9% 3.8%

GTX3 0.999 -3.6% 5.3% -0.7% -0.6% 106.2% 8.8% 7.2%

GTX5 0.994 -14.3% -6.4% -0.9% 3.6% 92.5% 5.7% 4.6%

dcGTX2 0.994 -17.1% -0.4% -2.1% 2.9% 57.3% 22.1% 12.9%

dcGTX3 0.994 -16.9% -5.6% 2.0% 2.4% 83.8% 13.8% 8.5%

C1 0.996 -9.5% -7.3% 4.1% 1.0% 81.9% 21.5% 8.3%

C2 0.979 -11.2% -21.9% 0.8% 6.4% 38.3% *6.0% 30.0%

* One outlier, tested with Grubbs test, removed from dataset

TABLE 33: VALIDATION RESULTS OF HYDROPHILIC PHYCOTOXINS IN TISSUE, MMS AFTER SAMPLES

Compound Linearity % Deviation from the back-calculated concentration Drift in

sensitivity Level 1 Level 2 Level 3 Level 4

Requirements >0.9900 <20% <20% <20% <20% <30%

DA 0.993 -27.1% 8.9% 16.3% -3.8% -15.2%

STX 0.988 -11.2% -16.0% 1.3% 4.6% -17.5%

dcSTX 0.997 -9.3% -5.0% 0.6% 2.0% -18.1%

NEO 0.968 -32.3% -16.6% 0.1% 7.7% -25.3%

dcNEO 0.969 -27.1% -16.1% -4.8% 9.2% -14.1%

GTX1&4 0.980 -20.1% -13.9% -3.2% 7.1% -10.4%

GTX2 0.994 -7.6% -10.6% -0.8% 3.8% -11.5%

GTX3 0.996 -6.6% -8.7% -0.6% 3.2% -5.9%

GTX5 0.995 -10.3% -9.3% 1.6% 2.7% -8.5%

dcGTX2 0.982 -26.5% -2.9% -6.7% 6.6% -4.6%

dcGTX3 0.994 -17.0% -4.3% 0.5% 2.8% -6.8%

C1 0.996 -5.8% 7.1% -7.3% 2.1% -15.8%

C2 0.991 -23.0% -2.0% -1.0% 3.5% -16.2%

a generic method for regulated and unregulated phycotoxins in various matrices … ·...

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