Ana M. Botana , Paz Otero , Paula Rodr í guez , Amparo ...

DOI 10.1515/revac-2012-0020 Rev Anal Chem 2013; 32(1): 15–34

Ana M. Botana , Paz Otero , Paula Rodr í guez , Amparo Alfonso and Luis M. Botana *

Current situation on analysis of marine toxins Abstract: Marine toxins are a food safety concern world-

wide. This review discusses current analytical methods

for those toxins that are legally regulated in Europe,

namely domoic acid, saxitoxin, okadaic acid, yessotoxin,

pectenotoxin and azaspiracids, and all their analogues,

and those that are not regularly monitored but are a threat

to humans, such as tetrodotoxin, ciguatoxins and cyclic

imines. Because of legislative changes that were imple-

mented in 2011, most legally required methods are chro-

matographic. Saxitoxin and domoic acid are monitored

by high-performance liquid chromatography with fluo-

rescent and ultraviolet detection, respectively. All other

toxins are monitored nowadays by means of chromato-

graphic separation and mass spectrometry detection.

Keywords: analytical standards; chromatography; marine

toxins; mass spectrometry.

*Corresponding author: Luis M. Botana, Department of

Pharmacology, Veterinary School , Faculty Veterinaria, Universidad

de Santiago de Compostela, Campus de Lugo, Lugo 27002 , Spain ,

e-mail: [email protected]

Ana M. Botana: Department of Analytical Chemistry , Universidad de

Santiago de Compostela, Campus de Lugo, Lugo 27002 , Spain

Paz Otero: Department of Pharmacology, Veterinary School , Faculty

Veterinaria, Universidad de Santiago de Compostela, Campus de

Lugo, Lugo 27002 , Spain

Paula Rodr í guez: Department of Pharmacology, Veterinary School ,

Faculty Veterinaria, Universidad de Santiago de Compostela,

Campus de Lugo, Lugo 27002 , Spain

Amparo Alfonso: Department of Pharmacology, Veterinary School ,

Faculty Veterinaria, Universidad de Santiago de Compostela,

Campus de Lugo, Lugo 27002 , Spain

Introduction Phycotoxins are an acute worldwide seafood safety

concern; hence, their detection and quantification in food

with reliable methods is important. The methods must be

validated according to international guidelines to ensure

adequate results and to protect public health and reduce

their economic impact.

Harmful algal blooms is the term used to describe

a proliferation of marine microalgae (dinoflagellates,

diatoms) that carries a toxic effect and, in many cases,

economic losses. The microalgae are usually present in

the plankton in low concentrations, but under some envi-

ronmental conditions they can rapidly multiply in dense

blooms. These microalgae produce phycotoxins, which

accumulate in the digestive organs of filter-feeding shell-

fish, zooplankton and herbivorous fishes and through

the trophic chain can produce human intoxications. The

toxins are generally considered to be secondary metabo-

lites and therefore not essential to the basic metabolism

and growth of the microorganism (Van Dolah 2000 ). These

episodes previously regarded as sporadic and localised

phenomena and restricted to certain geographic areas

have started to show up as recurrent and ubiquitous in

any coast of the world and sometimes cause great damage

to the local fishing industry and also to the natural com-

munities of marine organisms (Vieites and Cabado 2008 ).

Different countries have established standards about

the total amount of phycotoxins that can be present in

seafood products and also about the testing methods

for detecting these molecules. Legal requirements for

paralytic shellfish toxins [usually named paralytic shell-

fish poisoning (PSP)], amnesic shellfish toxins [amnesic

shellfish poisoning (ASP)], lipophilic toxins, ciguatoxins

(CTXs), brevetoxins or tetrodotoxins (TTXs) have been set

around the world (Rodr í guez-Velasco 2008 ). The Euro-

pean legislation establishes a maximum level of toxins

in bivalve molluscs, echinoderms, tunicates and marine

gastropods (the whole body or any part edible separately)

of 800 μ g of PSP equivalent/kg, 20 mg of domoic acid

(DA)/kg, 160 μ g of okadaic acid (OA) equivalents/kg, 160

μ g of azaspiracids (AZAs) equivalents/kg and 1 mg of yes-

sotoxin equivalent/kg, and the total absence of TTX and

CTX (European Commission 2004a ) (see structures in

Figure 1 ). To check compliance with the limits laid down,

several methods including mouse bioassay (MBA), high-

performance liquid chromatography (HPLC) with fluo-

rescence (FL), ultraviolet (UV) or mass spectrometry (MS)

detection, immune assays or functional assays have been

proposed. However, not all these methods are officially

accepted as a validation process is necessary (European

Commission 2007 ). MBA has been for many years consid-

ered as the reference monitoring method for phycotoxins,

although the European Commission established that to be

in accordance with animal health protection, all possible

replacement, refinement and reduction of animals must be

taken into account when laboratory animal methods are

16 A.M. Botana et al.: Analysis of marine toxins

Palytoxin

Azaspiracid1

Saxitoxin

Ciguatoxin3C Okadaic acid

Pectenotoxin

Yessotoxin

N

NH

O

R1

+NH2

R3

R4

OH

HO

H

R2 H

H

HH

HO

O

H

109

2

1

5

7

4

Tetrodotoxin

Brevetoxin

Figure 1 Structures of representative toxins.

A.M. Botana et al.: Analysis of marine toxins 17

used (Hess et al. 2006 ), and therefore alternative methods

should be applied. Any method proposed as an alternative

to the biological assay should not be less effective than

the biological method, and the implementation should

provide an equivalent level of public health protection.

Following these premises, several methods were validated

as alternatives to the MBA (Botana et al. 2009 , 2010, Camp-

bell et al. 2011 ). In the case of PSP toxins, the total content

of toxins in the edible parts of molluscs must be officially

detected by MBA (European Commission 2005 ) and also by

the HPLC Lawrence method with pre-column oxidation as

published in the Association of Official Analytical Chemi-

sts Official Method2205.06 (European Commission 2006 ).

If the results are challenged, the reference method shall be

the biological method. To detect PSP toxins, other analyti-

cal non-validated methods are available, such as an HPLC

method with post-column oxidation (Oshima et al. 1976 ,

Rodriguez et al. 2010 ), plasmon resonance biosensors

(Fonfria et al. 2007 ) or enzyme immunoassays [enzyme-

linked immunoassay (ELISA)] (Huang et al. 1996 , Garet

et al. 2010 ), based on both the use of antibodies and several

functional assays based on the mechanism of action of the

toxins (Vieytes et al. 1993 , Louzao et al. 2001 ). In the case of

ASP, the total content of toxins in edible parts of molluscs

may be detected by using either an HPLC method with UV

detection (HPLC-UV) or an ELISA method (European Com-

mission 2007 ). In this case, if results are challenged, the

reference method shall be the HPLC method. For the detec-

tion of lipophilic toxins, a series of MBA procedures, dif-

fering in the test portion of flesh and the solvents used for

extraction, were the official method for many years (Euro-

pean Commission 2005 ); however, after July 2011 a liquid

chromatography (LC) tandem MS (LC-MS/MS) method was

approved to be applied as the reference method for the

detection of these toxins (Commission Regulation 2011 ). In

addition to this, several methods based on the mechanism

of action of each lipophilic toxin group have been devel-

oped (Vieytes et al. 1997 , Alfonso et al. 2004 , Vilarino et al.

2009 , Otero et al. 2011b ). There are no official methods to

detect the presence of TTX or CTX even though several

LC-MS/MS methods can be used to detect these toxins

(Otero et al. 2010 , Rodr í guez et al. 2012 ).

As was mentioned earlier, the presence of phycotox-

ins is a dynamic and ubiquitous process in any coast of

the world and therefore the presence of new analogues,

new toxins or toxins from different locations are issues

that must be taken into account when any analytical

method is used. However, the legislation also established

that in addition to the amount of toxin, the total toxicity,

which is calculated by using conversion factors based

on the available toxicity data of each toxin, should be

calculated (Commission Regulation 2011 ). In this sense,

the toxicity equivalent factor (TEF) of each compound

compared with the potency of the reference compound

must be applied to calculate the total toxicity of a sample

(Botana et al. 2010 ). The Working Group on marine bio-

toxins, as part of the Contaminants Panel at the European

Food Safety Agency (EFSA), has defined, on the basis of

current knowledge, the TEF that should be used for the

conversion of analytical results into toxic concentrations

in each toxin group (European Commission Panel 2009b ).

Another essential item to implement and to validate any

chemical detection method is the availability of standards

as the identification of one toxin by using other analogues

can lead to gross errors (Otero et al. 2011a ). In addition,

pure toxins in high amount are necessary to calculate the

toxicity and TEF of each molecule to achieve results to

protect human health. It can be concluded that analytical

methods are useful tools to quantify the amount of known

marine toxins in seafood samples, but several important

details such as toxicity, calibrants and new toxins should

be considered.

Analysis of PSP toxins PSP is a fatal human syndrome produced by a naturally

occurring group of neurotoxic alkaloids present in several

species of dinoflagellates. This group of toxins constitutes

the most spread worldwide affecting the whole of Europe,

South Africa, India, Morocco, eastern Asian coast and

North and South America as well.

Saxitoxin (STX) is the first described and most studied

compound of the group (it is the representative com-

pound), and 57 other analogues also have been researched

(Wiese et al. 2010 ). Traditionally, STX analogues have been

composed only of hydrophilic compounds and divided

into subgroups on the basis of substituent side chains:

carbamate [STX, neo-STX and gonyautoxins (GTX1 – 4)],

decarbamoyl (dc-STX, dc-neo-STX and dc-GTX1 – 4) and N -

sulfocarbamoyl (GTX5 – 6 and C1 – 4). These groups present

different toxicities, with the carbamoyl analogues being

the most toxic, followed by decarbamoyl analogues with

intermediate toxicity, and N -sulfocarbamoyl analogues

the least toxic (Vale et al. 2008a,b ). Figure 1 shows the

chemical structure of the more often found compounds in

toxic dynoflagellates and poisoned seafood.

More recently, analogues from a novel family of PSP

toxins containing a hydrophobic side chain were isolated

and structurally characterised from Australian strains of

Gymnodinium catenatum and designated GC1-GC3 (Negri


et al. 2003 ). Other hydrophilic analogues of STX with an

acetate R 4 side chain were reported in the freshwater fila-

mentous cyanobacterium Lyngbya wollei but not in the

marine environment so far (Onodera et al. 1997 ).

PSP toxins specifically block the excitation current in

nerve and muscle cells by means of site one of the sodium

channel (Messner and Catterall 1986 ); therefore, the accu-

mulation of PSP toxins in shellfish creates a serious public

health problem and affects the fisheries industry. Sommer

and Meyer (1937) were the first to develop a method to

determine PSP toxins: their MBA method established the

basis for that. This is the reference method internationally

accepted to quantify PSP toxicity, and it is used worldwide

in monitoring programmes. The chemicals methods used

to determine PSP toxins are fluorimetric assays, HPLC

with fluorimetric detection (either pre-column or post-

column oxidation), LC/MS and capillary electrophoresis

(CE) methods.

The HPLC methods are widely used to quantify PSP

toxins present in seafood samples, and they are also

useful to provide the PSP profile because chromatographic

methods are identification methods as well. These toxins

have only a weak chromophore group, and it must be

modified before detection: when they are oxidised in alka-

line solution, a purine is formed that becomes fluorescent

at acidic pH. This reaction can be either a pre-column one

or a post-column one, and obtained purines are monito-

rised with an FL detector.

Bates and Rapoport (1975) first studied the oxidative

alkaline conditions of PSP toxins to get fluorescent com-

pounds; then, Buckley et al. (1978) added them to their

method and established the basis of the HPLC method for

these compounds with the post-column reaction.

In 1984, Oshima et al. (1984) proposed a method

based on alkaline oxidation to produce highly fluores-

cent derivatives, but with problems in separating toxins

such as GTX1, GTX4 and GTX3, GTX5. Afterwards, they

described a method that was able to separate almost all

the PSP toxins with three different eluents according to

the basicity of the three groups of toxins (group I: C1 – C4;

group II: GTX1,4, GTX5 (B1) and GTX6 (B2), dc-GTX1,4;

group III: NEO, dc-STX and STX) (Oshima et al. 1987 ).

In a later work, Oshima (1995) improved the system and

included an extract cleaning procedure. The first dis-

advantage is that it is a time-consuming method for the

analysis of PSP toxins.

In the group of pre-column methods, the method

developed by Lawrence and Menard (1991) needs to be

highlighted. One of the biggest disadvantages was the ina-

bility to easily distinguish between different toxin groups

with similar toxicity.

Following this, the method was modified by chang-

ing chromatographic conditions to reduce analysis time

and improve performance (Lawrence et al. 1995 ). In 2005,

the Lawrence method was adopted as the official method

to detect PSP toxins and then approved by the European

Union (EU) for monitoring these toxins (Association of

Official Analytical Chemists 2005, European Commis-

sion 2006 ). It is based on the pre-column oxidation of PSP

toxins with hydrogen peroxide and sodium periodate fol-

lowed by fluorimetric detection. It was validated for the

determination of STX, NEO, GTX2,3, GTX1,4, dc-STX, GTX5

(B1), C1,2 and C3,4 in molluscs (mussels, clams, oysters

and scallops). In 2009 a validation study was under-

taken for the extension of this method to two more toxins:

dc-NEO and dc-GTX2,3 (Turner et al. 2009 ).

Pre- and post-column HPLC methods, despite the

many benefits of each, which includes an increased sen-

sitivity to low concentrations of toxins and less variability

in results, also present some drawbacks that should be

resolved. In the case of hydrophobic analogues, they are

retained by C18 resins (Negri et al. 2003 ), because of which

the use of HPLC methods in monitoring programmes will

miss the presence of hydrophobic analogues (Vale 2008 ,

Vale et al. 2009 ). LC-MS methods are also being developed

to obtain a good characterisation of these compounds:

Electrospray ionisation MS (ESI-MS) is effective for the

detection of polar PSP toxins (Quilliam 2003 , Dell ’ Aversano

et al. 2005 ); however, variable retention times for PSP

toxins in different seafood matrixes were observed. At

present, it is not possible to achieve a complete separa-

tion of all PSP toxins, and the sensitivity of MS detection is

not good enough to control seafood toxin at the regulatory

limit established (Diener et al. 2007 ).

Although the analysis of PSP toxins provides a profile

of the composition of a given toxic episode, the use of

surface plasmon resonance biosensors has reached a con-

siderable level of development (Campbell et al. 2007 ). The

high sensitivity of this approach allows fast throughput,

as sample extraction and detection is rather simple, plus

the chips can be used many times (Fonfria et al. 2007 ).

For this techonology, an antibody-based method has been

described and validated both at the single laboratory level

(Campbell et al. 2010 ) and in an interlabotoratory study

(van den Top et al. 2011b ).

Analysis of DA DA, belonging to the kainoid class of compounds, is the

chemical responsible for ASP. It was originally isolated as


a product from the red algae Chondria armata and subse-

quently isolated from other red algae and several species of

diatoms, mainly of the genus Pseudonitzschia (Bates et al.

1989 , Lundholm et al. 1994 ). DA has also been detected

in several species of molluscs worldwide including the

United States, New Zealand, Mexico, several European

countries such as France, Portugal, Ireland and Spain and

the Croatian coast (Horner et al. 1993 , M í guez et al. 1996 ,

Vale, 2001, Ujevic et al. 2010 ). Although the DA is the main

toxin found in a variety of shellfish species, other minor

analogues, about 10 isomers of DA (isodomoic acids A – H

and DA 5 ′ diasteriomer), have been identified in marine

samples (Wright et al. 1990 , Zaman et al. 1997 ).

The first method developed to detect the compound

was LC-UV (Lawrence et al. 1989 ), and a protocol involv-

ing aqueous methanol extraction and strong anion-

exchange (SAX) clean-up has been applied extensively to

the analysis of DA and its known isomers in shellfish and

fish tissues on a regulatory basis. Nowadays, the LC-UV

remains the most widely used method, and it has been

validated and standardised as the reference method for

DA quantification (International 2000 , European Com-

mittee for Standardization 2003 ). Furthermore, ELISA

has also been validated (Kleivdal et al. 2007 ) and is used

officially in the EU for screening purposes. These methods

have limits of detection (LODs) low enough to detect DA at

a concentration of 4.5 mg/kg of shellfish meat. This dose

was established as the maximum amount that a portion of

400 g of meat should contain for not exceeding the acute

reference dose of 30 mg DA/kg body weight (European

Commission Panel 2009a ).

Several other biological methods have been deve-

loped to detect and quantify DA in seawater and tissue

samples, such as electrochemical ELISA (Micheli et al.

2004 ), radioimmunoassay (Lawrence et al. 1994 ), recep-

tor binging assay (Lefebvre et al. 2010 ), cytotoxicity assay

(Beani et al. 2000 , Leira et al. 2003 ) and surface plasmon

resonance (Le Berre and Kane 2006 , Traynor et al. 2006 ,

Stevens et al. 2007 ). Some of these techniques, such as

enzyme immunoassay and ELISA, sometimes may require

little clean-up of shellfish samples and have the advan-

tages of being easy and fast (Yu et al. 2004 ). However,

the cross-reactivity with similar toxins will result in false

positives and limit the demonstration of toxicity (He et al.

2010 ).

DA contains a characteristic conjugated diene chromo-

phore with strong absorbance at 242 nm, which permits

its detection by LC-UV at concentrations as low as 4 – 80

ng/ml, depending on the sensitivity of the detector (Mafra

et al. 2009 ). This is suitable for regulatory purposes;

however, false positives are frequently encountered

because of interferences from crude extracts (Hess et al.

2001 ). Mainly, tryptophan and its derivatives, normally

contained in shellfish and finfish tissues, may be eluted

close to DA with some columns and chromatographic con-

ditions. Thus, there are other alternatives, such as FL (James

et al. 2000 , Chan et al. 2007 ) and chemilumine scence

detection (Kodamatani et al. 2004 ). As DA concentra-

tions in Pseudonitzschia cultures and phytoplankton field

samples are often much lower, a more sensitive method

of detection is required. The most common technique for

the ana lysis of DA in seawater samples uses a derivatising

agent, which results in low LODs (Pocklington et al. 1990 ),

but this procedure involves some problems. Among them,

a poor selectivity because many compounds in the sample,

such as amino acids, can be derivatised and interfere with

DA detection. Also, the handling during the derivatisation

step is time consuming for the use of LC-FL on a large scale.

In addition to the LC-UV and LC-FL, various extraction and

screening techniques, such as thin-layer chromatography

(Quilliam et al. 1998 ), CE (Gago -Martinez et al. 2003 ), gas

chromatography coupled to MS (GC-MS) (Pleasance et al.

1990 ), LC-MS/MS (Wang et al. 2007 , Mafra et al. 2009 ) and

ultraperformance liquid chromatography with tandem MS

detection (UPLC-MS/MS) (de la Iglesia et al. 2008 ), have

allowed the detection of novel compounds inside of the DA

group in new coastal areas of the world.

As the seafood matrix can interfere in the detection

of trace levels, pre-treatment steps to concentrate and

purify DA are needed. Solid-phase extraction (SPE) is the

most common method used for the clean-up of shellfish

samples before using a chromatography or other analyti-

cal technique to quantify the toxin amount (Zhao et al.

1997 , Quilliam et al. 1998 , James et al. 2000 , Pardo et al.

2007 , He et al. 2010 ]. So, several methods have been based

on SAX and strong cation exchange (Pleasance et al. 1990 ,

Furey et al. 2001 , Gago -Martinez et al. 2003 , Ciminiello

et al. 2005 ). The purification with SPE cartridges results in

a valid approach for the routine monitoring of DA in shell-

fish to prevent the matrix effect and signal suppression.

However, this is a manual time-consuming procedure;

even some authors concluded that an additional step

with an SPE cartridge did not significantly improve the

recovery of DA from shellfish samples (Powell et al. 2002 ).

Therefore, there are many analytical methods that do use

a sample clean-up and could be avoided when the pH of

the mobile phase in the LC methods is properly adjusted

(He et al. 2010 ). Table 1 summarises several chemical

methods, specifying the sample clean-up procedure in

different matrices and the LOD obtained in each method.

MS techniques are being increasingly used in some

laboratories, and today, LC-MS/MS is perhaps the most


Method Matrices Treatment sample (clean-up) LODs References

LC-UV Mussels Molecularly imprinted polymer-

solid-phase extraction (MISPE)

0.1 mg/ml (Ciminiello et al. 2005)

LC-UV Fur seals (fluidics) SPE 8 ng/ml (European Commission Panel

2009a )

UPLC-MS/

MS

< 1 ng/ml

LC-UV Phytoplankton, seawater

samples

Without sample pre-

concentration and derivatisation

42 pg/ml

(on column)

(Traynor et al. 2006 )

LC-ESI-MS 15 pg/ml

LC-FL ∼ 15 pg/ml

LC-FL Mussels SPE and post-column

derivatisation

25 ng/ml (Powell et al. 2002 )

LC-FL Shellfish and phytoplankton SPE and pre-column

derivatisation (shellfish) Without

SPE (phytoplankton)

≤ 1 ng/ml (He et al. 2010 )

UPLC-MS/

MS

Seawater SPE-C18 (with Empore disks) 0.02 ng/ml (Gago -Martinez et al. 2003 )

UPLC-MS/

MS

Crude extracts Without clean-up 0.05 – 0.09 mg/kg (McNabb et al. 2005 )

LC-MS/MS Seawater and phytoplankton SPE-C18 cartridges 30 pg/ml (Quilliam et al. 1998 )

LC-MS/MS Mussels, clams, cockles Pressurised liquid extraction

(PLC) with purification inside the

extraction cell

0.2 μ g/ml (Wang et al. 2007 )

LC-UV Mussels and scallops Without SPE clean-up 25 ng/ml (Doucette et al. 2009 )

LC-ESI-MS 0.008 μ g/ml

LC-UV-CLD Mussels Without any pre-concentration or

derivatisation steps

8 pg/ml (Hess et al. 2001 )

LC-MS/MS Seawater, phytoplankton,

mammalian fluids and tissues

SPE-C18 cartridges without pre-

concentration

1 – 4 pg/ml

(on column)

(Mosher et al. 1965 )

TLC Mussels SAX clean-up ∼ 10 μ g/g (Miyazawa and Noguchi 2001 )

GC-MS Mussels SPE clean-up: reversed phase

and SCX cartridges

1 μ g/ml (Pocklington et al. 1990 )

LC-UV Clams and scallops SAX cartridges 0.47 μ g/ml (Kodamatani et al. 2004 )

CE-EIA Mussels, oysters, clams and

scallops

– 0.02 ng/ml (Noguchi and Arakawa 2008 )

CE Mussels, razor clams and

anchovy

SAX clean-up 0.15 μ g/g (Pleasance et al. 1990 )

Table 1 Some representative analytical chemical methods for the detection of DA in a range of matrices.

CE, capillary electrophoresis; CE-EIA, capillary electrophoresis-enzyme immunoassay; DA, domoic acid; EIA, enzyme immunoassay;

GC-MS, gas chromatography-mass spectrometry; LC-ESI-MS, liquid chromatography-enzyme immunoassay-tandem mass spectrometry;

LC-FL, liquid chromatography with fluorescence detection; LC-MS/MS, liquid chromatography with tandem mass spectrometry; LC-UV, liquid

chromatography with ultraviolet detection; LC-UV-CLD, liquid chromatography-ultraviolet-chemiluminescence detection; LODs, limits

of detection; SAX, strong anion exchange; SCX, strong cation exchange; UPLC-MS/MS, ultraperformance liquid chromatography with

tandem mass spectrometry; TLC, thin layer chromatography.

important and legally accepted confirmatory tool, provid-

ing a high sensitivity, accuracy and selectivity for DA and

its isomers in crude extracts (McNabb et al. 2005 ). Nev-

ertheless, LC-UV is often the only analytical instrument

available in many research institutes and regulatory agen-

cies responsible for monitoring the occurrence of marine

toxins. Although the LOD of DA can be improved by using

different injection volumes in the LC-UV, the matrix com-

plexity may result in some degree of uncertainty about

the peak ’ s identity at low DA levels ( < 3 ng/ml), when a

sensible UV spectrum cannot be obtained (Mafra et al.

2009 ). In this case, confirmation by LC-MS is sometimes

necessary and should also be required whenever DA is

reported in a new geographical area. Some studies on

algal production of DA to track the toxin in seawater, as an

early alert for toxin accumulation in marine organisms,

are available for remote, subsurface detection (Doucette

et al. 2009 ).


Overall, an improvement in techniques based on

LC and/or MS to achieve reliable, simple and low-cost

methods is essential for DA detection in any sample

matrix.

Analysis of TTX TTX, a well-known potent neurotoxin, was first isolated

from puffer fish (Mosher et al. 1965 ) and has also been

found in a wide variety of animals including arthropods,

echinoderms, mollusks, worms, newts or frogs (Miyazawa

and Noguchi 2001 , Noguchi and Arakawa 2008 ). Neither

its biochemical path nor its true origin is fully clarified, as

three hypotheses point to its origin: endogenous (Lehman

et al. 2004 ), through food chain (Lin and Hwang 2001 ,

Kono et al. 2008 ) or through symbionts (Narita et al. 1987 ,

Wang et al. 2008 ).

Although TTX exists mainly in tropical waters

worldwide, this toxin has appeared on European coasts

(Katikou et al. 2009 ). It is possibly due to the migration of

toxic species from the Red Sea to the Mediterranean Sea

through the Suez Canal, a phenomenon known as Lessep-

sian migration (Golani 1998 , Galil and Zenetos 2002 ).

This migration may happen because of the opening of

new corridors allied to the increase in water temperature

as a result of climate change. So, marine species typical

of tropical and subtropical waters are adapted to waters

less warm, and changes in environmental conditions

lead to poisoning incidents in new geographical areas. In

fact, recent intoxication cases caused by the consumption

of TTX bearers have been described in the Israeli coast

(puffer fish: Lagocephalus sceleratus ) and in Spain (gastro-

pod: Charonia lampas ) (Zaki and Mossa 2005 , Bentur et al.

2008 , Rodriguez et al. 2008 ). This last episode was the

first report of TTX occurrence in autochthonous species in

Atlantic and Mediterranean waters. It triggered an investi-

gation monitoring different invertebrate species in several

sites of Portuguese coast, where low amounts of TTXs were

found in two intertidal gastropod species (Silva et al. 2012 ).

As a consequence of the migration of TTX bearers, interna-

tional fisheries agreements in the EU could be modified.

At the moment, the importation of puffer fish and other

toxic species is not permitted in some countries, including

the United States and Europe, and no regulatory limits for

TTX and its analogues have been established yet (Gessner

and McLaughlin 2008 ). With regard to current EU legis-

lation, toxic fish belonging to the family Tetraodontidae

or their products should not be placed on the European

markets (European Commission 2004b ). However, there

are regulations for TTX in countries such as Japan and

Korea, where official control does not regulate the amount

of toxin in fish that can be placed on the market, but res-

taurants that want to serve species containing TTX require

special licenses. EFSA has not published any documents

related to the risks involving TTX, but some authors claim

that the minimum lethal dose in humans is 2 mg, though

it may vary with factors such as age, health and sensitivity

to the toxin (Noguchi and Ebesu 2001 , Cohen et al. 2009 ).

Currently, there is no official method for TTX detection,

but the MBA has been used in many cases to determine

TTX toxicity in food matrices (Kawabata 1978 , Yasumoto

1991 ). However, the MBA is not completely satisfactory,

due to its low sensibility. MBA is not suitable for meas-

uring TTX in biological samples as it cannot distinguish

between TTX and STX or TTX analogues. Other biological

methods have been developed for TTX detection, such as

ELISA, methods that use antibodies specific to the toxin

(Raybould et al. 1992 , Neagu et al. 2006 , Zhou et al. 2007 ),

tests with cultured neuroblastoma, hemolytic assays

(Hamasaki et al. 1996 ) and also techniques using biosen-

sors (Kreuzer et al. 2002 , Neagu et al. 2006 , Yakes et al.

2011 ). Nevertheless, to obtain specific information of a

sample, as the toxic profile or the amount of an individual

toxin, chemical methods have been developed, including

immunoaffinity chromatography (Kawatsu et al. 1999 ),

GC-MS (Man et al. 2010 ), HPLC with FL detection (HPLC-

FL) ( O ’ Leary et al. 2004 , Jen et al. 2007 ) and HPLC-UV

(Yu et al. 2010 ). Although these techniques ensure low

LODs and high sensitivity to detect TTX, they have some

limitations. In the case of immunoaffinity or ELISA, both

methods require a costly monoclonal antibody or reagent

(Leung et al. 2011 ), and the GC-MS requires a complex

extraction procedure (Jen et al. 2008 ). However, HPLC-FL

shows differences in the FL intensities of some TTX ana-

logues compared with TTX itself, which causes problems

in quantifying (Shoji et al. 2001 ). For these reasons, ana-

lytical methods based on LC-MS and LC-MS/MS have been

developed (Jang et al. 2010 , Chen et al. 2011 , Rodr í guez

et al. 2012 ). These LC methods, together with appropri-

ate extraction procedures, have been able to determine

the presence of TTX, not only in the remains of fish and

other animals but also in the blood and urine of poisoned

patients (Tsai et al. 2006 , Rodriguez et al. 2008 ). More-

over, these methods provide better LODs when compared

with HPLC-FL or UV (Jen et al. 2008 ). Most of the LC-MS

instruments are equipped with an atmospheric pressured

ionisation (API) fitted with an ESI source based on colli-

sion-induced dissociation (CID) of a triple quadrupole

mass analyser. For the detection of TTXs, the ESI interface

operates in the positive ionisation mode. So, TTX and its


analogues are analysed by MS, in which positive ionisa-

tion produces a typical molecular ion of [M + H] + for each

one. Because of the continuous emergence of new TTX

analogues, the most recent 8-epi-type TTX analogues

found in newts (Kudo et al. 2012 ), some authors prefer to

operate with the MS in the multiple reaction monitoring

(MRM) mode to quantify the individual toxins. However,

others use the MS operating in the single ion monitoring

mode or the single ion recording mode, such that only a

selected m/z value is detected in the analysis. On the con-

trary, the MRM mode not only allows detecting the precur-

sor molecule but also tracks multiple product ions from

the fragmentation pattern in the same run. In this case,

only one product ion is used in the quantification and the

others to confirm the toxin.

Today, the investigation is focused in developing

LC-MS methods that lead to a good separation and iden-

tification of all TTX analogues in any sample with low

LODs. So, different analytical columns and LC condi-

tions are tested by researchers to improve the detection

of TTX. Table 2 summarises several LC-MS/MS methods

to detect TTX and its analogues. The toxin separation is

typically achieved in reverse-phase columns and with

solvents containing an ion pair reagent (i.e., ammonium

heptafluorobutyrate). But this ion pair sometimes tends

to remain in the MS, causing spectral noise (Shoji et al.

2001 , Rodr í guez et al. 2012 ). Also, because of the pola-

rity of TTXs, some of them are not well retained in these

columns. To solve all these problems, the hydrophilic

interaction liquid chromatography (HILIC) is becoming

more useful in the analysis of TTXs (Chen et al. 2011 ,

Chulanetra et al. 2011 , Cho et al. 2012 , Kudo et al. 2012 ,

Rodr í guez et al. 2012 , Silva et al. 2012 ). The HILIC tech-

nique employs low aqueous/high polar organic solvents

without ion pair reagents, resulting in increased sensitiv-

ity compared with the reverse phase. So, TTX, which is

the more polar compound between its analogues, elutes

with the higher proportion of water in a gradient elution.

Also, problems with noise peaks are reduced in HILIC

columns. Almost all TTX analogues show a common

major product ion, which is usually 162 (m/z) formed

from the molecular ions in MS/MS, suggesting that this

product ion can be used in the quantification. However,

there are analogues such as TTX and 4-epi-TTX that have

the same molecular weight and the same fragmentation

pattern; therefore, a complete separation is needed to

quantify them. As a result, the HILIC separation system

is very useful for the simultaneous quantification of

TTXs by LC-MS/MS in the MRM mode. Figure 2 shows an

example of a chromatogram obtained by LC-ESI-CID-MS/

MS from puffer fish sample. The TTX analogues eluted in

a HILIC column and detected under the MRM mode were

the following: 5,6,11-trideoxy-TTX (m/z 272 > 254/162),

5-deoxy-TTX, and 11-deoxy-TTX (m/z 304 > 286/176),

4,9-anhydro-TTX (m/z 302 > 256/162), 11-nor-TTX-6(R)-ol

and 11-nor-TTX-6(S)-ol (m/z 290 > 272/162),4-epi-TTX and

TTX (m/z 320 > 302/162).

In summary, the LC-MS/MS technology is the most

appropriate and chosen by some laboratories world-

wide for detecting TTXs. One problem is the difficulty in

obtaining purified TTX analogues for calibration. TTX is

frequently used as an internal standard for their quantifi-

cation, and this could lead to errors in the toxin amount.

Nevertheless, because of the timely appearance of TTX in

new geographical areas, monitoring programmes coupled

to a suitable and standardised analytical method should

be developed and for this, certified standards of TTXs

would be necessary.

Detection of lipophilic marine toxins Marine lipophilic toxins are a heterogeneous group of

toxins with different molecular weights and specific

chemical characteristics (Table 3 ). This group includes

yessotoxins (YTXs), AZAs, pectenotoxins, gymnodimines

(GYMs), spirolides (SPXs), CTXs and diarrhetic shellfish

poisoning (DSP). Members of the DSP toxin group are OA

and its derivatives dinophysistoxin-1 (DTX-1), dinophysis-

toxin-2 (DTX-2) and dinophysistoxin-3. To detect them, a

number of analytical methods have been developed in the

last decades, comprising MBAs, in vitro assays and chemi-

cal assays (Botana et al. 2012 ). Each method has its merits

and drawbacks, and ultimately it is the responsibility of

regulators and analysts to decide upon the most appropri-

ate technology for their particular situation. The regula-

tion of lipophilic marine toxins worldwide differs widely.

In Japan, Canada and South America, MBA is the method

used for the official control of these toxins, while in New

Zealand, chromatographic methods have been used for

their monitoring programmes (McNabb 2008 , Gerssen

et al. 2011 ). In Europe, chromatographic techniques

coupled to MS were also the method of choice to replace

MBAs to detect lipophilic marine toxins (Commission Reg-

ulation 2011 ). MBA, which has been used for many years

as an exclusive reference method for lipohilic marine

toxin detection, was replaced by the LC-MS/MS approach

with the new regulation.

To date, more than 200 lipophilic marine toxins

have been described in the literature (Gerssen et al.


Separation Eluents Toxins [M + H] + (m/z) Matrices LODs References

Reverse

phase

30 m m ammonium heptafluorobutyrate in

1 m m ammonium acetate buffer (pH 5.0)

320, 336, 302, 290,

304 and unknown

analogues (348, 330)

Amphibian – (Stobo et al.

2005)

ZIC-HILIC (A) 10 m m ammonium formate and 10 m m

formic acid in water

(B) acetronitrile:water (80:20) in 5 m m

ammonium formate and 2 m m formic acid

320, 302, 304, 272 Puffer fish 0.09 – 0.2

ng/ml

(Villar -Gonzalez

et al. 2008 )

HILIC 16 m m ammonium formate/acetonitrile

(3:7 v/v)

256, 272, 288, 302,

304, 320, 336

Puffer fish 0.9 pmol/

injection

(Wang et al.

2008)

HILIC (A) 0.1 % formic acid/water and (B) 100 %

methanol

320 Blood serum 0.1 ng/ml (Jang et al. 2010)

Reverse

phase

1 % acetonitrile, 10 m m trimethylamine and

10 m m ammonia formate in water

320, 272 Gastropods and human

fluids (blood and urine)

< 10 ng/ml (Gessner and

McLaughlin 2008)

HILIC 16 m m ammonium formate/acetonitrile

(3:7 v/v)

320, 302, 304, 288,

272

Puffer fish < 0.5

nmol/g

(Cho et al. 2012)

ZIC-HILIC (A) 10 m m ammonium formate and 10 m m




320, 302, 304 Puffer fish – (McNabb 2008)

HILIC 10 m m ammonioum formate/acetonitrile

(22:78 v/v)

272, 288, 290, 302,

304, 320, 336

Puffer fish 0.10 ng/ml (Kudo et al. 2012)

HILIC 1 % acetic acid in acetonitrile/1 % acetic

acid in water (88:12 v/v, pH 4.1)

320 Blood 0.32 ng/ml (Gerssen et al.

2011)

Reverse

phase

(A) 1 % acetonitrile, 20 m m

heptafluorobutyric acid, 20 m m ammonium

hydroxide and 10 m m ammonium formate

in water (B) and same mixture (A) with 5 %

acetonitrile

320, 290, 304, 302 Puffer fish – (Rodr í guez et al.

2012)

HILIC (A) 10 m m ammonium formate and 10 m m




320, 290, 304, 302,

272

Puffer fish 16 ng/ml (Rodr í guez et al.

2012)

HILIC Not clear 254, 270, 272, 286,

288, 290, 302, 304,

320

Netws – (Botana et al.

2012)

HILIC (A) 10 m m ammonium formate and 10 m m




320, 272 Gastropods 1.7 ng/ml (Cohen et al.

2009)

Table 2 Summary of several LC-MS/MS methods to detect TTX and its analogues.

HILIC, hydrophilic interaction liquid chromatography; LC-MS/MS, liquid chromatography with tandem mass spectrometry; LODs, limits of

detection; TTX, tetrodotoxin; ZIC-HILIC, Highly polar Zwitterionic hydrophilic interaction liquid chromatography.

2011 ). However, most LC-MS/MS methods are focused

on the analysis of the 13 toxins that are legislated in the

EU (Table 3). GYMs and SPXs are usually included in the

multi-toxin detection methods despite the fact that they

are not legislated in the EU. Hence, LC-MS/MS methods

detect different marine toxin groups in a short period of

time (Stobo et al. 2005 , McNabb 2008 , Gerssen et al. 2011 ),

and they are classified according to the chromatographic

solvents used. Basic or acid mobile phases can be used

with an elution gradient. Table 4 summarises the LC char-

acteristics of two types of chromatography developed

for the monitoring of lipophilic marine toxin. If an acid

chromatography is employed, the mobile phase is com-

posed of water and ACN/water (95:5), both containing

50 m m formic acid and 2 m m ammonium formate (Alfonso

et al. 2008 , Villar -Gonzalez et al. 2008 , Otero et al. 2010 ,

Blay et al. 2011 ). Mobile phases for basic chromatogra-

phy are composed of water and ACN/water (90:10), both

containing 0.05 % ammonia, 2 m m bicarbonate or 6.7 m m

ammonium hydroxide (pH 11) (Gerssen et al. 2009 , 2011).

MRM data are detailed in Table 4. The transition with

the highest intensity is used for quantification, while the


100 TIC of +MRM

Max. 4.5 x 105

1: 5-deoxyTTX2: 11-deoxyTTX3: 11-norTTX-6(R)-ol4: 4,9-anhydroTTX5: 11-norTTX-6(S)-ol6: 4-epiTTX7: TTX

5,6,11-trideoxyTTX

02 4 6 8 10 12 14

Time (min)16

14 6

7

5

3

2

18 20 22 24

Rel

ativ

e in

tens

ity (%

)

Figure 2 Mass chromatogram of the LC-ESI-CID-MS/MS obtained

under MRM operation from the puffer fish sample. Toxins eluted

in an XBridge Amide column (i.d. 2.1 × 150 mm; 3.5 μ m) at 25 ° C,

0.2 ml/min. The LC was operated in gradient with eluent A consisting

of 10 m m ammonium formate and 10 m m formic acid in water. Eluent

B contained 95 % acetonitrile and 5 % water with a final concentration

of 5 m m ammonium formate and 2 m m formic acid. Minutes 16 to

22 represent a broadening of the chromatogram where the toxins

are eluted. CID, collision-induced dissociation; ESI, electrospray

ionisation; i.d., inner diameter; LC, liquid chromatography; MRM,

multiple reaction monitoring; MS, mass spectrometry; TIC, total ion

chromatogram.

Toxin groups Chemical class Main compounds ( M r )

Formula Toxin analogues covered by the EU legislation

Okadaic acid (OA) Polyether, spiro-keto

assembly

OA (804) C 44

H 68

O 13

OA, DTX-1, DTX-2, DTX-3

Azaspiracid (AZA) Polyether, second amine,

3-spiro ring

AZA-1 (841.5) C 47

H 71

NO 12

AZA-1, AZA-2, AZA-3

Petecnotoxin (PTX) Polyeter, ester

macrocycle

PTX-1 (874.5) C 47

H 70

O 14

PTX-1, PTX-2

Yessotoxin (YTX) Ladder-shaped polyether YTX (1141) C 55

H 82

O 21

S 2 YTX, 45-OH-YTX, homo-YTX,

45-homo-YTX

Gymnodimines (GYMs) Macrocycle; cyclic imine GYM (507) C 32

H 45

NO 4 None

Spirolides (SPXs) Macrocycle; cyclic imine SPX-1 (691.5) C 41

H 61

NO 7 None

Table 3 Chemical characteristics of lipophilic marine toxins.

EU, European Union.

transition with the lowest intensity is used for confirma-

tory purposes. With the exception of OA and YTX toxins

groups, which are ionised in the negative mode, the

remaining lipophilic toxins are preferably ionised in the

positive mode. If the instrument is able to simultaneously

operate in positive and negative ionisation modes, the MS

methods include transitions for the toxins that are ionised

in both ionisation modes. If it is not capable of detecting

all toxins in a single injection, compounds are detected in

two separated runs, one with the equipment operating in

the positive mode and the other with the equipment oper-

ating in the negative mode.

This technique has been evaluated, and it is consid-

ered to be successful by many laboratories (Gerssen et al.

2009 , Blay et al. 2011 ). The short, narrow-bore column

packed with 3 μ m Hypersil-BDS-C8 phase and the X-Bridge

C18 are two of the most widely used columns, which are

capable of separating a wide range of toxins by using rapid

gradient (Villar -Gonzalez et al. 2007 , Ciminiello et al. 2010 ,

Gerssen et al. 2011 ). The method with both basic and acid

mobile phases was also adjusted to the new technologies,

UPLC-MS/MS (Fux et al. 2007 , Rundberget et al. 2011 ), and

more than 20 analogues were separated in only 6.6 min

(Fux et al. 2007 ). One advantage of the acid against basic

chromatography is that the first one facilitates good sepa-

ration of acidic OA analogues by suppressing the ionisa-

tion of the carboxyl groups and preventing deleterious ion

exchange interactions with residual silanol groups in the

stationary phase (Suzuki and Quilliam 2011 ). Neverthe-

less, the chromatography of compounds included in the

YTX group can be problematic under acidic conditions

(Gerssen et al. 2009 ). LODs for lipophilic toxins achieved

by the LC-MS/MS approach are low (Table 4), and toxins

can be detected at levels below the current regulatory

limit. For both acid and basic chromatography conditions,

these LODs are lower for toxins ionised in the positive

mode than in the negative one, specially for SPXs (Blay

et al. 2011 , van den Top et al. 2011a ).

Although analytical methods are by far the target of

most of the international efforts for the detection of this

group of compounds, it should be highlighted that enzyme-

based assays are available for high-throughput detection

of these compounds (Vieytes et al. 1997 , Gonz á lez et al.

2002 , Rubiolo et al. 2012 ), and biosensor methods that use

the surface plasmon resonance technology (Llamas et al.


Crom

atog

raph

y un

der a

cid

cond

ition

sCr

omat

ogra

phy

unde

r bas

ic co

nditi

ons

Co

lum

nB

DS

-Hyp

ers

il C

8,

50

mm

×2 m

m,

3 μ

m p

art

icle

siz

eX

-Bri

dg

e C

18

, 1

50

mm

×3 m

m,

5 μ

m p

art

icle

siz

e

Flo

w0

.2 m

l/m

in0

.25

–0

.4 m

l/m

in

Inje

ctio

n v

ol.

5 μ

l5

–1

0 μ

l

Co

lum

n T

25

°C2

5°C

–4

0°C

Mo

bil

e p

ha

se

A:

Wa

ter

(bo

th c

on

tain

ing

2 m

m a

mm

on

ium

fo

rma

te

A:

Wa

ter

(bo

th c

on

tain

ing

6.7

mm

am

mo

niu

m h

ydro

xid

e)

B:

AC

N (

95

%)

an

d 5

0 m

m f

orm

ic a

cid

) B

: A

CN

(9

0%

)(p

H 1

1)

Gra

die

nt

Tim

e (

min

)M

ob

ile

ph

as

e A

(%

)M

ob

ile

ph

as

e B

(%

)Ti

me

(m

in)

Mo

bil

e p

ha

se

A (

%)

Mo

bil

e p

ha

se

B (

%)

07

03

00

–1

90

10

81

09

01

01

09

0

11

10

90

13

10

90

11

.57

03

01

59

01

0

17

70

30

19

90

10

LOD

sO

A:

22

.1 p

g,

YTX

: 6

.1 p

g,

AZ

A-1

: 1

.1 p

g;

PTX

-2:

6.9

pg

; S

PX

-1:

1.9

pg

; G

YM

: 1

4.1

pg

(Ge

rss

en

et

al.

20

09

)

OA

: 9

.1 p

g,

YTX

: 2

.2 p

g,

AZ

A-1

: 1

.1 p

g;

PTX

-2:

7.4

pg

; S

PX

-1:

0.8

pg

; G

YM

: 3

.7 p

g

(Ge

rss

en

et

al.

20

09

)

OA

: 1

0 μ

g/k

g,

AZ

A-1

: 0

.3 μ

g/k

g

(Kil

coyn

e,

20

10

#2

4)

OA

: 5

μg

/kg

, A

ZA

-1:

0.5

μg

/kg

(Kil

coyn

e,

20

10

)

OA

: 1

5 μ

g/k

g,

PTX

-2:

10

μg

/kg

, S

PX

-1:

4 μ

g/k

g

(Vil

lar-

Go

nza

lez

et

al.

20

07

)

OA

: 3

μg

/kg

, D

TX-1

: 5

μg

/kg

; D

TX-2

: 3

μg

/kg

;

AZ

A-1

: 2

μg

/kg

; A

ZA

-2:

2 μ

g/k

g,

AZ

A-3

:

3 μ

g/k

g,

PTX

-2:

2 μ

g/k

g,

YTX

: 1

6 μ

g/k

g

(va

n d

en

To

p e

t a

l.

20

11

)

Tabl

e 4

Ma

in c

ha

ract

eri

sti

cs o

f tw

o t

ype

s o

f li

qu

id c

hro

ma

tog

rap

hy

(LC

) fo

r th

e d

ete

ctio

n o

f li

po

hil

ic m

ari

ne

to

xin

s:

(1)

chro

ma

tog

rap

hy

un

de

r a

cid

ic c

on

dit

ion

s a

nd

(2

) ch

rom

ato

gra

ph

y

un

de

r b

as

ic c

on

dit

ion

s.

AC

N,

ace

ton

itri

le;

AZ

A,

aza

sp

ira

cid

; D

TX,

din

op

hys

isto

xin

; G

YM

, g

ymn

od

imin

e;

OA

, o

ka

da

ic a

cid

; P

TX,

pe

tecn

oto

xin

; S

PX

, s

pir

oli

de

; Y

TX,

yes

so

toxi

n.

aLi

mit

s o

f d

ete

ctio

n (

LOD

s)

of

se

vera

l

lip

op

hil

ic t

oxi

ns

are

als

o i

nd

ica

ted

on

it.

MS

/MS

de

tect

ion

wa

s p

erf

orm

ed

in

th

e m

ult

iple

re

act

ion

mo

nit

ori

ng

(M

RM

) m

od

e b

y u

sin

g t

wo

tra

ns

itio

ns

pe

r to

xin

: O

A a

nd

DTX

-2 (

m/z

80

3.5

/25

5.0

,

m/z

80

3.5

/11

3.0

), D

TX-1

(m

/z 8

17

.5/2

55

.0,

m/z

81

7.5

/11

3.0

), Y

TX (

m/z

11

41

.5/1

06

1.7

, m

/z 1

14

1.5

/85

5.0

), 4

5-O

H-Y

TX (

m/z

11

57

.5/1

07

7.7

, m

/z 1

15

7.5

/87

1.5

), h

om

o-Y

TX

(m/z

11

55

.5/1

07

5.5

, m

/z 1

15

5.5

/86

9.5

), 4

5-O

H-h

om

o-Y

TX (

m/z

11

71

.5/1

09

1.5

, m

/z 1

17

1.5

/86

9.5

), P

TX-1

(m

/z 8

92

.5/8

21

.0,

m/z

89

2.5

/21

3.2

), P

TX-2

(m

/z 8

76

.5/8

23

.4,

m/z

87

6.5

/21

3.2

), A

ZA

-1 (

m/z

84

2.5

/82

4.5

, m

/z 8

42

.5/8

06

.5),

AZ

A-2

(m

/z 8

56

.5/8

38

.5,

m/z

85

6.5

/82

0.5

) a

nd

AZ

A-3

(m

/z 8

28

.5/8

10

.5,

m/z

82

8.5

/79

2.5

).


2007 ), in combination with antibodies that match their

cross-reactivity to the relative toxicity of each analogue

(Stewart et al. 2009a,b ).

In general, methods based on LC-MS/MS are spe-

cific, have sufficient LODs and also have the possibility

for multi-toxin group detection in a single run. However,

one of the major problems found using this approach is

the standard availability. When toxins are not available,

the calibration curve of one toxin standard is often used

to quantify other toxins from the same group, assum-

ing a given ionisation conversion factor. This important

issue was checked by using the DSP toxin group (OA,

DTX-1 and DTX-2) (Otero et al. 2011a ), and no compara-

ble results were obtained. When each one of the three

analogues was quantified by using the calibration

curve of the other ones, the amounts were increased

or decreased; that is, wrong results were obtained. The

highest variability was obtained when DTX-2 was quanti-

fied by using a DTX-1 calibration curve. In this case, the

overestimation was up to 204 % . When DTX-2 was quanti-

fied by using an OA calibration curve, the overestimation

was 88 % . This last quantification type is very usual in

many laboratories as the OA standard is more available

than DTX-2. Therefore, the use of quality standards or

certified reference materials of each one of the marine

toxins is essential for the exact quantification of these

products by MS, and all compounds for which standards

are not available or unknown should not be monitored

in the samples.

Analysis of CTX This toxin group is the only one that does accumulate

in fish of certain species, such as G. javanicus , Lutjanus bohar , Plectropomus leopardus and Epinephelus fuscogut-tatus (Caillaud et al. 2010 ), not in bivalves. CTXs are potent

polyether toxins issued from Gambierdiscus species of

dinoflagellates. These toxins are usually found in areas

between 35 ° N and 35 ° S latitude, mainly Indo-Pacific and

Caribbean areas. However, in recent years, these toxins

are increasingly appearing in countries not expected for

their latitude such as waters close to the European and

African continents (Perez -Arellano et al. 2005 , Bentur and

Spanier 2007 , Otero et al. 2010 ). Its distribution is expected

to shift towards other countries, and in fact this seems to

have begun the trend. For this reason, some laboratories

that did not normally include these compounds in the

toxin analysis are now in need of improving the methods

for detecting them.

Nowadays, the most used chemical method used for

detecting CTXs in shellfish samples is LC-MS/MS. Methods

based on HPLC-UV and HPLC-FL have been also used for

many years, but if they are compared with LC-MS/MS, it

is obvious that both have disadvantages. As CTXs do not

have characteristic chromophore groups in their struc-

tures (like DA), the use of HPLC-UV results in methods

with poor sensitivity and selectivity (Caillaud et al. 2010 ).

Despite this, several authors have used this technique as a

strategy for the isolation and purification of CTXs prior to

their characterisation in fish tissues and extracts of dino-

flagellates (Lewis and Jones 1997; Lewis and Vernoux 1998 ,

Satake et al. 1998 , Hamilton et al. 2002a,b ). However, the

presence of primary hydroxy groups in some CTXs (P-CTX-

1, P-CTX-2, P-CTX-3 and 2,3-dihydroxy-CTX-3C) suggested

that these toxins could be derivatised into fluorescent

esters and thus become suitable for HPLC-FL (Yasumoto

et al. 1995 ). These analyses are performed in a Develosil

ODS-5 column (4.6 × 250 mm) using an isocratic elution

gradient with around 90 % acetonitrile (Yasumoto et al.

1995 ). This method was successfully applied to samples

of Gambierdiscus toxicus and to different carnivorous fish

species, including G. javanicus , Lutjanus bohar , Plectro-pomus leopardus and Epinephelus fuscoguttatus (Caillaud

et al. 2010 ). However, this approach lacks applicability

as many CTX analogues such as P-CTX-3C do not exhibit

this primary hydroxy group, and therefore they cannot be

derivatised.

Later, the application of MS played a critical role in the

structure elucidation of many CTX congeners (Lewis and

Jones 1997 , Hamilton et al. 2002a,b , Pottier et al. 2002a ).

Today, LC-MS/MS technology is widely used by many

laboratories for their detection in contaminated samples.

This technique is not the reference method, as happens in

the case of other lipophilic toxins. Each laboratory uses

LC conditions that are considered more properly and thus,

the bibliography describes different methods of analy-

sis. Prior to the chromatographic analysis of CTXs in fish

samples, strict clean protocols are used (Hamilton et al.

2002a,b , Pottier et al. 2002a , Otero et al. 2010 ). Although

these steps improve the sensitivity and reduce the matrix

effects, it is certain that they do not remove metallic impu-

rities, which result in the formation of adducts (Hamilton

et al. 2002a , Otero et al. 2010 ).

LC systems comprise C18 columns, such as 5- μ m

Phenomenex Luna (Lewis et al. 2009 ) or 3.5- μ m Zorbax

300SB (Hamilton et al. 2002a,b ) and C8 columns such

as 3- μ m Phenomenex Hyperclone (Roeder et al. 2010 )

or 3- μ m Phenomenex Luna (Dechraoui et al. 2005 ).

Mobile phases are composed generally by acetonitrile

and water, and the use of gradients of these organic


solvents buffered with 1 m m ammonium acetate improve

the separation and detection of CTXs compared with

an acetonitrile:water gradient modified with 0.1 % tri-

fluorocetic acid (Lewis and Jones 1997 ). MS detection

is usually performed in the positive MRM mode (Perez -

Arellano et al. 2005 , Roeder et al. 2010 ) or in the Q1 mode

selecting a range of mass (Hamilton et al. 2002a , Pottier

et al. 2002a ). In the last case, the MS spectrum will show

the characteristic pattern of ion formation for CTX poly-

ethers (multiple losses of water and the formation of

sodium and ammonium adducts) (Pottier et al. 2002a ).

Each CTX gives rise to prominent ammonium, potassium

and sodium adducts and losses of waters. An example

of a typical chromatogram and spectrum of a CTX stand-

ard is shown in Figure 3 . The compound is the synthetic

51-hydroxy-CTX-3C with M r 1038.5, and the identification

was performed in a UPLC system coupled to an Xevo TQ

MS mass spectrometer from Waters (Manchester, UK).

The column used was a Waters Acquity UPLC BEH C 18

(100 × 2.1 mm; 1.7 μ m), and the mobile phase consisted of

a gradient of acetonitrile/water. As can be observed, the

ion 1039.5 m/z due to the [M + H] + is not the most promi-

nent. However, the ions m/z 1077.5 [M + K] + and m/z 1061.5

[M + Na] + have a high intensity. In addition, the spectrum

shows two water losses, m/z 1021.5, which corresponds

to [M + H-H 2 O], and m/z 1003.5, which corresponds to

[M + H-H 2 O].

Another consideration to be taken into account in the

analysis of CTXs by LC-MS/MS is the big number of ana-

logues belonging to this toxin group and the few stand-

ards available. To date, more than 50 different congeners

have been identified (Litaker et al. 2010 ), and the pres-

ence of several CTXs with equal molecular weight in the

same extract is very frequent. Up to six different analogues

for the compound 1111.6 m/z (CTX-1B) eluting in different

retention times have been described (Lewis and Jones

1997 ). This issue makes the identification of CTX difficult.

Moreover, because of the lack of certified standards and

reference materials and the limited amounts of contami-

nated material available for method development, the vali-

dation status of LC-MS/MS methods is very restricted and

up to now no collaborative study has been undertaken.

Recently, a method with 14 reference toxins prepared

by either synthesis or isolation from natural sources was

used to identify and quantify 16 toxins from the CTXs

group (Yogi et al. 2011 ). LC separation was performed in

a Zorbax Eclipse Plus C18 column in < 14 min by using

a linear gradient of mobile phases: 5 m m ammonium

formate and 0.1 % formic acid in water and in methanol.

The mass spectrometer operates in the positive mode

for the monitoring of sodium adduct ions [M + Na] + . This

method has greatly reduced the time of analysis as up to

now almost all methods described use run times of around

50 min ( Hamilton et al. 2002a,b, 2009 , Pottier et al. 2002b ,

100A

B 100[M+H - 2H2O]+

1003.5

[M+H - 2H2O]+

1021.5

1077.5 [M+H]+ MS2 ES+1.09e5

[M+H]+

1039.5

[M+Na]+

1061.5

%

%

XEVO-TQMS51-OH-CTX-3C

0

01000 1020 1040 1060 1080 1100 1120 1140 1160

2.00 4.00 6.00

5.801039.5

SIR of 1 channel ES+TIC

6.36e4

8.00 10.00 12.00Time (min)

m/z

Figure 3 Chromatogram (A) and mass spectrum (B) of 51-OH-CTX-3C ( M r 1038.5) standard at 1000 ng/ml, using selected ion recording

(SIR) UPLC mode of 1039.5 m/z [M + H] + . Analysis was performed in an ACQUITY UPLC system coupled with Xevo TQ MS mass spectrometer.

Chromatographic identification was achieved in a Waters Acquity UPLC BEH C 18

column (100 × 2.1 mm; 1.7 μ m). The mobile phase consisted

of two components: acetonitrile/water (95:5) (A) and water (B), both containing 50 m m formic acid and 2 m m ammonium formate using a

gradient elution. 51-OH-CTX-3C, 51-hydroxy-CTX-3C; UPLC, ultraperformance liquid chromatography.


Dechraoui et al. 2005 , Roeder et al. 2010 ). This method pre-

sents limits of 0.25 pg for CTX detection. This means that

it can detect CTX-1B at 45 pg/g in fish flesh. However, an

obstacle to the widespread use and further validation of

this method is the difficulty in obtaining standard toxins.

In summary, the LC-MS/MS methodology has proven

to be extremely useful for identifying characteristic profiles

of CTXs in different strains of Gambierdiscus spp. (Rhodes

et al. 2010 , Roeder et al. 2010 ) and fish (Pottier et al. 2002a ,

Dechraoui et al. 2005 , Otero et al. 2010 ) worldwide. The LC

and MS methods and instruments have greatly improved

in recent years. Because of the high selectivity, accurate

mass spectra are obtained, susceptible to being compared

with spectra of known molecules or standard spectra in

the case they are available. These advances give rise to the

confirmation of many suspected cases of ciguateric fish

by selecting the transitions object study. Techniques such

as the UPLC-MS/MS improve sensitivity; however, at the

time of protecting the human health they are still far from

being used as a routine method in the laboratories for the

detection of CTX.

Conclusions Although there are many choices for the analysis and

detection of marine toxins, the current legal requirement

demands the use of LC-MS for most of the groups, with

the exception of the DA and saxitoxin groups, which can

be detected by HPLC. The chemical complexity of each

toxin group, with new analogues being added every year,

and their different profiles depending on the geographi-

cal area and the climate conditions highlight the impor-

tance of robust analytical methods and a good supply of

analytical standards. Along with this demand, it is very

important to continue with the development of high-

throughput methods that may provide quick results about

the presence of these very toxic compounds in food, as

food safety will always be a concern for industries that

deal with certain mollusks and certain types of fish.

Received June 7, 2012; accepted October 5, 2012; previously

published online November 23, 2012

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Ana M. Botana professor of Analytical Chemistry; 60 international

papers.

Luis M. Botana full professor of Pharmacology at the University

of Santiago de Compostela (USC), expert in marine toxin

pharmacology; 200 international papers, editor of 8 books and

author of 25 patents.

Paula Rodr í guez and Paz Otero Postdoctoral researchers;

20 international papers.

Amparo Alfonso Professor of Pharmacology at the USC;

90 International papers, and 6 patents.

Ana M. Botana , Paz Otero , Paula Rodr í guez , Amparo ...

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