Ana M. Botana , Paz Otero , Paula Rodr í guez , Amparo ...
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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: Luis.Botana@usc.es
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
18 A.M. Botana et al.: Analysis of marine toxins
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.M. Botana et al.: Analysis of marine toxins 19
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
20 A.M. Botana et al.: Analysis of marine toxins
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 ).
A.M. Botana et al.: Analysis of marine toxins 21
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
22 A.M. Botana et al.: Analysis of marine toxins
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.
A.M. Botana et al.: Analysis of marine toxins 23
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
formic acid in water
(B) acetronitrile:water (80:20) in 5 m m
ammonium formate and 2 m m formic acid
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
formic acid in water
(B) acetronitrile:water (95:5) in 5 m m
ammonium formate and 2 m m formic acid
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
formic acid in water
(B) acetronitrile:water (95:5) in 5 m m
ammonium formate and 2 m m formic acid
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
24 A.M. Botana et al.: Analysis of marine toxins
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.
A.M. Botana et al.: Analysis of marine toxins 25
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
).
26 A.M. Botana et al.: Analysis of marine toxins
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
A.M. Botana et al.: Analysis of marine toxins 27
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.
28 A.M. Botana et al.: Analysis of marine toxins
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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34 A.M. Botana et al.: Analysis of marine toxins
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.
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