Anal Bioanal Chem (2008) 391:117–134
DOI 10.1007/s00216-007-1778-x
REVIEW
Determination of marine biotoxins relevant for regulations:
from the mouse bioassay to coupled LC-MS methods
Bernd Christian & Bernd Luckas
Received: 18 September 2007 / Revised: 23 November 2007 / Accepted: 27 November 2007 / Published online: 15 December 2007
# Springer-Verlag 2007
Abstract The frequency of occurrence and intensity of
harmful algal blooms (HABs) appear to be increasing on a
global scale. Consequently, methods were established for
the evaluation of possible hazards caused by the enrichment
of algal toxins in the marine food chain. Different clinical
types of algae-related poisoning have attracted scientific
attention: paralytic shellfish poisoning (PSP), diarrhetic
shellfish poisoning (DSP), and amnesic shellfish poisoning
(ASP). In several countries fish specialties are consumed
which may be contaminated with algal toxins typical for the
respective region (e.g., ciguatera and tetrodotoxins). Bio-
assays are common methods for the determination of
marine biotoxins. However, biological tests are not com-
pletely satisfactory, due to the low sensitivity and the
absence of specialized variations. Moreover, there is
growing resistance against the use of animal experiments.
Therefore, many efforts have been made to determine algal
toxins with chemical methods. In this context LC-MS
methods replaced HPLC methods with optical detectors,
allowing both effective seafood control and monitoring of
phytoplankton in terms of the different groups of marine
biotoxins.
Keywords Marine biotoxins . LC-MS/MS . PSP toxins .
DSP toxins . Domoic acid . Tetrodotoxins . Ciguatera
B. Christian (*) : B. Luckas
Institute for Nutrition, Friedrich-Schiller-University of Jena,
Dornburger Str. 25,
07743 Jena, Germany
e-mail: b1chbe@uni-jena.de
Harmful algal blooms (HABs) and marine biotoxins
Anthropogenic contaminants such as polychlorinated
hydrocarbons accumulate in the tissue of warm-blooded
animals. It is less known, however, that natural contami-
nants may also accumulate along food chains. An example
for this phenomenon is the ingestion and storage of algal
toxins in mussels. Mussels filter approximately 20 L h−1
water. During algal blooms water contains several million
algae per liter. Although not all algae produce toxins, it is
plausible that a significant accumulation of toxins will
occur in mussels.
Monitoring of toxins in seafood and risk assessment for
human exposure is the main task of food control.
Consumption of seafood contaminated with marine biotox-
ins may cause serious diseases. Damage to the nervous
system (paralytic shellfish poisoning, PSP), the intestinal
system (diarrhetic shellfish poisoning, DSP), and loss of
memory (amnesic shellfish poisoning, ASP) have been
observed subject to the type of algal bloom. In addition,
regionally specific algal toxins (e.g. ciguatera and tetrodo-
toxins) have been identified in seafood specialties from
several countries.
The worldwide harvesting and transport of marine
organisms, in order to maintain the non-seasonal production
and import of seafood, has caused a potentially serious
threat for the consumer. Therefore, the analysis of marine
food for marine biotoxins is an important task that must be
conducted according to international regulations and in
strict compliance with the respective restrictions.
The residue analyst specialized in the determination of
marine biotoxins by chemical methods has to determine
rapidly the toxins in complex biomatrices with high
118
sensitivity and, most importantly, unambiguously. The
simultaneous realization of those demands was often
accompanied with difficulties. However, the introduction
of LC-MS-based methods during the last decade has
allowed the analysis of algal toxins, especially the
quantitative determination of those relevant for regulations,
to be established in routine analysis.
Occurrence, chemical structure, toxicity, and analysis
of marine biotoxins
Empirical scientific observations of harmful algal blooms
and toxic mussels were reported as early as 1937. At that
time, some algae were described which seemed to be
responsible for the production of toxins and a biological
test, the mouse bioassay, was developed to identify toxins
produced by those algae. Up to now, the mouse bioassay
has been the most important international method for the
detection of algal toxins, and all analytical methods
described below have to be evaluated against bioassays.
However, such biological tests reveal only the total toxicity
of a sample. The limited validity resulted in the need for
physicochemical methods for the detection of each individ-
ual algal toxin. A further important argument against the
mouse bioassay is the growing resistance to the use of
animal experiments [1].
Paralytic shellfish poisoning (PSP) toxins
The main producers of paralytic shellfish poisoning (PSP)
toxins are dinoflagellates of the genus Alexandrium.
Alexandrium occurs along the Atlantic and Pacific coast-
lines where it may grow in great quantities, particularly
during the summer period. Toxins produced by Alexandrium
were named PSP toxins due to the observation that their
consumption caused symptoms of poisoning in warm-
blooded species similar to paralytic phenomena such as
cramp, signs of paralysis, and blocking of respiration. PSP
toxins are potential neurotoxins which specifically block the
Fig. 1 Chemical structure,
toxicity, and molecular weight
of PSP toxins [3]
Anal Bioanal Chem (2008) 391:117–134
excitation current in nerve and muscle cells resulting in signs
of paralysis.
Consequently, the development of analytical methods for
the determination of poisoning caused by PSP toxins was
an important task. The mouse bioassay unambiguously
gives evidence of the toxic potential of a sample, since the
application of higher toxin concentrations yields a shorten-
ing of the time until death of the animals. However, those
biological tests only reveal the total PSP toxicity of a
sample expressed in MU (mouse units) kg−1 or in PSP kg−1.
Thus, the determination of individual PSP toxins was only
possible after isolation and structure elucidation [2].
In 1957, a PSP toxin was isolated from Saxidomus
giganteus (clams) from Alaska, and in 1975 the chemical
structure was assigned to the so-called saxitoxin (STX).
Later on, more PSP toxins were identified which were all
related to saxitoxin (STX) or N-1-hydroxy-saxitoxin (neo-
saxitoxin, NEO). Today, it is convenient to distinguish
between three groups of PSP toxins: carbamoyl toxins,
N-sulfocarbamoyl toxins, and decarbamoyl toxins.
N-Sulfocarbamoyl toxins exhibit only low toxicity,
whereas the toxicity of the carbamoyl and decarbamoyl
toxins is significantly higher. During the manufacturing of
canned seafood contaminated with PSP toxins, the
N-sulfocarbamoyl toxins may be hydrolyzed to the more
toxic carbamoyl or decarbamoyl toxins, thus resulting in an
increase of the total PSP toxicity (Fig. 1).
In 1975, a fluorometric method was recommended for
PSP determination in samples in addition to the mouse
bioassay [4]. PSP toxins which exhibit neither UV
absorption nor fluorescence were oxidized in alkaline
solution to fluorescent purine derivatives. After acidifica-
tion the oxidation products′ intensity of fluorescence was
measured in solution. However, each PSP toxin differs both
in toxicity and fluorescence intensity after oxidation.
Therefore, a chromatographic separation of the PSP toxins
was suggested prior to determination of the fluorescence
activity, since the total PSP toxicity has to be calculated
from the individual toxin concentration and the absolute
toxicities of each PSP toxin. Hence, the unambiguous
Anal Bioanal Chem (2008) 391:117–134 119

assignment of the peaks in the chromatograms to definite
PSP toxins is necessary for a proper quantification of PSP
toxins [3].
A breakthrough in the use of HPLC methods for PSP
determination took place in 1984 when Sullivan et al. [5]
succeeded in the separation of underivatized PSP toxins by
ion-pair chromatography with addition of alkylsulfonic
acids followed by a post-column oxidation with periodic
acid. A disadvantage of that method was the co-elution of
STX and dc-STX resulting in a wrong total PSP toxicity,
since the toxicity of STX is double that of dc-STX.
Therefore, a separate determination of STX/dc-STX is
essential for a comparison of the HPLC results with the
results from the mouse bioassay [6].
Oshima et al. [7] described an entire separation of PSP
toxins. However, that method requires three separate
isocratic runs to analyze most of the PSP toxins and is
slow and labor-intensive. As a consequence, the develop-
ment of a new method allowing complete separation of all
relevant PSP toxins by a single chromatographic run
followed by exact quantification with high sensitivity was
lations using one chromatographic run followed by selec-
tive and sensitive quantification by a mass spectrometric or
fluorimetric detection was required.
This challenge was solved by using a zwitterionic (ZIC)
hydrophilic interaction chromatography (HILIC) column.
In 2007, Diener et al. [12] published the application of a
ZIC-HILIC column (SeQuant, Haltern, Germany) for the
separation of underivatized PSP toxins including a new LC-
MS/MS method which enabled the separation of all three
groups of PSP toxins in a single chromatographic run (Fig. 2).
In order to prove the applicability of this method for
various sample materials, mussel extracts and the marine
dinoflagellate Gymnodinium catenatum were analyzed by
application of the new ZIC-HILIC column in combination
with MS/MS detection (SRM mode). Independent of the
sample matrix, mussels or algae, all present PSP toxins
could be determined (Fig. 3).
Although the limits of detection (LODs) are sometimes
higher with MS detection than with fluorescence detection

GTX 1
necessary.
Recently, an HPLC method for PSP determination was
developed based on ion-pair chromatography with post-
column oxidation and fluorescence detection. The resulting
C 1/ dcGTX 2

B1
GTX 2
chromatograms clearly showed a complete chromatograph-
ic separation of all relevant PSP toxins such as the GTXs

dcSTX
and dc-GTXs [8].
On the other hand, it is recommended that positive

NEO
STX
GTX 4

findings of PSP toxins should be confirmed by application TIC

GTX 3
dcGTX 3
of mass spectrometry. However, eluents containing phos- C2
phate and ion-pair-forming reagents prevented an efficient
application of the LC-MS technique. Therefore, Jaime et al.
[9] proposed the application of ion-exchange chromatogra-
phy with eluents containing only volatile compounds to
determine PSP toxins with both fluorescence and MS
detection. 493/298
Electrospray ionization mass spectrometry (ESI-MS) is
353/255
effective for detection of the polar PSP toxins, which are
quite basic and therefore form stable [M+H]+ ions. Thus, 396/298
the direct detection of underivatized PSP toxins is possible 257/126
using a mass spectrometer as detector [10]. 412/314
300/282
Aversano et al. [11] examined hydrophilic interaction
353/273
liquid chromatography (HILIC) mass spectrometry for the
316/298
analysis of PSP toxins. However, the GTXs were not 493/316
separated completely using a TSK-gel Amide-80 column,
e.g., GTX-1 co-elutes with GTX-2. For that reason the 396/316
380/300
method can be applied only in combination with MS, but
412/332
lacks in the combination with a fluorescence detector.
Furthermore, inconstant retention times for PSP toxins in
different seafood matrices were observed. As a conse- 010 20 30 40
quence, the development of a new method allowing a Time (min)
complete separation of all PSP toxins relevant for regu- Fig. 2 HILIC-MS/MS analysis of a PSP standard mixture [12]
120
dcGTX 2
C1/
dcGTX 3
GTX 2
dcSTX
GTX 3
C2
TIC
493/298
396/298
353/255
493/316
353/273
257/126
396/316
010 20 30 40
Time (min)
Fig. 3 HILIC-MS/MS analysis of a Gymnodinium catenatum extract
(0.03 M HAc), Baja California, Mexico [12]
the sensitivity of the MS detection is sufficient to control
seafood at the regulatory limit for a PSP toxin content at
800 μg STX equivalents kg−1 wet weight of soft tissues.
Diarrhetic shellfish poisoning (DSP) toxins
Since the first reports of diarrheic shellfish poisoning (DSP)
in Japan, 1978, the illness is now recognized as a threat to
public health throughout the world. All DSP toxins are
relatively nonpolar, with molecular weights higher than
500, and easily extractable by organic solvents such as
methanol and chloroform. Most DSP toxins are polyether
compounds with distinctive chemical structures and widely
varying functional groups resulting in different toxicolog-
ical and chemical characteristics [13].
Marine biotoxins in shellfish have traditionally been
categorized and regulated according to the poisoning syn-
dromes that they evoke in human beings or animals. For
example, okadaic acid (OA) and dinophysistoxins (DTXs)
have been classified as diarrhetic shellfish poisoning (DSP)
toxins due to their production of symptoms of diarrhea.
Unfortunately, when pectenotoxins (PTXs) and yessotoxins
(YTXs) were discovered and before their toxicity was
Anal Bioanal Chem (2008) 391:117–134
understood, they were placed into the DSP category because
they often occur together with OA and DTXs and were
detected by the mouse bioassay procedure first used generally
to detect DSP toxins in lipophilic extracts.
With the continuing discovery of a series of new
lipophilic toxins, such as azaspiracids (AZAs), gymnodi-
mines, and spirolides, it became clear that a better approach
would be to categorize the toxins strictly according to their
chemical classes rather than their toxic symptoms. This
would allow seafood safety to be regulated according to
allowable levels of specific toxins rather than to the result
of a specific assay. This is the path on which all other
contaminants in food, such as mycotoxins and pesticides,
are controlled [14].
Okadaic acid and dinophysistoxins (OA and DTXs)
Dinophysistoxin-1 (DTX-1) was identified as the causative
toxin for incidents of diarrhetic shellfish poisoning in Japan,
and it originates from the dinoflagellate Dinophysis fortii.
Although DTX-1 is found in Europe to a limited extent, it
was identified as the major toxin in shellfish from Norway.
Most studies have implicated okadaic acid (OA) as the
responsible toxin arising from a variety of Dinophysis
species. DTX-2, an isomer of OA, was isolated from Irish
mussels. In addition, DTX-3 has been observed in plankton
and shellfish (Fig. 4).
The term DTX-3 was originally coined to describe a
group of compounds in which saturated or unsaturated
C14–C18 fatty acid moieties are attached through the 7-
hydroxyl group of DTX-1. Subsequently, it was shown that
any of the parent toxins, OA, DTX-1, and DTX-2, can be
acylated and are shellfish metabolites [15].
All lipophilic toxins can be extracted from plankton or
shellfish tissues by organic solvents such as acetone,
methanol, and acetonitrile. The use of acetone for extraction
is required if the mouse bioassay is applied for the
determination of OA, DTXs, PTXs, and YTXs [16].
However, 80% methanol in water is recommended as an
effective extraction solvent for application of HPLC
methods with fluorescence detection or the LC-MS/MS
technique [17]. After addition of more water to reduce the
percentage of methanol to approximately 50%, toxins can
be partitioned between chloroform or dichloromethane
which are then evaporated to dryness and the residue can
be redissolved in 0.5–1.0 mL methanol.
In addition, the different ester derivatives, e.g., DTX-3,
can be hydrolyzed quantitatively to their parent toxins by
treatment of the aqueous methanol extract with 0.25 M
NaOH [18]. This procedure enables the unambiguous
determination of both the DSP content of a sample material
as OA equivalents and the percentage of DTX-3, indepen-
dent of the method used for analysis [19].
Anal Bioanal Chem (2008) 391:117–134 121

Fig. 4 Chemical structures of Me

the DSP toxins OA and DTXs
O
OH
O R1
HO O
O O O
Me OH
OR3 Me
H
O H O
OH Me R2

okadaic acid (OA) R1 = CH3 R2 = H R3 = H

dinophysistoxin-1 (DTX-1) R1 = CH3 R2 = CH3 R3 = H

dinophysistoxin-2 (DTX-2) R1 = H R2 = CH3 R3 = H

dinophysistoxin-3 (DTX-3) R1, R2 = H or CH3 R3 = acyl

In this context it is important that the DSP profile of
mussels can consist of a higher percentage of OA esters and
their analogs than of other DSP toxins (see Fig. 5).
Pectenotoxins (PTXs)
The pectenotoxins (PTXs), named after the genus of scallop
from which they were first isolated, Patinopecten yessoensis,
are a group of DSP toxins originating from Dinophysis
species throughout the world (Fig. 6).
PTX-2 is the main toxin in this group. When accumu-
lated in shellfish, the methyl group at position C-43 is
oxidized to the corresponding alcohol (PTX-1), aldehyde
(PTX-3), and carboxylate (PTX-6) forms. The spiroketal
system in rings A and B can also undergo rearrangement
and/or epimerization under acidic conditions to produce
PTX-4 and PTX-7 through PTX-9. The lactone ring in
PTX-2 may be opened to yield pectenotoxin seco acid
(PTX-2sa), epimerization yields 7-epi-PTX-2sa [20].
PTXs appear to be highly toxic by intraperitoneal
injection, leading to positive responses in the mouse
bioassay for lipophilic marine biotoxins. Because of that,
the maximum level of PTXs (PTX-1 and PTX-2) permitted
in European shellfish has been set at 160 μg kg−1 OA
equivalents [16, 21]. However, PTXs appear to be of low
toxicity orally and, unlike OA, they do not cause diarrhoea
[22, 23].
All pectenotoxins absorb between 235 nm and 239 nm
(UV). Detailed stability studies have not been performed,
but it is known that rearrangements can occur under acidic
conditions [24]. In addition, PTXs are easily destroyed
under strongly basic conditions. This fact has to be

Fig. 5 SIM chromatograms of edible parts of razor clams: a before NaOH hydrolysis, b after hydrolysis; OA=0.9 and 50.1 μg OA/100 g,
respectively [18]
122
Fig. 6 Chemical structures of
PTX homologs
OH
Me
Pectenotoxin-1
Pectenotoxin-2
Pectenotoxin-3
Pectenotoxin-6
Pectenotoxin-2 seco acid
considered in analyses of samples containing PTXs and,
consequently, sample preparation has to be performed at a
pH value near 7.
On the other hand, comparison of the PTX profiles of the
toxic dinoflagellate Dinophysis acuta, of Greenshell mussels
(Perna canalicus), and Blue mussels (Mytilus galloprovin-
cialis) from New Zealand by application of an LC-MS
technique revealed that the major PTX homolog in D. acuta
was PTX-2, whereas both Greenshell and Blue mussels
contained PTX-2sa as the predominant toxin. More than
90% of PTX-2 isolated from D. acuta was rapidly converted
to PTX-2sa and its epimer 7-epi-PTX-2sa in the Greenshell
mussel extract (Fig. 7). The conversion from PTX-2 to PTX-
2sa and 7-epi-PTX-2sa was not observed in phosphate
buffers at various pH ranging from 4.1 to 9.1. These findings
indicate that PTX-2sa and 7-epi-PTX-2sa are not artifact
toxins resulting from hydrolysis of PTX-2, but they arise
from the conversion of PTX-2 by mussel tissues [25, 26].
Yessotoxins (YTXs)
Yessotoxins (YTXs) are sulfated polyethers (Fig. 8) pro-
duced by the dinoflagellates Protoceratium reticulatum and
Gonyaulax polyedrum in many parts of the world [27–30].
YTXs are toxic by intraperitoneal injection in mice and
therefore give an increase to positive results in the
traditional mouse bioassay for DSP toxins [31].
A number of oxidized YTXs, apparently arising from
metabolism of YTX and 1-homo-YTX present in ingested
algae, were subsequently isolated from shellfish by mouse
bioassay guided fractionation [32].
Anal Bioanal Chem (2008) 391:117–134
Me
O
O O Me
1
O O
O O
33 OH
OH Me
O O
O 43
R
O Me Me
R C1-C33
CH2OH -O-
CH3 -O-
CHO -O-
COOH -O-
CH3 -OH HO-
Antibodies to yessotoxin (YTX) have been produced and
used to develop an ELISA. Its application for analyses of
algal samples gave results two to three times higher than
with LC-MS for known YTX, and for shellfish the results
by ELISA were three to nine times higher than the results
obtained from LC-MS. Those results suggested that algae
could produce a series of YTXs which were recognized by
the antibody and that those analogs had been metabolized
in shellfish [33].
In spite of recent evidence that YTXs may have no
significantly acute toxicity when administered orally to
mice [30], the actual quarantine level of YTX, 45-
hydroxyYTX, 1-homo-YTX, and 45-hydroxy-1-homoYTX
in shellfish is 1 mg kg−1 in the European Union [16, 21].
Recently, a number of LC-MS methods have been
demonstrated as powerful techniques for the quantification of
YTXs analogs and structure elucidation of novel toxins in
plankton and shellfish [34–37]. Figure 9 shows the determi-
nation of YTXs in mussel tissue obtained using LC-MS3 [37].
Azaspiracid poisoning (AZP) toxins
A shellfish poisoning event occurred in the Netherlands in
1995 when at least eight severe gastrointestinal illnesses in
humans were reported after the consumption of mussels
(Mytilus edulis). These shellfish were traced to Killary
Harbor, Ireland, where subsequent investigations revealed
that a number of local intoxications had also occurred.
Unfortunately, this toxicity was not covered within the DSP
toxin-monitoring program in Ireland, which involved
biweekly sampling in this region and utilized a recently
Anal Bioanal Chem (2008) 391:117–134 123

Fig. 7 LC-MS chromatogram

of PTX homologs obtained
from Greenshell mussels col-
lected from Wedge Point, Queen
Charlotte Sound, New Zealand
in 1998 [24]: a TIC obtained by
negative mode with full-scan
monitoring (m/z 850–910), b
extracted ion mass chromato-
gram (m/z 857–858) (PTX-2), c
extracted ion mass chromato-
gram (m/z 875–876) (PTX-2sa
and 7-epi-PTX-2sa)

modified rat bioassay. In subsequent chemical analyses of
those shellfish, only low levels of OA and DTX-2 were
detected. In the DSP mouse test, a slow progressive
paralysis was observed using extracts of mussels, and these
neurotoxic symptoms were quite different from those
typical for DSP toxins. In addition, the extracts were
subjected ESI-MS and 1H NMR analysis. The skeletal
structures of a toxin group named azaspiracids (AZAs)
were elucidated [38]. It was apparent that they contained
novel spiro-linked five- and six-membered rings, with one
of those containing nitrogen (Fig. 10).
In toxicity studies using azaspiracids, mice became
progressively paralyzed with labored breathing. Diarrhea
was not observed and, at low doses, mice died within 2–
3 days. Histopathological changes were observed in mice
which were induced by both intraperitoneal and oral
administration of azaspiracids. Subsequently, azaspiracid
analogs have been isolated from Irish mussels, of which
AZA-1, AZA-2, and AZA-3 seem to be the main
compounds responsible for contamination. Methylazaspir-
acid (AZA-2) and desmethylazaspirazid (AZA-3) occur in
shellfish at lower concentrations than azaspiracid (AZA-1),
but they are more toxic in the mouse bioassay [39]. The
chemical structure of AZA-4 and AZA-5 suggests that they
are oxidized metabolites of AZA-3 [40, 41].
Although AZAs were first identified in mussels that
were cultivated in Ireland, a widespread European distri-
bution of these toxins has recently been confirmed [42, 43],
and the EU regulatory limit for the combined AZA-1,
AZA-2, and AZA-3 content has been set at 0.16 μg g−1
total shellfish tissue [16, 21].
Most regulatory testing only examines the digestive
glands (DG). That practice was a consequence of an early
report mentioning that DSP toxins are concentrated in these
124 Anal Bioanal Chem (2008) 391:117–134

Fig. 8 Chemical structures of R1

selected yessotoxins shown as
their sulfonic acid forms HO
Me
H
O

O
Me
O
OH

O
O
Me
Me
HO3SO O O O
n
O Me
HO3SO O O Me

Toxin n m/z R1

YTX 1 1141

1-homo-YTX 2 1155

45-hydroxyYTX 1 1157
OH

45-hydroxy-1-homoYTX 2 1171
OH

45,46,47-trinorYTX 1 1101
H

45,46,47-trinor-1-homoYTX 2 1115
H

tissues. However, it was demonstrated recently that AZAs
can migrate to other tissues and this fact was a contributory
factor in false negative bioassay results [44, 45].
The determination of various AZAs in shellfish relies
upon the usage of LC-MS methods. LC-MS methods have
been developed for the analysis of AZAs in shellfish using
an ion-trap mass spectrometer [46, 47]. It was found that the
ubiquitous “benign” marine dinoflagellate Protoperidinium
is the progenitor of azaspiracides [44].
Figure 11 shows the LC-MS3 analysis of an extract of the
hepatopancreas (HP) of mussels (Mytilus edulis) containing
the azaspiracides AZA-1, AZA-2, and AZA-3 [39].
Amnesic shellfish poisoning (ASP) toxins
Quilliam and Wright [48] reported on big efforts to
elucidate the cause of the so-called Prince Edward Island
Disease in 1987 in which more than 100 people were
poisoned after consumption of mussels harvested on this
eastern Canadian island. Three people died and the
survivors suffered memory loss. The underlying toxin was
therefore named amnesic shellfish poisoning (ASP) toxin.
During the isolation of the ASP toxin from mussels,
several toxic and non-toxic mussel extracts were investi-
gated. The respective HPLC chromatograms showed
Anal Bioanal Chem (2008) 391:117–134 125

algae and mussels at concentrations of 1.0 mg DA kg−1 by

application of an automated HPLC system with column-
switching system and UV detection. The mass spectrometric
determination is another sensitive and selective method for
domoic acid analysis [53]. However, a thorough purification
of the sample extracts is necessary for an unambiguous
determination of DA [54].
Both LC-UV and LC-MS methods were applied for the
determination of DA concerning EC legislation [55]. Figure 12
shows an ESI mass spectrum (positive mode) of DA [56].

Tetrodotoxins (TTXs)

Puffer fish (Takifugu spp.) is a delicacy in Japan and some

other Asiatic countries. Unfortunately, every now and then
serious poisonings have been reported after consumption of
that fish species [57]. Consequently, a so-called tetrodotox-
in (TTX) was isolated from puffer fish followed by
structure elucidation [58–62]. Later on, it was discovered
that TTX and its analogs (Fig. 13) also occur in other
marine organisms and some terrestrial vertebrates [63, 64].
TTX and its derivatives are neither produced by these
fish species nor by algae but from bacteria [66–68].
However, several serious intoxications after consumption

R2
8
O R1
A H
B O H
Fig. 9 Determination of YTX (0.20 μg g−1) and 45-OH-YTX HO
1 3 10

(0.11 μg g−1) in mussel tissue using LC-MS3 [37] O O D C 20 OH

14
H
H3C O R3
39 NH H HO
H3C O E
22
I H
characteristic differences compared with non-toxic frac- H
23
R4
tions. On the basis of the UV spectrum the corresponding H O
37 26 24
O G
compound was identified as domoic acid. The amino acid H3C O CH3
H
domoic acid (DA) has an effect on the nervous system and F
acts as a glutamic acid agonist, which explains the amnesia 30

since glutamic acid plays an important role for the storage CH3
of information [49]. In the case of the DA, it was surprising
that a diatom (Pseudonitzschia spp.) widely spread along R1 R2 R3 R4 MW
the Canadian coast and in European waters was the
producer of DA. Therefore, mussels harvested in Europe
may be also contaminated with DA [50].
Azaspirazid (AZA-1) H H CH3 H 841.5
Domoic acid became a subject of interest for food control
laboratories after introduction of a limit of 20 mg DA kg−1 Azaspirazid-2 (AZA-2) H CH3 CH3 H 855.5
flesh of mussels [21]. HPLC separation of the underivatized
domoic acid on reversed-phase (RP) columns followed by Azaspirazid-3 (AZA-3) H H H H 827.5
UV detection at 242 nm was suggested. In addition, several
Azaspirazid-4 (AZA-4) OH H H H 843.5
methods involving MS have also been used for the
determination of DA and its analogs in shellfish, following Azaspirazid-5 (AZA-5) H H H OH 843.5
the solid-phase extraction (SPE) cleanup of extracts [51]. Fig. 10 Chemical structures and molecular weights of various
Hummert et al. [52] succeeded in the determination of DA in azaspiracid toxins
126 Anal Bioanal Chem (2008) 391:117–134

Fig. 11 Chromatogram obtained

from the LC-MS3 analysis of
mussels from Sognefjord:
a m/z 828.5→810.5→792.5;
b m/z 842.5→824.5→806.5;
c m/z 856.5→838.5→820.5
corresponding to AZA-1,
AZA-2, and AZA-3,
respectively [39]

of puffer fish and further species led to the result that
analysis of tetrodotoxins is a task similar to the determina-
tion of algal toxins [69]. The toxic effect of tetrodotoxins is
based on blocking of the sodium channels in nerve cells
and, consequently, the mouse bioassay is suitable for
determination of those potential neurotoxins [70].
Moreover, it was discovered that puffer fish may contain
both TTXs and PSP toxins [71–73]. Therefore, specific
methods were required for the determination of TTXs and
PSP toxins and, consequently, the application of chromato-
graphic methods was suggested [74].
The detection of fugu toxin was a major problem since
TTXs neither show UV absorption nor fluorescence activity
[75]. Therefore, the HPLC system was equipped with a
reaction unit for post-column derivatization, enabling a
selective and sensitive determination of TTX and its deriva-
tives. After chromatographic separation TTX is converted into
a fluorescent derivative by addition of NaOH [76]. Further-

Fig. 12 Positive ion ESI mass

spectrum of domoic acid,
MW 311.3 [56]
Anal Bioanal Chem (2008) 391:117–134 127

Fig. 13 Chemical structures of OH

TTXs [65] R1 R2 R3 R4 [M+H]+
O O
TTX H OH OH CH2OH 320
HO R4 4-epiTTX OH H OH CH2OH 320
R1
6-epiTTX H OH CH2OH OH 320
OH 11-deoxyTTX H OH OH CH3 304
R2
R 3 norTTX-6(S)-ol H OH OH H 290
HN NH
norTTX-6(R)-ol H OH H OH 290
+
H2N
norTTX-6, 6-diol H OH OH OH 306
OH

O O R1 R2 [M+H]+
R2
O anhydroTTX OH CH2OH 302
6-epianhydroTTX CH2OH OH 302
OH
H 1
NH R
HN
+
H2N

R1 R2 [M+H]+
H O
HO 2
H
R 5-deoxyTTX OH CH2OH 304
trideoxyTTX H CH3 272
OH
HO
NH R1
HN
+
H2N

more, it is possible to use such an HPLC system for the
simultaneous determination of TTX and PSP toxins [77].
Since ESI-MS was introduced for analysis of marine
biotoxins [78], this technique also proved to be a very
effective tool in the analysis of TTXs. Reliable methods
using LC-ESI-MS for the determination of tetrodotoxin in
puffer fish have been developed, too [61, 79].
TTXs are generally extracted with an aqueous or
methanolic solution of acetic acid because TTXs are stable
in weakly acidic aqueous conditions and under heating.
TTX is present at higher concentrations than its derivatives
in samples of puffer fish and newt, such that 5-deoxyTTX,
5,6,11-trideoxyTTX, and 4,9-anhydroTTX are considered
to be almost non-toxic analogs [80].
Recently, the HILIC column TSKgel Amide-80 was
applied successfully for ESI-LC-MS determination of
TTXs. It was found that in eggs of the puffer fish Fugu
poecilonotus the concentration of 5,6,11-trideoxyTTX was
higher than that of TTX, whereas that analog was not
detected in the skin of the newt Cynops ensicauda [81].
Diener et al. [65] used a ZIC-HILIC column for the
determination of TTXs in separated tissues of Bangladeshi
marine puffer, Takifugu oblongus (Fig. 14). TTX was
predominant in skin, muscle, and liver, whereas 5,6,11-
trideoxyTTX was preponderant in the ovary.
In the Annex III, Section VII, Chapter V of the Regulation
(EC) No 853/2004 of the European Parliament and of the
Council of 29 April 2004 [21], health standards for live
bivalve mollusks concerning the marine biotoxins were
established. This regulation also contains (in Section VIII,
Chapter V) health standards for fishery products in which the
following specific requirements are defined with regard to
toxins harmful to human health: fishery products derived from
poisonous fish of the families Tetraodontidae, Molidae,
Diodontidae, and Canthigasteridae as well as fishery products
containing biotoxins such as ciguatera or muscle-paralyzing
toxins must not to be brought onto the market [82].
Ciguatera (CTXs)
The term ciguatera fish poisoning was first used in the
Caribbean to describe an intoxication induced by ingestion
of a marine snail, Turbo pica (called cigua by the Cuban
natives). Now the world is used this term to describe the
intoxication caused by consumption of certain fish, primar-
ily reef fish from the tropical and subtropical areas of the
Caribbean Sea and the Pacific Ocean, which have accumu-
lated specific toxins (CTXs) via their diet.
The more than 175 ciguateric symptoms that have been
reported can be classified into four categories: gastrointes-
128
Fig. 14 HILIC-MS analysis (SIM mode) of extracts of T. oblongus:
a skin, b ovary [65]
tinal, neurological, cardiovascular, and general [83]. This
multiphase intoxication is thought to be due to the presence
of different ciguatera-related compounds at different ratios
[84, 85].
Although Scheuer et al. [86] isolated ciguatera in 1967,
the structure was not elucidated until 1989 [87]. Further
ciguatoxins (CTXs) were later discovered in algae [88] and
fish [89, 90].
CTXs are cyclic polyethers with similar structures to DSP
toxins and brevetoxins [91]. Therefore, a fast and unambig-
uous determination of ciguatoxins with simple chemical tests
is difficult [83].
Anal Bioanal Chem (2008) 391:117–134
The mouse bioassay was applied first to determine a
contamination of fish with ciguatera [92]. Later, bioassays
with chicken [93] and mosquitos [94] and an enzyme
immunoassay, the so-called stick test, were applied for
detection of ciguatera [95].
Despite the further development of immunological and
enzymatic methods, doubtful or false positive results were
obtained during the control of tropical fish on ciguatera. It
was the introduction of a solid-phase immunobead assay
(S-PIA) which, for the first time, resulted in ciguatera
values correlating with data obtained with other detection
methods [96]. On the basis of this immunoassay a test kit
named Ciguatect™ was introduced for the determination of
ciguatoxins. However, Ciguatect™ can only be used for the
determination of ciguatoxins in the absence of DSP toxins
since okadaic acid, for example, gives also positive reaction
with the kit. Therefore, application of Ciguatect™ has to be
accompanied with a second detection method for DSP
toxins [97]. This is of great importance, not only with
respect to the aforementioned food control regulation [82],
but also with concern to the Regulation (EC) No. 854/2004
of the European Parliament and the Council which
stipulates (in chapter II) that checks have to be done to
ensure that fishery products containing biotoxins such as
ciguatera are not placed on the market [98].
Intensive research was ongoing to develop LC-MS/MS-
based methods for the determination of CTXs to overcome the
lack of suitable physicochemical methods [99, 100]. It was
evident that differences exist between ciguatoxins from the
Pacific (P-CTXs) produced by certain strains of the dinofla-
gellate Gambierdiscus toxicus as well as from the Caribbean
(C-CTXs) and the Indian Ocean (I-CTXs) [101–103].
Figure 15 shows the chemical structures of P-CTXs and C-
CTXs, and Fig. 16 clearly demonstrates the formation of ions
characteristic for CTXs. Interestingly, the retention time and
masses for I-CTX and C-CTX-1 were virtually indistinguish-
able under the conditions applied for the ESI-LC-MS
measurements [104].
Application of LC-MS/MS methods for determination
of assorted marine biotoxins in compliance
with legislation
Due to the cases of human intoxication frequently observed
after mussel consumption, governmental institutions and
the fishery industry have established control methods to
ensure that seafood contaminated by biotoxins does not
reach the consumer [105]. The first step was to establish
monitoring programs for harmful algae blooms (HABs)
followed by restrictions concerning the harvest of mussels
in areas with an unacceptable amount of toxin-producing
algae per liter [106, 107]. Furthermore, efforts were made
Anal Bioanal Chem (2008) 391:117–134 129

Fig. 15 Structures of Pacific

ciguatoxin-1 (P-CTX-1) and
Caribbean ciguatoxin-1
(C-CTX-1) [104]

to harmonize legal regulations and monitoring methods, to both mouse bioassay and an LC-MS-based method to
provide a common basis for risk assessment [108], whereby determine the lipophilic toxins associated with diarrhetic
in 1958 USA and Canada were the first countries in the shellfish poisoning in Japanese bivalves demonstrated the
world to establish the mouse bioassay and residue limits for good comparability of the results obtained with the mouse
PSP toxins with 400 MU and 80 μg PSP/100 g mussel bioassay and LC-MS [118].
tissue, respectively [109, 110].
The European Union established reference laboratories to
monitor the shellfish production in the community [111].
Moreover, specific rules for official controls concerning live
bivalve molluscs from classified production areas were
established. Classified relaying and production areas have to
be periodically monitored to check the presence of toxin-
producing plankton. The sampling frequency for toxin analysis
in the molluscs is, as a general rule, to be weekly during the
periods at which harvesting is allowed. If any changes in toxin
populations that may lead to toxin accumulation are detected,
the sampling frequency of molluscs is to be increased or
precautionary closures of the areas are to be established until
the results of toxin analysis are obtained [112].
To fulfill the tasks concerning the monitoring of
phytoplankton in classified production areas, LC-MS-based
methods were developed for simultaneous determination of
various algal and cyanobacterial toxins extracted from
phytoplankton [113, 114], and, during recent years, espe-
cially mussels from different marine regions have to be
controlled in compliance with the newsworthy legislation
[115, 116]. The latter was focused on ASP and DSP toxins
which should be determined simultaneously by application
of a LC-MS/MS multiresidue method (Figs. 17 and 18).
A newly developed LC-MS/MS method allowing the
determination of various marine biotoxins in shellfish was
subjected to a full single-laboratory validation and a limited
interlaboratory study. The single-laboratory validation
proved that the LC-MS/MS method is suitable for routine
determination of ASP, DSP, and other lipophilic algal toxins Fig. 16 Mass spectra showing the different ratio of pseudomolecular
in shellfish with high specificity, good precision/accuracy, ([M+Na]+, [M+NH4]+, [M+H]+) and product ([M+H-nH2O]+) ions
and low detection limits [117]. Furthermore, a study using within the fragmentation patterns of a I-CTX and b C-CTX-1 [104]
130 Anal Bioanal Chem (2008) 391:117–134

Fig. 17 Reversed-phase gradi-

ent elution LC-MS analysis of a
range of toxins in a blend of
contaminated mussel tissue
extracts. Selected ion monitor-
ing was carried out on either
[M+H]+ or [M+NH4]+ ions,
which are displayed as individual
mass chromatograms. The toxins
present include domoic acid
(DA), spirolides (SpiroB/D),
okadaic acid (OA), dinophysis-
toxins (DTX1/2), pectenotoxins
(PTX2 and PTX2sa), azapsirac-
ids (AZA1, AZA2 and AZA3),
and acyl esters of OA and DTX2
(DTX3) [10]

Conclusions and enumeration of hazardous algal species in growing waters

and the testing of flesh from shellfish samples [117].
The safety of shellfish as food can be compromised by There is currently a high degree of dependence on
contamination with marine biotoxins. This risk is managed by mouse-based bioassays but there is growing acceptance for
monitoring programs that generally combine the identification the need to develop and implement non-animal-based

Fig. 18 LC-MS/MS multiple reaction monitoring chromatogram with positive and negative ESI. Greenshell mussel flesh from site J02, collected
7 February 2001. Each trace is normalized to the largest peak [115]
Anal Bioanal Chem (2008) 391:117–134
methods. Recent advances in analytical instrumentation
have enabled the development of alternative methods such
as LC-MS [119].
Comparison of the quantitative results obtained for
bivalve samples with the mouse bioassay and LC-MS
indicates that LC-MS is suitable for routine monitoring of
marine biotoxins and therefore this technology is becoming
the method of choice for the detection and quantification of
several marine biotoxins [120].
Moreover, further development and validation of analyt-
ical methodology from algal toxins is highly desirable,
because the enforcement of biotoxin legislation is ultimately
based on the ability of analysts to identify and quantify those
toxins accurately in seafood products.
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