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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) and marine biotoxins
harmful algal blooms (HABs) appear to be increasing on a
global scale. Consequently, methods were established for Anthropogenic contaminants such as polychlorinated
the evaluation of possible hazards caused by the enrichment hydrocarbons accumulate in the tissue of warm-blooded
of algal toxins in the marine food chain. Different clinical animals. It is less known, however, that natural contami-
types of algae-related poisoning have attracted scientific nants may also accumulate along food chains. An example
attention: paralytic shellfish poisoning (PSP), diarrhetic for this phenomenon is the ingestion and storage of algal
shellfish poisoning (DSP), and amnesic shellfish poisoning toxins in mussels. Mussels filter approximately 20 L h−1
(ASP). In several countries fish specialties are consumed water. During algal blooms water contains several million
which may be contaminated with algal toxins typical for the algae per liter. Although not all algae produce toxins, it is
respective region (e.g., ciguatera and tetrodotoxins). Bio- plausible that a significant accumulation of toxins will
assays are common methods for the determination of occur in mussels.
marine biotoxins. However, biological tests are not com- Monitoring of toxins in seafood and risk assessment for
pletely satisfactory, due to the low sensitivity and the human exposure is the main task of food control.
absence of specialized variations. Moreover, there is Consumption of seafood contaminated with marine biotox-
growing resistance against the use of animal experiments. ins may cause serious diseases. Damage to the nervous
Therefore, many efforts have been made to determine algal system (paralytic shellfish poisoning, PSP), the intestinal
toxins with chemical methods. In this context LC-MS system (diarrhetic shellfish poisoning, DSP), and loss of
methods replaced HPLC methods with optical detectors, memory (amnesic shellfish poisoning, ASP) have been
allowing both effective seafood control and monitoring of observed subject to the type of algal bloom. In addition,
phytoplankton in terms of the different groups of marine regionally specific algal toxins (e.g. ciguatera and tetrodo-
biotoxins. toxins) have been identified in seafood specialties from
several countries.
Keywords Marine biotoxins . LC-MS/MS . PSP toxins . The worldwide harvesting and transport of marine
DSP toxins . Domoic acid . Tetrodotoxins . Ciguatera 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
B. Christian (*) : B. Luckas strict compliance with the respective restrictions.
Institute for Nutrition, Friedrich-Schiller-University of Jena,
The residue analyst specialized in the determination of
Dornburger Str. 25,
07743 Jena, Germany marine biotoxins by chemical methods has to determine
e-mail: b1chbe@uni-jena.de rapidly the toxins in complex biomatrices with high
118 Anal Bioanal Chem (2008) 391:117–134

sensitivity and, most importantly, unambiguously. The excitation current in nerve and muscle cells resulting in signs
simultaneous realization of those demands was often of paralysis.
accompanied with difficulties. However, the introduction Consequently, the development of analytical methods for
of LC-MS-based methods during the last decade has the determination of poisoning caused by PSP toxins was
allowed the analysis of algal toxins, especially the an important task. The mouse bioassay unambiguously
quantitative determination of those relevant for regulations, gives evidence of the toxic potential of a sample, since the
to be established in routine analysis. 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
Occurrence, chemical structure, toxicity, and analysis sample expressed in MU (mouse units) kg−1 or in PSP kg−1.
of marine biotoxins Thus, the determination of individual PSP toxins was only
possible after isolation and structure elucidation [2].
Empirical scientific observations of harmful algal blooms In 1957, a PSP toxin was isolated from Saxidomus
and toxic mussels were reported as early as 1937. At that giganteus (clams) from Alaska, and in 1975 the chemical
time, some algae were described which seemed to be structure was assigned to the so-called saxitoxin (STX).
responsible for the production of toxins and a biological Later on, more PSP toxins were identified which were all
test, the mouse bioassay, was developed to identify toxins related to saxitoxin (STX) or N-1-hydroxy-saxitoxin (neo-
produced by those algae. Up to now, the mouse bioassay saxitoxin, NEO). Today, it is convenient to distinguish
has been the most important international method for the between three groups of PSP toxins: carbamoyl toxins,
detection of algal toxins, and all analytical methods N-sulfocarbamoyl toxins, and decarbamoyl toxins.
described below have to be evaluated against bioassays. N-Sulfocarbamoyl toxins exhibit only low toxicity,
However, such biological tests reveal only the total toxicity whereas the toxicity of the carbamoyl and decarbamoyl
of a sample. The limited validity resulted in the need for toxins is significantly higher. During the manufacturing of
physicochemical methods for the detection of each individ- canned seafood contaminated with PSP toxins, the
ual algal toxin. A further important argument against the N-sulfocarbamoyl toxins may be hydrolyzed to the more
mouse bioassay is the growing resistance to the use of toxic carbamoyl or decarbamoyl toxins, thus resulting in an
animal experiments [1]. increase of the total PSP toxicity (Fig. 1).
In 1975, a fluorometric method was recommended for
Paralytic shellfish poisoning (PSP) toxins PSP determination in samples in addition to the mouse
bioassay [4]. PSP toxins which exhibit neither UV
The main producers of paralytic shellfish poisoning (PSP) absorption nor fluorescence were oxidized in alkaline
toxins are dinoflagellates of the genus Alexandrium. solution to fluorescent purine derivatives. After acidifica-
Alexandrium occurs along the Atlantic and Pacific coast- tion the oxidation products′ intensity of fluorescence was
lines where it may grow in great quantities, particularly measured in solution. However, each PSP toxin differs both
during the summer period. Toxins produced by Alexandrium in toxicity and fluorescence intensity after oxidation.
were named PSP toxins due to the observation that their Therefore, a chromatographic separation of the PSP toxins
consumption caused symptoms of poisoning in warm- was suggested prior to determination of the fluorescence
blooded species similar to paralytic phenomena such as activity, since the total PSP toxicity has to be calculated
cramp, signs of paralysis, and blocking of respiration. PSP from the individual toxin concentration and the absolute
toxins are potential neurotoxins which specifically block the toxicities of each PSP toxin. Hence, the unambiguous

Fig. 1 Chemical structure,


toxicity, and molecular weight
of PSP toxins [3]
Anal Bioanal Chem (2008) 391:117–134 119

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

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 Anal Bioanal Chem (2008) 391:117–134

dcGTX 2
understood, they were placed into the DSP category because

C1/
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
dcGTX 3 chemical classes rather than their toxic symptoms. This
would allow seafood safety to be regulated according to
GTX 2

dcSTX
allowable levels of specific toxins rather than to the result
GTX 3

C2

of a specific assay. This is the path on which all other


TIC
contaminants in food, such as mycotoxins and pesticides,
493/298 are controlled [14].

396/298 Okadaic acid and dinophysistoxins (OA and DTXs)


353/255
Dinophysistoxin-1 (DTX-1) was identified as the causative
493/316 toxin for incidents of diarrhetic shellfish poisoning in Japan,
and it originates from the dinoflagellate Dinophysis fortii.
353/273 Although DTX-1 is found in Europe to a limited extent, it
257/126 was identified as the major toxin in shellfish from Norway.
396/316 Most studies have implicated okadaic acid (OA) as the
responsible toxin arising from a variety of Dinophysis
010 20 30 40 species. DTX-2, an isomer of OA, was isolated from Irish
Time (min) mussels. In addition, DTX-3 has been observed in plankton
Fig. 3 HILIC-MS/MS analysis of a Gymnodinium catenatum extract and shellfish (Fig. 4).
(0.03 M HAc), Baja California, Mexico [12] 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-
the sensitivity of the MS detection is sufficient to control hydroxyl group of DTX-1. Subsequently, it was shown that
seafood at the regulatory limit for a PSP toxin content at any of the parent toxins, OA, DTX-1, and DTX-2, can be
800 μg STX equivalents kg−1 wet weight of soft tissues. acylated and are shellfish metabolites [15].
All lipophilic toxins can be extracted from plankton or
Diarrhetic shellfish poisoning (DSP) toxins shellfish tissues by organic solvents such as acetone,
methanol, and acetonitrile. The use of acetone for extraction
Since the first reports of diarrheic shellfish poisoning (DSP) is required if the mouse bioassay is applied for the
in Japan, 1978, the illness is now recognized as a threat to determination of OA, DTXs, PTXs, and YTXs [16].
public health throughout the world. All DSP toxins are However, 80% methanol in water is recommended as an
relatively nonpolar, with molecular weights higher than effective extraction solvent for application of HPLC
500, and easily extractable by organic solvents such as methods with fluorescence detection or the LC-MS/MS
methanol and chloroform. Most DSP toxins are polyether technique [17]. After addition of more water to reduce the
compounds with distinctive chemical structures and widely percentage of methanol to approximately 50%, toxins can
varying functional groups resulting in different toxicolog- be partitioned between chloroform or dichloromethane
ical and chemical characteristics [13]. which are then evaporated to dryness and the residue can
Marine biotoxins in shellfish have traditionally been be redissolved in 0.5–1.0 mL methanol.
categorized and regulated according to the poisoning syn- In addition, the different ester derivatives, e.g., DTX-3,
dromes that they evoke in human beings or animals. For can be hydrolyzed quantitatively to their parent toxins by
example, okadaic acid (OA) and dinophysistoxins (DTXs) treatment of the aqueous methanol extract with 0.25 M
have been classified as diarrhetic shellfish poisoning (DSP) NaOH [18]. This procedure enables the unambiguous
toxins due to their production of symptoms of diarrhea. determination of both the DSP content of a sample material
Unfortunately, when pectenotoxins (PTXs) and yessotoxins as OA equivalents and the percentage of DTX-3, indepen-
(YTXs) were discovered and before their toxicity was 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 PTX-4 and PTX-7 through PTX-9. The lactone ring in
mussels can consist of a higher percentage of OA esters and PTX-2 may be opened to yield pectenotoxin seco acid
their analogs than of other DSP toxins (see Fig. 5). (PTX-2sa), epimerization yields 7-epi-PTX-2sa [20].
PTXs appear to be highly toxic by intraperitoneal
Pectenotoxins (PTXs) injection, leading to positive responses in the mouse
bioassay for lipophilic marine biotoxins. Because of that,
The pectenotoxins (PTXs), named after the genus of scallop the maximum level of PTXs (PTX-1 and PTX-2) permitted
from which they were first isolated, Patinopecten yessoensis, in European shellfish has been set at 160 μg kg−1 OA
are a group of DSP toxins originating from Dinophysis equivalents [16, 21]. However, PTXs appear to be of low
species throughout the world (Fig. 6). toxicity orally and, unlike OA, they do not cause diarrhoea
PTX-2 is the main toxin in this group. When accumu- [22, 23].
lated in shellfish, the methyl group at position C-43 is All pectenotoxins absorb between 235 nm and 239 nm
oxidized to the corresponding alcohol (PTX-1), aldehyde (UV). Detailed stability studies have not been performed,
(PTX-3), and carboxylate (PTX-6) forms. The spiroketal but it is known that rearrangements can occur under acidic
system in rings A and B can also undergo rearrangement conditions [24]. In addition, PTXs are easily destroyed
and/or epimerization under acidic conditions to produce 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 Anal Bioanal Chem (2008) 391:117–134

Fig. 6 Chemical structures of Me


PTX homologs O
O O Me
1
O O
OH O O
33 OH
OH Me
Me O O
O 43
R
O Me Me

R C1-C33

Pectenotoxin-1 CH2OH -O-

Pectenotoxin-2 CH3 -O-

Pectenotoxin-3 CHO -O-

Pectenotoxin-6 COOH -O-

Pectenotoxin-2 seco acid CH3 -OH HO-

considered in analyses of samples containing PTXs and, Antibodies to yessotoxin (YTX) have been produced and
consequently, sample preparation has to be performed at a used to develop an ELISA. Its application for analyses of
pH value near 7. algal samples gave results two to three times higher than
On the other hand, comparison of the PTX profiles of the with LC-MS for known YTX, and for shellfish the results
toxic dinoflagellate Dinophysis acuta, of Greenshell mussels by ELISA were three to nine times higher than the results
(Perna canalicus), and Blue mussels (Mytilus galloprovin- obtained from LC-MS. Those results suggested that algae
cialis) from New Zealand by application of an LC-MS could produce a series of YTXs which were recognized by
technique revealed that the major PTX homolog in D. acuta the antibody and that those analogs had been metabolized
was PTX-2, whereas both Greenshell and Blue mussels in shellfish [33].
contained PTX-2sa as the predominant toxin. More than In spite of recent evidence that YTXs may have no
90% of PTX-2 isolated from D. acuta was rapidly converted significantly acute toxicity when administered orally to
to PTX-2sa and its epimer 7-epi-PTX-2sa in the Greenshell mice [30], the actual quarantine level of YTX, 45-
mussel extract (Fig. 7). The conversion from PTX-2 to PTX- hydroxyYTX, 1-homo-YTX, and 45-hydroxy-1-homoYTX
2sa and 7-epi-PTX-2sa was not observed in phosphate in shellfish is 1 mg kg−1 in the European Union [16, 21].
buffers at various pH ranging from 4.1 to 9.1. These findings Recently, a number of LC-MS methods have been
indicate that PTX-2sa and 7-epi-PTX-2sa are not artifact demonstrated as powerful techniques for the quantification of
toxins resulting from hydrolysis of PTX-2, but they arise YTXs analogs and structure elucidation of novel toxins in
from the conversion of PTX-2 by mussel tissues [25, 26]. plankton and shellfish [34–37]. Figure 9 shows the determi-
nation of YTXs in mussel tissue obtained using LC-MS3 [37].
Yessotoxins (YTXs)
Azaspiracid poisoning (AZP) toxins
Yessotoxins (YTXs) are sulfated polyethers (Fig. 8) pro-
duced by the dinoflagellates Protoceratium reticulatum and A shellfish poisoning event occurred in the Netherlands in
Gonyaulax polyedrum in many parts of the world [27–30]. 1995 when at least eight severe gastrointestinal illnesses in
YTXs are toxic by intraperitoneal injection in mice and humans were reported after the consumption of mussels
therefore give an increase to positive results in the (Mytilus edulis). These shellfish were traced to Killary
traditional mouse bioassay for DSP toxins [31]. Harbor, Ireland, where subsequent investigations revealed
A number of oxidized YTXs, apparently arising from that a number of local intoxications had also occurred.
metabolism of YTX and 1-homo-YTX present in ingested Unfortunately, this toxicity was not covered within the DSP
algae, were subsequently isolated from shellfish by mouse toxin-monitoring program in Ireland, which involved
bioassay guided fractionation [32]. 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 analogs have been isolated from Irish mussels, of which
those shellfish, only low levels of OA and DTX-2 were AZA-1, AZA-2, and AZA-3 seem to be the main
detected. In the DSP mouse test, a slow progressive compounds responsible for contamination. Methylazaspir-
paralysis was observed using extracts of mussels, and these acid (AZA-2) and desmethylazaspirazid (AZA-3) occur in
neurotoxic symptoms were quite different from those shellfish at lower concentrations than azaspiracid (AZA-1),
typical for DSP toxins. In addition, the extracts were but they are more toxic in the mouse bioassay [39]. The
subjected ESI-MS and 1H NMR analysis. The skeletal chemical structure of AZA-4 and AZA-5 suggests that they
structures of a toxin group named azaspiracids (AZAs) are oxidized metabolites of AZA-3 [40, 41].
were elucidated [38]. It was apparent that they contained Although AZAs were first identified in mussels that
novel spiro-linked five- and six-membered rings, with one were cultivated in Ireland, a widespread European distri-
of those containing nitrogen (Fig. 10). bution of these toxins has recently been confirmed [42, 43],
In toxicity studies using azaspiracids, mice became and the EU regulatory limit for the combined AZA-1,
progressively paralyzed with labored breathing. Diarrhea AZA-2, and AZA-3 content has been set at 0.16 μg g−1
was not observed and, at low doses, mice died within 2– total shellfish tissue [16, 21].
3 days. Histopathological changes were observed in mice Most regulatory testing only examines the digestive
which were induced by both intraperitoneal and oral glands (DG). That practice was a consequence of an early
administration of azaspiracids. Subsequently, azaspiracid 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 Amnesic shellfish poisoning (ASP) toxins
can migrate to other tissues and this fact was a contributory
factor in false negative bioassay results [44, 45]. Quilliam and Wright [48] reported on big efforts to
The determination of various AZAs in shellfish relies elucidate the cause of the so-called Prince Edward Island
upon the usage of LC-MS methods. LC-MS methods have Disease in 1987 in which more than 100 people were
been developed for the analysis of AZAs in shellfish using poisoned after consumption of mussels harvested on this
an ion-trap mass spectrometer [46, 47]. It was found that the eastern Canadian island. Three people died and the
ubiquitous “benign” marine dinoflagellate Protoperidinium survivors suffered memory loss. The underlying toxin was
is the progenitor of azaspiracides [44]. therefore named amnesic shellfish poisoning (ASP) toxin.
Figure 11 shows the LC-MS3 analysis of an extract of the During the isolation of the ASP toxin from mussels,
hepatopancreas (HP) of mussels (Mytilus edulis) containing several toxic and non-toxic mussel extracts were investi-
the azaspiracides AZA-1, AZA-2, and AZA-3 [39]. 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 PSP toxins and, consequently, the application of chromato-
analysis of tetrodotoxins is a task similar to the determina- graphic methods was suggested [74].
tion of algal toxins [69]. The toxic effect of tetrodotoxins is The detection of fugu toxin was a major problem since
based on blocking of the sodium channels in nerve cells TTXs neither show UV absorption nor fluorescence activity
and, consequently, the mouse bioassay is suitable for [75]. Therefore, the HPLC system was equipped with a
determination of those potential neurotoxins [70]. reaction unit for post-column derivatization, enabling a
Moreover, it was discovered that puffer fish may contain selective and sensitive determination of TTX and its deriva-
both TTXs and PSP toxins [71–73]. Therefore, specific tives. After chromatographic separation TTX is converted into
methods were required for the determination of TTXs and 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 In the Annex III, Section VII, Chapter V of the Regulation
simultaneous determination of TTX and PSP toxins [77]. (EC) No 853/2004 of the European Parliament and of the
Since ESI-MS was introduced for analysis of marine Council of 29 April 2004 [21], health standards for live
biotoxins [78], this technique also proved to be a very bivalve mollusks concerning the marine biotoxins were
effective tool in the analysis of TTXs. Reliable methods established. This regulation also contains (in Section VIII,
using LC-ESI-MS for the determination of tetrodotoxin in Chapter V) health standards for fishery products in which the
puffer fish have been developed, too [61, 79]. following specific requirements are defined with regard to
TTXs are generally extracted with an aqueous or toxins harmful to human health: fishery products derived from
methanolic solution of acetic acid because TTXs are stable poisonous fish of the families Tetraodontidae, Molidae,
in weakly acidic aqueous conditions and under heating. Diodontidae, and Canthigasteridae as well as fishery products
TTX is present at higher concentrations than its derivatives containing biotoxins such as ciguatera or muscle-paralyzing
in samples of puffer fish and newt, such that 5-deoxyTTX, toxins must not to be brought onto the market [82].
5,6,11-trideoxyTTX, and 4,9-anhydroTTX are considered
to be almost non-toxic analogs [80]. Ciguatera (CTXs)
Recently, the HILIC column TSKgel Amide-80 was
applied successfully for ESI-LC-MS determination of The term ciguatera fish poisoning was first used in the
TTXs. It was found that in eggs of the puffer fish Fugu Caribbean to describe an intoxication induced by ingestion
poecilonotus the concentration of 5,6,11-trideoxyTTX was of a marine snail, Turbo pica (called cigua by the Cuban
higher than that of TTX, whereas that analog was not natives). Now the world is used this term to describe the
detected in the skin of the newt Cynops ensicauda [81]. intoxication caused by consumption of certain fish, primar-
Diener et al. [65] used a ZIC-HILIC column for the ily reef fish from the tropical and subtropical areas of the
determination of TTXs in separated tissues of Bangladeshi Caribbean Sea and the Pacific Ocean, which have accumu-
marine puffer, Takifugu oblongus (Fig. 14). TTX was lated specific toxins (CTXs) via their diet.
predominant in skin, muscle, and liver, whereas 5,6,11- The more than 175 ciguateric symptoms that have been
trideoxyTTX was preponderant in the ovary. reported can be classified into four categories: gastrointes-
128 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].
Fig. 14 HILIC-MS analysis (SIM mode) of extracts of T. oblongus:
a skin, b ovary [65]
Application of LC-MS/MS methods for determination
tinal, neurological, cardiovascular, and general [83]. This of assorted marine biotoxins in compliance
multiphase intoxication is thought to be due to the presence with legislation
of different ciguatera-related compounds at different ratios
[84, 85]. Due to the cases of human intoxication frequently observed
Although Scheuer et al. [86] isolated ciguatera in 1967, after mussel consumption, governmental institutions and
the structure was not elucidated until 1989 [87]. Further the fishery industry have established control methods to
ciguatoxins (CTXs) were later discovered in algae [88] and ensure that seafood contaminated by biotoxins does not
fish [89, 90]. reach the consumer [105]. The first step was to establish
CTXs are cyclic polyethers with similar structures to DSP monitoring programs for harmful algae blooms (HABs)
toxins and brevetoxins [91]. Therefore, a fast and unambig- followed by restrictions concerning the harvest of mussels
uous determination of ciguatoxins with simple chemical tests in areas with an unacceptable amount of toxin-producing
is difficult [83]. 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) 391:117–134 131

methods. Recent advances in analytical instrumentation column for determination of paralytic shellfish poisoning toxins.
J Sep Sci 30:1821–1826
have enabled the development of alternative methods such
13. Yasumoto T, Murata M (1985) Diarrhetic shellfish toxins.
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16. EC (2002) Commission decision 2002/225/EC of 15. March
ical methodology from algal toxins is highly desirable, 2002 laying down detailed rules for the implementation of
because the enforcement of biotoxin legislation is ultimately Council Directive 91/492/EEC as regards the maximum levels
based on the ability of analysts to identify and quantify those and the methods of analysis of certain marine biotoxins in
bivalve molluscs, echinoderms, tunicates and marine gastropods.
toxins accurately in seafood products.
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