occurrence of azaspiracids, spirolides, yessotoxins, pectenotoxins

Not to be quoted without prior reference to the authors Fisheries Research Services Contract Report No 08/03

OCCURRENCE OF AZASPIRACIDS, SPIROLIDES, YESSOTOXINS, PECTONOTOXINS AND FREE FATTY ACIDS IN PLANKTON AND SHELLFISH

L Stobo, L Webster and S Gallacher May 2003 Fisheries Research Services Marine Laboratory 375 Victoria Road Aberdeen AB11 9DB

Table of Contents Executive Summary ..................................................................................................... 1 1. Introduction ............................................................................................................ 1 2. Azaspiracids ........................................................................................................... 4 3. Pectenotoxins ......................................................................................................... 9 4. Yessotoxins ............................................................................................................ 16 5. Spirolides ............................................................................................................... 22 6. Free Fatty Acids ..................................................................................................... 26 7. Conclusions ............................................................................................................ 31 8. References ............................................................................................................. 33

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OCCURRENCE OF AZASPIRACIDS, SPIROLIDES, YESSOTOXINS, PECTONOTOXINS AND FREE FATTY

ACIDS IN PLANKTON AND SHELLFISH

L Stobo, L Webster and S Gallacher

FRS Marine Laboratory, PO Box 101 375 Victoria Road, Aberdeen, AB11 9DB

EXECUTIVE SUMMARY

The aim of this literature review was to summarise the available information on the recently discovered compounds shown to cause a positive result using typical methodology for the monitoring of diarrhetic shellfish poisoning (DSP) toxins. The specific groups of compounds summarised were azaspiracids (AZAs), pectenotoxins (PTXs), yessotoxins (YTXs), spirolides and free fatty acids. In this review the occurrence, toxicology, isolation, extraction/clean up procedures and methods of determination in phytoplankton and shellfish are discussed for each of these compound groups. In particular, liquid chromatography mass spectrometry (LC-MS) methodology was outlined for the analysis of the compound groups. The review highlighted that additional work is required if LC-MS techniques are to be applied to routine, high throughput, shellfish monitoring of algal toxins. This included the need for evaluation and development of extraction and clean-up techniques for a range of shellfish matrices and validation of the methodology, in addition to optimising the LC-MS procedures. The information presented was originally summarised in January 2002 and has subsequently been used to support research for project B04004 (funded by the Food Standards Agency).

1. INTRODUCTION

Human intoxication due to the consumption of contaminated shellfish has been evident over the centuries. An early instance was recorded in Canada in 1798, where human deaths were later attributed to intoxication with paralytic shellfish poisoning (PSP) toxins (Wright, 1995). In more recent times compounds such as okadaic acid (OA), and structurally related compounds named dinophysistoxins, (DTXs; Yasumoto et al., 1978), domoic acid (DA; Quilliam and Wright, 1989) and azaspiracids (AZAs; James et al., 2000) were found to cause human illness upon consumption of shellfish. The toxic syndromes were named diarrhetic shellfish poisoning (DSP), amnesic shellfish poisoning (ASP) and azaspiracid shellfish poisoning (AZP) respectively, although the latter was initially described as DSP. Some confusion exists on the use of acronyms to describe both the toxins and the toxic syndrome. In this literature review the toxins are described with the syndrome abbreviation followed by the word toxin(s) e.g. DSP toxins. Other chemicals from shellfish extracts have been shown to be acutely toxic to mice. These include: • free fatty acids (FFAs), first noted in 1982 (Takagi et al., 1982) • pectenotoxins (PTXs), discovered in 1984 (Yasumoto et al., 1985) • yessotoxins (YTXs), discovered in 1987 (Murata et al., 1987) • spirolides, discovered in 1995 (Hu et al., 1995).

Occurrence of Azaspiracids, Spirolides, Yessotoxins, Pectenotoxins

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Until recently none of these compounds noted above have been implicated in human illness and the risk to human health has been a matter of much debate. However, in a recent published report, PTXs were implicated in a food poisoning incident in Australia after the consumption of shellfish commonly known as pipis (Plebidonax spp.; Burgess and Shaw 2001). It has been suggested that the occurrence of toxins in shellfish is increasing although the reason is unknown (Hallegraeff, 1985). Shellfish monitoring programmes exist in several countries world-wide. Within Europe legislation exists for “laying down the health conditions for the production and placing on the market of live bivalve molluscs” (Directive 91/492/EEC and amendment 97/61/EC). This Directive and it’s amendment includes the maximum limits for ASP, DSP and PSP toxins. When the Directive was first formulated, DSP toxins only included OA and the related DTXs. Latterly however, PTXs, YTXs and AZAs were incorporated into this toxin group primarily because they are lipophilic and are extracted by the solvent commonly used in extraction of shellfish for the determination of DSP (see below). Recent discussions within Europe have lead to proposals to monitor for these compounds as separate toxin groups with associated regulatory limits (Table 1.1). These plans are described in a draft EC Council Decision SANCO/2227/2001 that is likely to be adopted during 2002. Spirolides and free fatty acids (FFAs) are not included; spirolides, a group of macrocyclic imines, are currently not considered a hazard within Europe although this may change in the future (see sections 5 and 7) and free fatty acids are thought of more as a contaminant than a health threat.

TABLE 1.1 Proposed regulatory limits for recently discovered shellfish toxins

Toxin Group Toxins included Proposed

Regulatory Limit (µg 100 g-1)

DSP toxins Okadaic acid and Dinophysistoxins 16 Azaspiracids Azaspiracid-1, -2, -3 16

Pectenotoxins Pectenotoxin-1 & -2 16

Yessotoxins Yessotoxin, hydroxyyessotoxin, homoyessotoxin and hydroxyhomoyessotoxin 100

EU Directive 91/492 EEC states that the “customary biological testing method” should be used in monitoring shellfish for toxins. This is generally taken to mean the mouse bioassay described by Yasumoto et al. (1978) commonly called the DSP mouse bioassay. It involves extracting a homogenate of hepatopancreas tissue with acetone, followed by evaporation under reduced pressure to produce a residue which is re-suspended in 1% Tween 60. An aliquot of this suspension is then injected into the intraperitoneal cavity of a mouse. If the mouse dies within a set period (5 or 24 hours are commonly used) the sample is considered positive for DSP toxins although the chemical nature remains undefined. A later modification included an additional extraction step using diethyl ether which removed interfering PSP toxins and free fatty acids from the suspension (Yasumoto et al., 1984). Over the years EU Member States have used variations of this technique (Community Reference Laboratory, 2001). In attempts to clarify the methodology for the detection of the different lipophilic toxin groups, the draft Council Decision details a number of solvents, which can be used. Additionally, this decision allows for the first time an option to implement fully validated chemical methods and in vitro assays for the analysis of OA, DTXs, PTXs, YTXs and AZAs as alternatives to the mouse bioassay. With this Decision, methods can now be developed which remove the ethical issues currently faced by the countries employing the mouse bioassay.


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Liquid chromatography mass spectrometry (LC-MS) is a technique, which has seen considerable development, and in recent years increased interest has been shown in its application in the analysis of marine biotoxins in shellfish. The value of this technique is its ability to confirm the presence of a molecule from its molecular weight and fragmentation pattern. In most cases, a molecule will produce very distinctive mass fragments upon dissociation, which allows structural information to be obtained. It is this structural information, along with the molecular weight combined with the separation of the molecule using chromatography, which allows unrivalled confirmation of a particular compound. Additional use of a further MS stage, as in LC-MS/MS, allows selection of a particular ion or ions that are then subsequently fragmented. This selective acquisition of a particular ion or ions allows the effect of interfering ions to be reduced and the sensitivity and specificity of the analysis to be further increased. As LC-MS methodology is the prime focus of this literature review variations in the technique are described in more detail below. Acquisition of data, for LC-MS and LC-MS/MS, can be achieved in one of three modes; selective reaction monitoring (SRM), single ion monitoring (SIM) and full scan. Full scan allows the analysis of all ions over a specific mass range and is normally used in method development for the determination of suitable target and qualifier ions for SIM. Target ions are used for quantification of the compounds and qualifier ions are used for confirmation purposes. SIM acquires data on specified masses, enabling the operator to monitor for a number of particular ions in a single run. Sensitivity is increased with the use of SIM instead of full scan, as the instrument is focused only a few mass-to-charge ratios instead of many as in full scan. SRM monitors particular collisionally induced dissociation reactions. These reactions produce particular mass fragments from a parent ion that has been selected in the initial MS stage of LC-MS/MS. Only SIM and full scan modes can be used by LC-MS unlike LC-MS/MS, which can use all the modes. The mode and method of sample ionisation, generally through an interface, is also important, as it can influence the fragmentation of molecules. For a molecule to become ionised, one or more protons or electrons must be attached or removed. Attachment of protons occurs in positive mode and removal of protons (or attaching of an electron) occurs in negative mode. This generates the molecular ion, often used for quantification, and in some cases fragmentation may also occur. This produces the MS spectra which will be characteristic of the particular compound. Use of positive and negative ionisation modes can result in different fragmentation patterns for the same molecule. Two main interface types for LC-MS include atmospheric pressure chemical ionisation (APCI) and electrospray ionisation (ESI). APCI is used for molecules that are not easily ionised. In this process ions are produced by nebulising the sample in a heated tube causing the finely dispersed sample drops to vaporise. The mobile phase present in the spray is initially ionised via inducement by a corona discharge needle. The compounds are subsequently ionised by proton transfer from the mobile phase ions. This interface allows higher flow rates than ESI (up to 2 ml min-1) without affecting the sensitivity of the analysis. With ESI, ions are produced in solution as the sample passes through a needle charged with a high voltage. The sample molecules are then nebulised; producing droplets which evaporate causing the ions to enter the gas phase by a low energy process called ion evaporation. A modification of ESI is thermospray ionisation (TSI) which uses the same principles as ESI with the addition of a heated probe gas to increase the droplet evaporation resulting in an increase in signal. LC-MS interfaces result in ‘soft’ ionisation of compounds, which means less fragmentation, than observed for ionisation techniques such as electron impact. Fragmentation is, therefore, difficult with certain molecules. However, by increasing the mass spectrometer voltages (e.g. cone and ring voltages) it may be possible to induce fragmentation.


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Various combinations of the above have been applied to LC-MS analysis of shellfish toxins. This literature review summarises published information in terms of LC-MS methods for AZAs, PTXs, YTXs, spirolides and FFAs as well as general information on the toxin groups. It highlights that much work is required if these techniques are to be applied to routine, high throughput, shellfish monitoring. This includes the need for evaluation and development of extraction and clean-up techniques for a range of shellfish matrices and validation of the methodology, as we’ll as refining details of the LC-MS procedures. The information summarised will be used to support research on these areas as described in project B04004 (funded by the Food Standards Agency).

2. AZASPIRACIDS In November 1995 a shellfish poisoning event occurred in the Netherlands following the consumption of cultured mussels (Mytilus edulis) from Ireland. Azaspiracids (AZAs) were identified as the cause of this human intoxication (Satake et al. 1998b). AZAs, similar to other DSP toxins, are a group of polycyclic ethers. They contain a trispiro ring linkage that is a unique characteristic of this particular toxin group. At present only five AZAs have been characterised (Figure 2.1, Table 2.1). However, three additional analogues of AZA have been identified (James et al., 2000a) but have not yet undergone full structural elucidation. Ito et al. (2002) recently highlighted the potential of AZAs to induce tumours. In contrast, okadaic acid and the dinophysistoxins have been reported to have tumour promoting properties only in the presence of a known initiator (Ito et al., 2002). Figure 2.1. Structure of azaspiracids (unique trispiro ring linkage encircled).


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TABLE 2.1 Molecular weight and nomenclature of AZAs.

Abbreviation Chemical name Mol weight (g mol-1)

Structural elucidation reference

Azaspiracid 1 (AZA1) Azaspiracid 841.5 Satake et al., 1998a Azaspiracid 2 (AZA2) 8-methylazaspiracid 855.5 Ofuji et al., 1999a Azaspiracid 3 (AZA3) 22-demethylazaspiracid 827.5 Ofuji et al., 1999a

Azaspiracid 4 (AZA4) 3-hydroxy- demethylazaspiracid 843.5 Ofuji et al., 2001

Azaspiracid 5 (AZA5) 24-hydroxy- demethylazaspiracid 843.5 Ofuji et al., 2001

Occurrence AZAs, to date, have been found in oysters (Crassostrea gigas) and mussels (Mytilus edulis) (James et al., 2000) as well as scallops (Pecten maximus) (Hess et al., 2001). The toxins are widespread on the west and southwest Irish coasts. Reports also exist of their occurrence in shellfish from Norway, France and the United Kingdom (K. James, Pers. Comm.). Outbreaks of foodborne intoxication caused by AZAs from Irish mussels have been reported in the Netherlands (1995), Arranmore Island (1997), France (1998), Italy (1998) and United Kingdom (2000) (S. Morris, Pers. Comm.; James et al., 2000b). The occurrence of AZAs in phytoplankton has been investigated by Roden (2001) and the causative organism was thought to be the dinoflagellate, Protoperidinium crassipes. This was ascertained by hand picking a large number of cells of P. crassipes from a sample of phytoplankton taken while shellfish were contaminated with AZAs, and analysing the cells for AZAs using LC-MS. This experiment was repeated on four separate occasions with the same positive results (Roden, 2001). Nevertheless, doubts still exist within the scientific community on whether this dinoflagellate species is the main producer of the toxins (E. Bresnan, Pers. Comm.). Toxicology The published oral and intraperitoneal toxicities of the AZA1 and its analogues are given in Table 2.2, the OH derivatives (AZA4 and AZA5) are the most toxic AZAs. Ito et al. (2002) proposed further experimentation to confirm the tumourogenic properties of AZAs, although in general research is hampered by a lack of pure AZA standards.

TABLE 2.2 Summary of azaspiracids toxicity in mice.

Oral Toxicity (µg kg-1)*

Intraperitoneal Toxicity (µg kg-1)* Reference

900 200 Satake et al., 1998a AZA1 500 200 Ito et al., 2000 AZA2 No data given <200 Ofuji et al., 1999 AZA3 No data given <200 Ofuji et al., 1999 AZA4 No data given 0.47 Ofuji et al., 2001 AZA5 No data given 1.0 Ofuji et al., 2001


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*refer to references for definition of toxicity Isolation Azaspiracid 1 (AZA1) was first isolated from mussel tissue by Satake et al. (1998a). Ofuji et al. (1999a and 2001) isolated the analogues, azaspiracid 2 (AZA2), azaspiracid 3 (AZA3), azaspiracid 4 (AZA4) and azaspiracid 5 (AZA5). These isolations used acetone extraction with successive chromatographic separation on preparative columns (silica, Toyopearl HW-40, CM-Toyopearl 650M and DEAE-Toyopearl). Small amounts of AZAs were recovered from 20 kg of shellfish material; 11 mg AZA1, 2.5 mg AZA2, 0.6 mg AZA3, 0.5 mg AZA4 and 0.3 mg AZA5 (M. Satake, Pers. Comm.). Extraction and Clean Up Methods Only limited data has been published on the extraction of AZAs from tissue and phytoplankton. Two techniques, which use LC-MS, have used acetone extraction (Draisci et al., 2000 and James et al., 2000) despite recoveries been reported as “low” (James et al., 2000). Precise detail on recoveries and validation of the methods are lacking from both of these studies. The only published recovery data available for the AZAs from any matrix is for shellfish tissue (Ofuji et al., 1999b). Recoveries of > 97% were observed for tissue spiked with 0.5 µg g-1 of AZA1-3 using exhaustive methanol extraction, chloroform partition and solid phase extraction. Methods of Determination The most frequently used techniques for the detection of AZAs are the mouse bioassay (Satake et al., 1998a; Ito et al., 2000; Ofuji et al., 1999a, 1999b, 2001) and LC-MS (Table 2.3), although a cytotoxicity test has also been proposed by Flanagan et al. (2001). The structural properties of the AZAs do not allow for HPLC analysis utilising common detection methods such as photo diode (HPLC-UVDAD) and fluorescent detection (HPLC-FLD) with precolumn derivatisation (which require either the compound to have a chromophore or contain a functional group which can be derivatised to produce a fluorescent moiety). Hence only HPLC methods using MS detection for the analysis of AZAs have been reported. At present LC-MS is the most promising chemical detection technique for the analysis of AZAs. Analytical Methods The published LC-MS methods for the determination of AZAs in shellfish and phytoplankton are variable in the amount of detail given, these are summarised in Table 2.3. The ions used for the analysis are also listed in Table 2.3 and from this summary, it is clear that the molecular ion is commonly used as the target ion. For single LC-MS, both full scan (Ofuji et al., 1999a, 1999b) and single ion monitoring (SIM) have been used. The methods that use tandem MS or LC-MSn to detect the product ions have utilised selective reaction monitoring (SRM) to select for the molecular ion and produce the confirmatory product ions, through successive loss of water from the molecule. Analysis of AZAs by single LC-MS (Ofuji et al., 1999b) and tandem LC-MS (Draisci et al., 2000) has shown both techniques to have good calibration ranges and linearity with the latter having a lower limit of detection (0.020 µg ml-1 AZA1) than LC-MS (0.050 µg ml-1 AZA1). Although LC-MSn has also been used (James et al., 2000) no data was reported on the limit of detection or the calibration range/linearity. All of the methods used the positive ion mode with electrospray ionisation (Table 2.3). The LC-MS methods summarised above all require significant further development if they are to be applied to routine shellfish monitoring and this is discussed in more detail in Section 7.


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Other Methods The mouse bioassay (Yasumoto et al., 1978) has been applied to routine monitoring of shellfish for AZAs, particularly in the Republic of Ireland (J. Silke, Pers. Comm.). Symptoms observed in the mice, upon intraperitoneal exposure to shellfish extracts, include hopping, scratching and progressive paralysis leading to death in 60-90 minutes (Flanagan et al., 1999). However, the mouse bioassay (Yasumoto et al., 1978) has not been validated for quantification of AZAs and the efficiency of the extraction method applied (e.g. acetone) has been brought into question (James et al., 2000). The Community Reference Laboratory for Marine Biotoxins has recently recommended that acetone extraction followed by use of ethyl acetate, or dichloromethane or diethyl ether, may be applied in mouse bioassays for AZA detection (M. Fernandez, Pers. Comm.) Again validation studies on the different extraction solvents are lacking. The cytotoxicity assay (Flanagan et al., 1999 and 2001) is in early stages of development and is, at present, untried with pure AZAs. The assay examines cell viability and morphological changes in the cell and through a combination of these two observations, professes the ability to distinguish between okadaic acid and AZAs. LC-MS analysis of samples that gave a positive result using the assay confirmed the presence of AZAs. The cell assay was found not to be affected by saxitoxin, tetrodotoxin and domoic acid. However, when tested against YTX there was a decrease in cell viability, which could lead to the misidentification of the presence of AZAs. This technique also has the disadvantage of being laborious given the logistics of high-throughput monitoring.

TABLE 2.3 Summary of published AZA LC-MS methods

Reference Azaspiracids & ions

determined Ionisation technique

and mode LC conditions Comments

Ofuji et al., 1999a

AZA2 & AZA3 [M+H]+ m/z 856.5 AZA3 [M+H]+ m/z 828.5

LC-MS (Full scan) ESI Positive

Asahipak ODP-50 (Showa Denko, Tokyo, Japan) Linear gradient from 50% methanol to 100% methanol (with 0.1% acetic acid).

No column dimensions, flow rate or gradient program given

Ofuji et al., 1999b

AZA1 [M+H]+ m/z 842.5 AZA2 [M+H]+ m/z 856.5 AZA3 [M+H]+ m/z 828.5

LC-MS (Full scan) ESI Positive

Capcell Pak C18 UG-120 (Shiseido, Tokyo, Japan), 2 x 150 mm @ 35°C Isocratic ethanol/water/acetic acid (700:300:1) @ 0.2 ml min-1, 1 µl injected

Linearity: 0.05 – 100 µl ml-1 R2 >0.992 Limit of detection (S/N = 3): AZA1 0.05 µg ml-1 AZA2 & AZA3 0.075 µg ml-1

Draisci et al., 2000

AZA1 [M+H]+ m/z 842.5 [M+H-nH20]+ (n=1-3) m/z 794.5-824.5

LC-MS/MS (SRM) ESI Positive

Vydac 218TP51 (Separations Group, Hesperia, USA), particle size 5 µm, 250 x 1 mm @ ambient temperature Isocratic acetonitrile-water (85:15) with 0.03% TFA @ 0.030 ml min-1, 1 µl injection.

Linearity: 0.1 – 1.0 µl ml-1 R2 >0.995 Limit of detection: AZA1 0.020 µg ml-1

James et al., 2000

AZA1 [M+H]+ m/z 842.5 [M+H-nH20]+ (n=1-3) m/z 794.5-824.5 AZA2 [M+H]+ m/z 856.5 [M+H-nH20]+ (n=1-3) m/z 838.5-808.5 AZA3 [M+H]+ m/z 828.5 [M+H-nH20]+ (n=1-3) m/z 780.5-810.5

LC-MS3 (SRM) ESI Positive

Luna (2) (Phenomenex, Macclesfield, UK), particle size 5 µm, 150 x 2.0 mm @ 40°C Isocratic acetonitrile-water (65:35 or 70:30) with 0.05% TFA @ 0.2 ml min-1 (flow injection 3 µl min-1)

Quilliam et al., 2001a

AZA1 [M+H]+ m/z 842.5 AZA2 [M+H]+ m/z 856.5 AZA3 [M+H]+ m/z 828.5

LC-MS (SIM) ESI Positive

Hypersil BDS C8 (Thermo Hypersil, Runcorn, UK), particle size 3 µm, 50 x 2.0 mm Gradient 5-100% acetonitrile (50mM formic acid, 2 mM ammonium formate) over 10 mins, held at 100% for 10 mins @ 0.2 ml min-1.

SIM – single ion monitoring SRM – selective reaction monitoring S/N – signal-to-noise ratio ESI – electrospray ionisation

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3. PECTENOTOXINS Pectenotoxins (PTXs) are cyclic polyether macrolides. At present, there are eleven known structures (Fig. 3.1, Table 3.1). They differ from other polyether toxins, such as okadaic acid, in that they have a dioxabicyclo ring structure, a lactone ring and a longer carbon backbone. PTX-1 and PTX-2 were first isolated from shellfish in 1984 (Yasumoto, et al., 1985). PTX-2 was identified as the main toxin produced in the dinoflagellate, Dinophysis fortii (Lee et al., 1989). In scallops, this compound has been shown to rapidly undergo successive oxidations at the C-43 methyl group to yield the alcohol (PTX-1), the aldehyde (PTX-3) and the carboxylic acid (PTX-6) (Suzuki et al., 1998). Under acidic conditions, PTX-4, PTX-7, PTX-8 and PTX-9 are produced by rearrangement and/or epimerisation of the spiroketal ring at C-7 (Sasaki et al., 1998). PTX seco acids (PTX-2sa and 7-epi-PTX-2sa) have been found in Greenshell mussels and the dinoflagellate, Dinophysis acuta (Daiguji et al., 1998a). These are formed by the cleavage of the lactone ring to produce the carboxylic acid, PTX-2sa followed by epimerisation to give 7-epi-PTX-2sa however, little is known about these compounds. It is thought that the PTX-1, PTX-2sa, epi-PTX-2sa and PTX-2 to PTX-9 are all derivatives of PTX-2 (Sasaki et al., 1998). There is little information in the literature regarding PTX-5. Human illness has occurred as a result of the ingestion of shellfish contaminated with PTX-2sa and 7-epi-PTX-2sa (Burgess and Shaw, 2001). Figure 3.1. Structures of PTXs

(1) Carbonyl at C14 reduced to hydroxyl

Cleavage of lactone ring

Lactone ring dioxabicyclo ring


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TABLE 3.1 Molecular weights of PTXs

Abbreviation Name Mol weight

(g mol-1) Structural elucidation

reference(s) PTX-1 Pectenotoxin-1 874.5 Yasumoto et al., 1985 PTX-2 Pectenotoxin-2 858.5 Murata et al., 1986

PTX-3 Pectenotoxin-3 872.5 Yasumoto et al., 1989 PTX-4 Pectenotoxin-4 874.5 Yasumoto et al., 1985 PTX-5 Pectenotoxin-5 876.5 Quilliam et al., 2002 PTX-6 Pectenotoxin-6 888.5 Sasaki et al., 1997 PTX-7 Pectenotoxin-7 888.5 PTX-8 Pectenotoxin-8 874.5 PTX-9 Pectenotoxin-9 888.5

Sasaki et al., 1998

PTX-2sa & 7-epi-PTX-2sa Pectenotoxin-2 seco acids 876.5 Daiguji et al., 1998a and

James et al., 1999 Occurrence PTX-2 has been found in extracts of Dinophysis fortii and Dinophysis acuta (Draisci et al., 1996; Daiguji et al., 1998a), which have also been shown to produce OA and DTXs (Lee et al., 1989). PTX-2 was detected in scallops from Japan (Yasumoto et al., 1985), mussels from Italy (Draisci et al., 1999) and scallops and mussels from New Zealand (Suzuki et al., 2001a and 2001b). PTX-2 was also identified in two marine sponges, Poecillastra sp. and Jaspis sp.; it was surmised that the toxin was present due to the sponges consuming toxic dinoflagellates (Jung et al., 1995). Although the seco acids have been detected in shellfish and phytoplankton (Daiguji et al., 1998a; Suzuki et al., 2001a and 2001b), they are thought to be produced by enzymatic degradation of PTX-2 (Suzuki et al., 2001a; M. Quilliam, Pers. Comm). There have been two recorded incidents of PTX related human illness. The first incident occurred in New South Wales (Australia) during December 1997. This resulted in 56 hospitalisations when 100 people became ill, with symptoms of vomiting and diarrhoea, after consuming pipis (Plebidonax sp.). The shellfish were found to contain PTX-2sa and epi-PTX-2sa along with a number of unidentified possible metabolites and degradation products. The role of the latter in the illness has not been defined. Examination of the pipis digestive system led to the suggestion that two dinoflagellates, D. acuminata and D. tripos, were the toxin source (Quilliam et al., 2000). In the second incident in Australia, consumption of pipis caused an elderly woman to become seriously ill, on North Stadbroke Island, Queensland. Again PTX-2sa and epi-PTX-2sa were identified in the pipis (Burgess and Shaw, 2001). Toxicology PTX-1 and PTX-2 are reported to be hepatotoxic (Terao et al., 1986, Shimada et al., 1994; Ishige et al., 1988). Additionally, PTX-2 was observed to be a potent cytotoxin of several human carcinomas cell lines (Jung et al., 1995). By intraperitoneal exposure, PTX-1 is nondiarrhogenic in mice (Terao et al., 1986) although oral ingestion of PTX-2 induced diarrhoea (Ishige et al., 1988). The intraperitoneal toxicity of PTXs in mice are summarised in Table 3.2; there is insufficient information on the oral toxicities of PTXs, although research is ongoing (Burgess et al., 2001a).


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TABLE 3.2 Summary of pectenotoxin toxicity in mice

Intraperitoneal

Toxicity (mg kg-1)* Reference

PTX-1 0.25 PTX-2 0.23

Yasumoto et al., 1985

PTX-3 0.35 Murata et al., 1986 PTX-4 0.77 Sasaki et al., 1998 PTX-5 No data available PTX-6 0.5 Yasumoto et al., 1989

PTX-7,8,9 >5 Sasaki et al., 1998 PTX2SA & 7-epi-PTX2SA No data available

*refer to references for definition of toxicity Isolation Initial attempts to isolate PTXs from toxic scallops, Patinopecten yessoensis, resulted in Yasumoto and co-workers (1985) obtaining PTX-1 and PTX-2. Subsequent work with this tissue lead to the discovery of PTX-3 to PTX-7 and PTX-10 (Yasumoto et al., 1989). The structure of PTX-10 has not been fully elucidated and there is limited data on this compound in the literature. Elution of the PTXs, from the preparative columns used, was monitored using UV at wavelengths between 235 nm and 239 nm. Suzuki et al. (2001) has also isolated PTX-2 with a method similar to that of Yasumoto et al. (1985). This involved two chromatographic steps, using alumina and reversed phase columns successively, and yielded PTX-2 with a purity of greater than 95%. The isolation of PTX-2 seco acids was achieved from the digestive glands of Greenshell mussels, Perna canaliculus (Daiguji et al., 1998a). This involved extraction of the tissue with 80% aqueous methanol followed by partitioning between ethyl acetate and water. The organic layer was subjected to a sequence of five preparative chromatographic steps, these included separations on alumina, silica gel and reversed phase columns. Elution of the seco acids was monitored using UV at 235 nm. Information on the isolation of PTX-8 and PTX-9 in not currently available in the published literature. Extraction and Clean Up Methods The extraction of the PTXs from phytoplankton and shellfish tissue is facilitated by the use of organic solvents. Draisci et al. (1996), Sasaki et al. (1999) and Suzuki et al. (1998, 2001) used unadulterated methanol to extract PTXs from phytoplankton while James et al. (1999) used 8:2 (v/v) methanol-water. Combinations of freeze thawing and sonication (James et al., 1999) were used to rupture the phytoplankton cell membranes as well as sonication alone (Draisci et al., 1996). At this time, only one paper gives recovery data for the extraction of PTXs from shellfish tissue (Goto et al., 2001). Using aqueous methanol, a solid phase extraction clean up and LC-MS detection, recoveries of 134, 128, 79 and 68% were obtained for PTX-1, PTX-2, PTX-2sa and PTX-6 from scallop digestive glands, respectively. The recovery of PTX-2sa and PTX-6 from scallop adductor muscle was found to be 79 and 68%, respectively. This


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extraction and solid phase clean up technique was based on Yasumoto and Takizawa’s (1997) method and is summarised in Table 3.3. Good recoveries (>99%) of PTX-2 and PTX-6 were also achieved from a methanol extract of phytoplankton using C18 solid phase clean up (Suzuki et al., 1998). Briefly, the extract was passed through the conditioned C18 cartridge, washed with distilled water and eluted with methanol. The methanol eluant of the phytoplankton was evaporated and reconstituted in 80% (v/v) aqueous methanol, defatted using hexane, and extracted twice with chloroform after addition of 0.2% (v/v) acetic acid. The chloroform extract was then evaporated to dryness, reconstituted in methanol and subsequently analysed by HPLC with fluorescence detection (HPLC-FLD, PTX-6) or LC-MS (PTX-2).

TABLE 3.3 Summary of sample preparation stages for extraction and clean up of PTXs (adapted from Goto et al., 2001).

Sample preparation stage Description

Tissue extraction

2 g tissue extracted with 18 ml of extraction solvent (90% (v/v) aqueous methanol) Centrifuged (2500 rpm for 10 minutes) Supernatant partitioned extracted twice with chloroform after addition of 0.5% acetic acid solution (contained 2% sodium chloride for adductor muscle extraction) Chloroform evaporated

Solid phase extraction

Sample loaded onto a preconditioned silica gel cartridge washed with acetone PTX-1 and PTX-2 collected in acetone PTX-2sa and PTX-6 collected in 3:7 (v/v) acetone-methanol

Additional liquid partitioning

Fraction (containing PTX-1 and PTX-2) was dissolved in 8:2 (v/v) methanol-water and partitioned with hexane Hexane layer discarded and the methanolic layer evaporated and reconstituted in methanol

Analysis LC-MS details given in Table 3.4. Methods of Determination LC-MS is again the instrumental “tool of choice” when it comes to analysis of PTXs, due to the versatility and sensitivity of the method and the ability to detect a number of PTXs (Table 3.4). Other methods reported include mouse bioassay, cytotoxicity assays, ELISA and HPLC with fluorescence detection (HPLC-FLD). Analytical Methods The HPLC-FLD method for the analysis of PTXs is the ADAM (9-anthryldiazomethane) technique (Lee et al., 1989), originally developed for okadaic acid compounds. This requires the presence of a carboxylic acid group and therefore has limited effectiveness toward PTXs, as only PTX-6, PTX-7, PTX-9 and PTX-2sas have this functional group. Reports exist on its use to detect PTX-6 (Suzuki et al. 1998) and PTX-2sas (James et al., 1999) although validation details were not stated. In the analysis of PTX-2sas James et al. (1999) have successfully applied the BAP (1-bromoacetylpyrene) derivatising reagent; this method however, still requires carboxylic acid functionality.


13

The HPLC-FLD method used in the detection of YTXs (Yasumoto and Takizawa, 1997) has also been applied to the analysis of PTX-2 from algae by Sasaki et al. (1999). Briefly, methanol extraction of the algae was followed by partitioning between equivolumes of methanol-water (8:2 (v/v)) and hexane, with subsequent separation of the evaporated aqueous methanol phase between chloroform and water. An aliquot of the chloroform layer was then evaporated and derivatised with DMEQ-TAD (4-[2,-(6, 7-dimethoxy-4-methyl-3-oxo-3, 4-dihydroquinoxalinyl) ethyl]-1, 2, 4-triazoline-3,5-dione). The derivatised sample was subsequently evaporated and subjected to C18 solid phase extraction. After elution of the derivatised PTX-2 it was again evaporated and reconstituted in mobile phase. Analysis was carried out by HPLC-FLD using a mobile phase of acetonitrile:water (6:4 v/v) on a C18 reversed phase column. LC-MS was used to confirm the findings of the HPLC-FLD method. As with YTX, two stereoisomeric peaks were present in the chromatograms. Good linearity of the calibration (R2 = 0.998) in the range of 0.1-20 µg ml-1 was reported with a limit of detection of 0.1 µg ml-1 (PTX-2). The suitability of this method for the analysis of PTXs in shellfish is under further investigation by the authors of this report although no details have been published to date. Nevertheless, in its current format this method can be considered time consuming and laborious and not suitable for application to a monitoring programme. The available LC-MS methods (Table 3.4) use both positive and negative ionisation modes, the positive ion mode being more popular. Electrospray ionisation is frequently implemented although thermospray ionisation has also been used (Suzuki et al., 2001a, 2001b). PTXs form molecular, sodium adduct and ammonium adduct ions under analysis conditions. The ammonium adduct has been reported to give the highest response and was used as the target ion in positive mode single ion monitoring (Suzuki et al. 1998; Quilliam et al., 2001a, Goto et al., 2001). Suzuki and Yasumoto (2000) found that ionisation efficiency was lower using positive ion mode, although the extent was not indicated, and that in negative ion mode, sodium and ammonium adducts were not formed. Draisci et al. (1999a) gave no indication of which mode was better and Goto et al. (2001) used both ion modes with individual chromatographic conditions depending on which PTX was under investigation (Table 3.4). LC-MS/MS experiments have indicated successive losses of water (Draisci et al., 1996), and cleavage of the lactone ring for PTX-2 and PTX-2sas in positive ion mode as the major fragments (James et al., 1999). In negative ion mode, PTX-2sas showed fragments not found in the PTX-2 spectrum, at m/z 367 and 645. These fragments represented cleavage at C-17 and C-10 respectively. The low mass region (m/z 100-200) of spectra showed a similar fragment pattern. In both positive and negative ion modes, the molecular ion was the most intense peak (James et al., 1999). Details of validation data, as with other recently discovered shellfish toxins, are not generally available for these methods. Information which is available has shown the LC-MS method is ten times more sensitive, at the limit of detection, than the DMEQ-TAD fluorescence method (Sasaki et al., 1999; Suzuki and Yasumoto, 2000). Other Methods The DSP mouse bioassay (Yasumoto et al., 1978) is reported to be capable of detecting PTXs. However, thorough and rigorous experimentation into the recovery of the toxins using this method has not been reported in the literature (S. Gallacher, Pers. Comm.). Two cytotoxicity assays have been published; one was able to detect PTX-1 at 5 µg ml-1 (Aune et al., 1991) and another could detect PTX-1 at 2 µM (Fladmark et aI., 1998). PTX-2 (0.05 µg ml-1) showed cytotoxicity against KB cells however, neither PTX-2-sa nor epi-PTX-2-sa (1.8 µg ml-1) resulted in cell death (Daiguji et al., 1998a). Efforts are being made to develop an ELISA technique capable of quantifying PTXs although further details are not available at this stage (I. Garthwaite, Pers. Comm.).

TABLE 3.4 Summary of some available PTX LC-MS methods published to date

Reference Pectenotoxins &

ions determined Ionisation technique

and mode Conditions Comments

Suzuki et al, 1998

PTX-2 [M+H]+ m/z 859 [M+NH4]+ m/z 876 [M+Na]+ m/z 881

LC-MS (Full scan/SIM) ESI Positive

Mightysil RP-18 (Kanto, Tokyo, Japan) particle size 3 µm, 100 x 2 mm Isocratic: 0.1% (v/v) acetic acid in 80:20 (v/v) acetonitrile-water. Flow rate 200 µl min-1@ 35ºC

Recovery data from spiked seawater and phytoplankton samples >99% (200-800 ng PTX-2)

Pavela-Vrancic et al., 2001 7-epi-PTX-2sa [M+NH4]+ m/z 876

LC-MS/LC-MS/MS (Full scan) ESI Positive

Vydac 218TP51 (Separations Group, Hesperia, USA) particle size 5 µm, 250 x 1 mm Isocratic: 0.1% TFA in 60:40 (v/v) acetonitrile-water. Flow rate 40 µl min-1

Flow injection analysis conditions given for both positive and negative modes for LC-MS/MS.

James et al., 1999

PTX-2 [M+H]+ m/z 859 PTX-2sa [M+H]+ m/z 877

LC-MS/LC-MS/MS (SIM) ESI Positive

Vydac 218TP51 (Separations Group, Hesperia, USA) particle size 5 µm, 250 x 1 mm Isocratic: 0.1% TFA in 60:40 (v/v) acetonitrile-water. Flow rate 40 µl min-1

Flow injection analysis conditions given for both positive and negative modes for LC-MS/MS.


PTX-2 [M+H]+ m/z 859 [M+H-nH2O]+ (n=1-5) m/z 841-769

LC-MS/LC-MS/MS (Full scan/SIM/SRM) ESI Positive

Supelcosil LC18-DB (Bellefonte, PA, USA) particle size 5 µm, 250 x 4.6 mm, Isocratic: 0.1% TFA in 60:40 (v/v) acetonitrile-water Flow rate 1ml min-1 (split 40 µl min-1)

Draisci et al., 1999a

PTX-2 [M+H]+ m/z 859 [M-H]- m/z 857 PTX-2sa [M+H]+ m/z 877 [M-H]- m/z 875

LC-MS (SIM) ESI Positive/Negative

Supelcosil LC18-DB (Bellefonte, PA, USA) particle size 5 µm, 300 x 1 mm Isocratic: 0.1% TFA in 80:20 (v/v) acetonitrile-water (Positive ion mode) 2 mM ammonium acetate in 80:20 (v/v) acetonitrile-water (Negative ion mode) Flow rate 40 µl min-1@ Room temperature

Suzuki & Yasumoto, 2000

PTX-6 [M-H]- m/z 887 [M+H]+ m/z 889 [M+NH4]+ m/z 900 [M+Na]+ m/z 911


Mightysil RP-18 (Kanto, Tokyo, Japan) particle size 3 µm, 150 x 2 mm Isocratic: 0.1% (v/v) acetic acid in 70:30 (v/v) acetonitrile-water. Flow rate 200 µl min-1@ 35ºC

Flow injection analysis conditions given Recovery from spiked extracts >86% (0.2, 1 & 4 µg g-1 Scallop and mussel midgut glands ) Linearity: 10 ng ml-1 to 30 µg ml-1 R2: >0.999 LOD: 10 ng ml-1

14

Table 3.4 (contd)

Reference Pectenotoxins & ions determined

Ionisation technique and mode Conditions Comments

Suzuki et al, 2001a/b

PTX-2 [M-H]- m/z 857 PTX-2sa [M-H]- m/z 875

LC-MS (Full scan) TSI TurboIonspray Negative

Luna C18 (2) (Phenomenex, Torrence, USA) particle size 5 µm, 150 x 2 mm Isocratic: 50 mM formic acid, 2 mM ammonium formate in 67:33 (v/v) acetonitrile-water. Flow rate 100 µl min-1 @ Room temperature

Goto et al., 2001

PTX-1 [M+H]+ m/z 875 [M+NH4]+ m/z 892 [M+Na]+ m/z 897 PTX-2 [M+H]+ m/z 859 [M+NH4]+ m/z 876 [M+Na]+ m/z 881 PTX-2sa [M-H]- m/z 875

PTX-6 [M-H]- m/z 887


PTX-1 and PTX-2: Develosil ODS-MG-5 (Nomura Chemicals, Seto, Japan) particle size 5 µm, 150 x 2.0 mm 7:3 (v/ v) acetonitrile–0.05% acetic acid Flow rate 100 µl min-1@ 40ºC PTX-2sa and PTX-6 Symmetry C18 (Waters, USA) particle size 3.5 µm, 150 x 2.1 mm 7:3 (v/ v) acetonitrile–0.05% acetic acid Flow rate 100 µl min-1@ 40ºC

Linear range used: 0.040-1.6 µg ml-1 Correlation >0.997 Limit of detection 40 ng g-1 & 80 ng g-1 (Scallop muscle and digestive gland respectively)

Ito and Tsukada, 2002 PTX-6 [M-H]- m/z 887

LC-MS (Full scan) SSI Negative

Inertsil ODS-2 (GL Science, Tokyo, Japan) particle size 5 µm, 15 x 2.1 mm 10 µl injected Linear Gradient: A = 1 mM ammonium acetate B = MeOH 40-100% B over 20 mins. Hold 100%B for 10 mins. Return to 40% B, hold 10 mins to equilibrate. Flow rate 200 µl min-1

Linear range used: 50-1000 ng ml-1 Correlation >0.999 Limit of detection 6 ng ml-1


PTX-2 [M+NH4]+ m/z 876 PTX-2sa [M+H]+ m/z 877

LC-MS (SIM) ESI Positive [M+H]+

Hypersil BDS (Keystone Scientific, Bellefonte, USA) particle size 3 µm, 50 x 2.1 mm Gradient: A=H2O B=AcN (2 mM ammonium formate, 50 mM formic acid) 95% A to 100% B over 10 minutes Hold for 10 minutes Re-equilibrate for 7 minutes Flow rate 0.2 ml min-1

SIM – single ion monitoring SRM – selective reaction monitoring S/N – signal to noise ratio ESI – electrospray ionisation TSI – thermospray ionisation

15


16

4. YESSOTOXINS

Yessotoxins (YTXs) are a group of polycyclic ethers that resemble brevetoxin and ciguatoxin (Daranas et al., 2001) in structure. They are unusual, in comparison to other marine biotoxins, in that they boast more than one sulphate group per molecule. YTX was first isolated from Patinopecten yessoensis, a Japanese scallop, in the late 1980s (Murata et al., 1987). Subsequent research discovered a number of analogues of YTX (Fig. 4.1), including adriatoxin, and all of the compounds have been structurally elucidated (Table 4.1). Figure 4.1. Structure of yessotoxins (see Table 4.1 for abbreviation definitions)

YTX

45-OH YTX

NorYTX

HomoYTX

45-OH homoYTX

CarboxyYTX

CarboxyhomeYTX

ATX

DesulfoYTX

YTX ATX


17

TABLE 4.1 Molecular weights of YTXs

Abbreviation Name Mol

weight (g mol-1)

Structural elucidation reference(s)

YTX Yessotoxin 1186

Murata et al., 1987+, Takahashi et al.,

1996 & Naoki et al., 1993

45-OH YTX 45-hydroxyyessotoxin 1202 NorYTX 45,46,47-trinoryessotoxin 1146 Satake et al., 1996+

HomoYTX 2-homoyessotoxin 1200 45-OH homoYTX 2-homo-45-hydroxyyessotoxin 1216 Satake et al., 1997a+

CarboxyYTX 44-carboxylyessotoxin 1218 Ciminiello et al., 2000+

DesulfoYTX 1-desulfoyessotoxin 1084 Daiguji et al., 1998b+

ATX Adriatoxin 1116 Ciminiello et al., 1998+

+Reference for isolation procedure for particular yessotoxin compound Occurrence YTXs have been found in Norway (Ramstad et al., 2001), Italy (Ciminiello et al., 1997), Japan, New Zealand and Chile (Yasumoto and Takizawa, 1997) in mussels as well as in Japanese scallops (Murata et al., 1987), although there have been no reported incidents of related human illness. The source of YTX and trinoryessotoxin was reported as the dinoflagellate, Protoceratium reticulatum (Satake et al., 1997b and 1999). Another dinoflagellate, Lingulodinium polyedrum, has been identified as producing 2-homoyessotoxin (Draisci et al, 1999b, Tubaro et al, 1998). It was proposed that YTX oxidised in mussel tissue to produce hydroxyyessotoxin, as this analogue was not detected in a P. reticulatum culture (Satake et al., 1997). Toxicology The toxicological data available for YTXs (Table 4.2) is more detailed than that of other toxins such as the azaspiracids and spirolides. YTXs have been shown to cause cardiac, pancreatic and hepatic damage (Terao et al., 1990). Some researchers (Ogino et al., 1997 and Aune et al., 2002) have suggested that YTX is more toxic to mice upon intraperitoneal compared to oral exposure (Table 4.2). De la Rosa et al. (2001) has proposed that the pharmacological mode of action is similar to that of brevetoxin and maitotoxin.


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TABLE 4.2 Summary of yessotoxin group toxicity in mice

Oral Toxicity (mg kg-1)*

Intraperitoneal Toxicity

(mg kg-1)* Reference

YTX > 10 0.1 Ogino et al., 1997 & Aune et al., 2002

HydroxyYTX 0.5 NorYTX 0.22 Satake et al., 1996

HomoYTX 0.1 Hydroxy-homoYTX 0.5 Satake et al., 1997a

CarboxyYTX 0.5 Ciminiello et al., 2000a DesulfoYTX 0.5 Daiguji et al., 1998b

ATX

No data available

>0.1 Ciminiello et al., 1998

*refer to references for definition of toxicity Isolation The isolation of YTX, from phytoplankton and shellfish, has been largely carried out using acetone extractions, with subsequent preparative reversed phase and ion exchange chromatography steps for the removal of interfering compounds (references given in Table 4.1). YTX was originally isolated from Japanese scallops (Murata et al., 1987), along with hydroxyyessotoxin and trinoryessotoxin. Homoyessotoxin and hydroxyhomoyessotoxin were isolated and identified after an incident of unusual mouse bioassay deaths in the Adriatic region of Italy (Satake et al., 1997a). Adriatoxin was isolated from the digestive glands of Mytilus galloprovincialis (Ciminiello et al., 1998) and desulfoyessotoxin from Norwegian Mytilus edulis (Daiguji et al., 1998b). All of the isolation procedures used in these studies were based on the original method of Murata et al., (1987). This isolation procedure used acetone to extract the digestive glands of scallops (Patinopecten yessoensis). This extract was then partitioned between petroleum ether and methanol-water and the consequent methanolic layer was re-extracted using butanol. This was loaded onto an alumina column and dichloromethane-methanol was used to wash lipidic compounds and PTXs from the column. The YTXs were eluted with 1% (v/v) ammonia in methanol (1:1). Further purification was using a series of four preparative chromatographic steps (Murata et al., 1987). Extraction and Clean Up Methods As well as acetone, aqueous methanol has been used as an extraction solvent for YTXs in shellfish. Yasumoto and Takizawa (1997) reported that extraction of scallop and mussel digestive glands with 8:2 (v/v) methanol-water recovered ~90% of spiked YTX into the solvent. Another study, using 9:1 (v/v) methanol-water and a C18 solid phase extraction clean up with LC-MS detection, obtained recoveries of 69% and 70% (1.6 µg g-1 YTX/45-OH YTX, n = 2) for yessotoxin and hydroxyyessotoxin from scallop digestive glands, respectively (Goto et al., 2001). A silica solid phase extraction clean up was also applied to the samples after extraction into chloroform. This was based on Yasumoto and Takizawa’s (1997) HPLC-FLD method for the clean up of shellfish sample prior to derivatisation with DMEQ-TAD. The resultant recoveries of YTX (only) from adductor muscle and digestive glands, were 90% (0.8 µg g-1, n = 2) and 80% (1.6 µg g-1, n = 5) respectively. Recovery of hydroxyyessotoxin was considered poor at 79% (1.6 µg g-1, n = 2) (Goto et al., 2001). Information from the Goto study is summarised in Table 4.3.


19

TABLE 4.3 Summary of recoveries of YTX and YTXOH after clean up (Goto et al., 2001).

Recovery Tissue Spiking level

(µg g-1) SPE YTX YTXOH

1.6 C18 69 %

(n = 2) 70 %

(n = 2) Scallop digestive glands 1.6 Silica 80 %

(n = 5) -

Scallop adductor muscle 0.8 Silica 90 % (n = 2) -

n = number of replicates Methods of Determination There are a number of methods for the detection of YTXs available in the literature. These include the mouse bioassay, a cytotoxicity test, an enzyme linked immunosorbent assay (ELISA) and HPLC with FLD, UV or MS detection. These methods are described in more detail below. Analytical Methods YTXs, with the exception of carboxyyessotoxin, contain a conjugated diene that allows ultraviolet detection. Unfortunately, the sensitivity is low, due to the diene being a very weak chromophore. Greater sensitivity can be achieved by derivatising the YTXs with a dienophile and using a fluorescent detector. One such method involved the use of DMEQ-TAD (4-[2,-(6, 7-dimethoxy-4-methyl-3-oxo-3, 4-dihydroquinoxalinyl) ethyl]-1, 2, 4-triazoline-3,5-dione) as a reagent and was shown to be effective and sensitive for the analysis of shellfish for YTX (Yasumoto and Takizawa, 1997). The method involves two solid phase extraction stages (C18 and silica chemistries) with an intermediary derivatisation step in dichloromethane at room temperature for two hours. Derivatisation gave two adducts (two epimers) both of which could be detected by HPLC-FLD. The method was able to distinguish between the YTX, hydroxyyessotoxin and trinoryessotoxin adducts chromatographically. However, the homoyessotoxin adducts were shown to co-elute with the YTX adducts using a C18 column with 40 mM phosphate buffer (pH 5.8) in methanol (3:7 (v/v)) as the mobile phase. The recovery of YTX, from the digestive glands, had a mean value of 94% (0.2-20 µg g-1, n = 3). Good linearity of the calibration (R2 = 0.998) was observed over the range 1-100 ng YTX on column. An estimated detection limit of 1 ng on column indicated the method was 2000 times more sensitive than the mouse bioassay. This validation information is summarised in Table 4.4. The derivatisation method has been successfully applied to the analysis of YTX in mussels (Ramstad et al., 2001) in Norway making it possible to evaluate the monthly variability of YTX in the mussels.


20

TABLE 4.4 Summary of validation details reported for YTX determination by HPLC-FLD (Yasumoto and Takizawa, 1997).

Tissue Spiking range

(µg g-1) SPE Recovery

Linear Calibration

Range Limit of

detection

Scallop digestive glands 0.2 - 20 C18 / silica

94 % (n = 3)

1 –100 ng on column (R2 = 0.998)

1 ng on column

Analysis of YTXs has also been undertaken using LC-MS and the methods published to date are summarised in Table 4.5. The majority of the cited references utilised the negative ion mode and electrospray ionisation. Ito and Tsukada (2002) used a sonic spray interface (SSI) which is similar to an electrospray interface. Draisci et al. (1998) found that YTX does not ionise in positive ion mode using a mixture of trifluoroacetic acid, acetonitrile and water as the mobile phase. No additional information is available in the literature to support this finding. The most intense peaks observed for YTXs in single ion monitoring (SIM) mode were [M-2Na+H]- (m/z 1141) and [M-Na]- (m/z 1163) (Draisci et al., 1998a; Goto et al., 2001). Using selective reaction monitoring (SRM), LC-MS/MS detected [M-SO3Na+H-Na]-, [M-SO3Na+H-Na-137]- and [M-SO3Na+H-Na-206]- fragments with the transitions m/z 1141→1061, m/z 1061→924 and m/z 1061→855. These were the most intense peaks (Draisci et al., 1998). The LC-MS and LC-MS/MS studies which report calibration data showed good linearity (R2 > 0.995) over a range of ~0.040-1.6 µg ml-1 (Table 4.5). In comparison to Yasumoto and Takizawa’s HPLC-FLD method (1997), LC-MS (Ito and Tsukada, 2002) is up to 16 times more sensitive as HPLC-FLD and up to 8 times as sensitive as LC-MS/MS (Draisci et al., 1998). LC-MS/MS is twice as sensitive as HPLC-FLD (Draisci et al., 1998). Nevertheless, more investigative work, especially in the area of method development of the other YTX analogues, is required but lack of commercially available standards hinders progress. Other Methods Use of the modified DSP mouse bioassay (Yasumoto et al., 1984), using a secondary extraction with diethyl ether, is considered inadequate for the detection of YTX, due to poor extraction of YTX in diethyl ether (Draisci et al., 1998; Ramstad et al., 2001). A false negative result has been demonstrated by Draisci et al. (1998) after comparison of the modified DSP mouse bioassay with the LC-MS/MS method. Ethyl acetate has also been shown to be an inefficient solvent for the extraction of YTXs (Goto et al., 2001). The original DSP mouse bioassay, using acetone extractions, (Yasumoto et al., 1978) is reported to facilitate YTX extraction, but leads to an increase in the number of “false positives” due to the effects of PSP toxins and high levels of free fatty acids (Draisci et al., 2001). The detection of YTXs can be potentially accomplished by a cytotoxicity test (Aune, 1998) however this has yet to be applied to shellfish samples. An ELISA technique is under development (Lyn Briggs, Pers. Comm.).

TABLE 4.5 Summary of published YTX LC-MS methods

Reference Yessotoxins & ions determined

Ionisation technique and mode Conditions Comments


YTX [M-2Na+H]- m/z 1141 [M-SO3Na+H-Na]- m/z 1061 [M-SO3Na+H-Na-137]- m/z 859 [M-SO3Na+H-Na-206]- m/z 859

LC-MS/LC-MS/MS (SIM/Full scan/SRM) ESI Negative

Supelcosil LC18-DB (Bellefonte, PA, USA) particle size 5 µm, 300 x 1 mm Isocratic: 80:20 (v/v) Acetonitrile-4 mM ammonium acetate. Flow rate 30 µl min-1

Linear range used: 0.2-1.5 µg ml-1 Correlation >0.996 Limit of detection 50 ng ml-1 (~25 ng g-1)

Draisci et al., 1999a

YTX [M-Na]- m/z 1163 [M-SO3Na+H-Na]- m/z 1061 [M-SO3Na+H-Na-137]- m/z 924 [M-SO3Na+H-Na-206]- m/z 854

homoYTX [M-Na]- m/z 1177 [M-SO3Na+H-Na]- m/z 1075 [M-SO3Na+H-Na-137]- m/z 938 [M-SO3Na+H-Na-206]- m/z 868

LC-MS/MS (SIM/Full scan/SRM) ESI Negative

Supelcosil LC18-DB (Bellefonte, PA, USA) particle size 5 µm, 300 x 1 mm Isocratic: 80:20 (v/v) acetonitrile-4 mM ammonium acetate. Flow rate 30 µl min-1

Linear range used: 0.1-1.5 µg ml-1 Correlation >0.996 Limit of detection 50 ng ml-1 (~25 ng g-1)

Draisci et al., 1999b YTX [M-2Na+H]- m/z 1141

LC-MS (SIM) ESI Negative

Supelcosil LC18-DB (Bellefonte, PA, USA) particle size 5 µm, 300 x 1 mm Isocratic: 80:20 (v/v) acetonitrile-2 mM ammonium acetate. Flow rate 40 µl min-1

Goto et al., 2001

YTX & [M-Na]- m/z 1163

45-OH YTX [M-Na]- m/z 1179

LC-MS (SIM) ESI Negative

Inertsil ODS-3 (GL Science, Tokyo, Japan) particle size 5 µm, 15 x 2.1 mm, 1 µl injected Isocratic: 80:20 (v/v) methanol-0.2M ammonium acetate. Flow rate 100 µl min-1 @ 40°C

Linear range used: 0.040-1.6 µg ml-1 Correlation >0.997 Limit of detection 40 ng g-1 & 80 ng g-1 (muscle and digestive gland respectively)

Ito and Tsukada, 2002 YTX [M-2Na+H]- m/z 1141

LC-MS (Full scan) SSI Negative

Inertsil ODS-2 (GL Science, Tokyo, Japan) particle size 5 µm, 15 x 2.1 mm, 10 µl injected Linear Gradient: A = 1 mM ammonium acetate B = MeOH 40-100% B over 20 mins. Hold 100%B for 10 mins. Return to 40% B, hold 10 mins to equilibrate. Flow rate 200 µl min-1

Linear range used: 0.050-1 µg ml-1 Correlation >0.999 Limit of detection 6 ng ml-1

SIM – single ion monitoring SRM – selective reaction monitoring SSI – sonic spray ionisation ESI – electrospray ionisation

21


22

5. SPIROLIDES Spirolides are lipid soluble macrocyclic imines (Fig. 5.1), which resemble the marine biotoxins known as pinnatoxins in structure (Falk et al., 2001). Other cyclic imine marine biotoxins include prorocentrolides, gymnodimines and pteriatoxins (Quilliam, 2002). At present, seven structures have been elucidated (Table 5.1; Fig. 5.1) and an additional five analogues have been proposed (Cembella et al., 1999). These analogues are thought to be two isomers of spirolide C and three isomers of spirolide D, one being the desmethyl analogue. At this time, no human intoxication incidents have been reported due to spirolides, however the structurally related pinnatoxins, have been associated with human illness (Otofugi et al., 1981). Figure 5.1. Structures of Spirolides

TABLE 5.1 Molecular weights of Spirolides

Name Mol Weight

(g mol-1) Structural elucidation

reference(s) Spirolide A 691.5 13-desmethyl spirolide C 691.5 Hu et al., 2001

Spirolide B 693.5 Hu et al., 1995 Spirolide C 705.5 Hu et al., 2001 Spirolide D 707.5 Hu et al., 1995 Spirolide E 709.5 Spirolide F 711.5

Hu et al., 1996

denotes a double bond between C2 and C3


23

Occurrence Spirolides A to F have been found in the digestive glands of both scallops (Placopecten magellianicus) and mussels (Mytilus edulis) (Hu et al., 1995). The dinoflagellate, Alexandrium ostenfeldii, has been identified in the field as producing spirolides A to D and their related isomers (Cembella et al., 1999). Recently, this dinoflagellate has been identified in Scottish waters and the North Atlantic (Ruhl et al., 2001), and has been previously found in Galician waters (Taylor, 1995). Spirolides E and F have not yet been encountered in phytoplankton samples and it has been suggested that these compounds are produced by spirolides A and B, respectively, undergoing chemical or enzymatic hydrolysis in shellfish (Hu et al., 2001). To date, reported occurrences of spirolides are limited to shellfish from aquaculture sites on the Nova Scotia coast of Canada. However, these occurrences have been shown to recur annually on a seasonal basis at these sites, highlighting the need for regular monitoring (Cembella et al., 1999). Toxicology Spirolides A to D have been observed by Hu et al. (1996) to cause rapid mouse death (3-20 minutes) upon both oral and intraperitoneal administration (i.p.). Symptoms include piloerection, abdominal muscle spasms, hyperextension of the back, convulsions and arching of the tail. The pharmacological mode of action is unknown although the presence of the cyclic imine is thought to be important as toxicity is significantly reduced upon reduction of the imine to an amine. There is some data indicating a different mode of action to those of ASP, DSP and PSP toxins (Hu et al., 1996; Richard et al., 2001). In mice it is possible to detect a dose of 5 µg of purified spirolide A to D given intraperitoneally (Hu et al., 1996) and a lethal dose (LD100) of 0.25 µg kg-1 has been proposed (Hu et al., 1995). Spirolides E and F are not detectable by intraperitoneal injection of mice at four times the aforementioned dose, this is believed to be due to the absence of the cyclic imine (Hu et al., 1996). A further study (Richard et al., 2000) proposed LD50 values of 40 µg kg-1 and 1 mg kg-1 for intraperitoneal (i.p.) and oral toxicity respectively. These values were based on an experiment, which involved injecting a plankton extract, containing mainly desmethyl spirolide C, into mice and observing the reaction. In previous experiments with spirolides A to D similar i.p. toxicities were observed (Hu et al., 1996). Isolation Spirolides A to F were first isolated from the digestive glands of shellfish in small quantities by Hu et al. (1995), using aqueous methanol extraction of the tissue with successive partitioning between hexane and chloroform. The residue of the chloroform extract was purified by a series of normal phase silica gel, reversed phase C18, and gel permeation chromatography. From this initial isolation, spirolides B, D, E and F were characterised (Hu et al., 1995 and 1996), however insufficient material was recovered to characterise spirolides A, C and desMe-C. These were subsequently isolated from scallop digestive glands and phytoplankton biomass and their structures elucidated (Hu et al., 2001). Extraction and Clean Up Methods Extraction of the spirolides from shellfish and phytoplankton, has been facilitated by the use of methanol, with partitioning with hexane and chlorinated solvents (Hu et al., 2001; Quilliam et al., 2001b). A solid phase extraction procedure, that combines the use of cation exchange and hydrophilic-lipophilic interaction, has been developed (Quilliam et al., 2001b), although no validation data was reported. There are no other published extraction methods available. Due to the limited availability of pure spirolides, method development has been hindered, although culturing of spirolide-producing Alexandrium ostenfeldii for production of


24

standards is possible (Cembella et al., 1999). No recovery data for the spirolides is available. Methods of Determination LC-MS is the only technique cited in the literature for the detection of these compounds. Analytical Methods LC-MS and LC-MS/MS have been used for the detection and accurate molecular weight measurement of spirolides (Hu et al., 1995; 1996; 2001). The published LC-MS methods are summarised in Table 5.2. The three methods reported, all use positive ion mode and electrospray ionisation. Quilliam et al. (2001b) obtained a limit of detection of ~3 ng g-1 for scallop tissue while Cembella et al. (1999) achieved a limit of detection of 2 ng ml-1 for a phytoplankton sample. Both researchers used LC-MS and detailed validation data was not reported. Full scan spectra of spirolide A by LC-MS indicated successive loss of water from the molecule (Quilliam et al., 2001b). The MS/MS of spirolides (Hu et al. 1995; 1996; 2001; Quilliam et al., 2001b) found fragment ions due to the cleavage of the lactone ring from the molecular ion to produce intense and distinctive fragments found at m/z 164 and 150 for spirolides C/D and A/B, respectively. This indicated the imine ring fragment produced upon collision induced dissociation. Other intense and distinctive fragments had mass to charge ratios of 444 and 458. Validation data for LC-MS/MS of spirolides is not available.

TABLE 5.2

Summary of the published spirolide LC-MS methods

Reference Spirolides & ions determined Ionisation technique and mode Conditions Comments

Cembella et al., 1999

Spirolides A to D and isomers Spirolide A [M+H]+m/z 692.5 Spirolide B [M+H]+m/z 694.5 Spirolide C [M+H]+m/z 706.5 Spirolide D [M+H]+m/z 708.5

LC-MS (SIM) ESI Positive

Method 1: Vydac 201TP (Separations Group, Hesperia, USA) particle size 3 µm, 250 x 2 mm Isocratic: 0.1% (v/v) trifluoroacetic acid in 35:65 (v/v) acetonitrile-water Flow rate 0.2 ml min-1 Method 2: Zorbax Rx C18 (Agilent Technologies, Ontario, Canada), particle size 5 µm, 150 x 2.1 mm Isocratic: 2 mM ammonium formate, 50 mM formic acid in 35:65 (v/v) acetonitrile-water. Flow rate 0.2 ml min-1 (split 20 µl min-1)

Method 1 LOD for spirolide B: 2 ng ml-1 (50 pg on column)


Spirolides A to Spirolide A [M+H]+m/z 692.5 Spirolide B [M+H]+m/z 694.5 Spirolide C [M+H]+m/z 706.5 Spirolide D [M+H]+m/z 708.5

LC-MS/LC-MS/MS (SIM/Full scan) ESI Positive

Hypersil BDS C8 (Keystone Scientific, Bellefonte, USA) particle size 3 µm, 50 x 2.1 mm Gradient: A=H2O B=AcN (2 mM ammonium formate, 50 mM formic acid) 95% A to 100% B over 10 minutes Hold for 10 minutes Re-equilibrate for 7 minutes Flow rate 0.2 ml min-1

Quilliam et al., 2001b

Spirolides A to D and isomers Spirolide A [M+H]+m/z 692.5 [M-nH20]+ (n = 1 to 3) m/z 644.5-674.5 Spirolide B [M+H]+m/z 694.5 [M-nH20]+ (n = 1 to 3) m/z 646.5-676.5 Spirolide C [M+H]+m/z 706.5 [M-nH20]+ (n = 1 to 3) m/z 658.5-688.5 Spirolide D [M+H]+m/z 708.5 [M-nH20]+ (n = 1 to 3) m/z 660.5-690.5

LC-MS/LC-MS/MS (SIM/Full scan) ESI Positive

Hypersil BDS C8 (Keystone Scientific, Bellefonte, USA) particle size 3 µm, 50 x 2.1 mm 2 mM ammonium formate, 50 mM formic acid in 35:65 (v/v) acetonitrile-water. Flow rate 0.2 ml min-1 (split 20 µl min-1)

LOD:~3 ng g-1 (shellfish using SPE clean up)

SIM – single ion monitoring ESI – electrospray ionisation

25


26

6. FREE FATTY ACIDS Fatty acids are found in every living organism in nature as components of waxes, mono-, di- and triglycerides, phospholipids, lipoproteins and lipopolysaccharides as well as other natural compounds. Common fatty acid molecules can easily have over 40 carbon atoms. Their diverse range of structures may be saturated/unsaturated, branched, cyclic, cis/trans in configuration and can have additional functional groups such as keto, epoxy and hydroxy groups. The simplest structure of a fatty acid is ethanoic acid (C2H4O2). Fatty acids can exist in bound or free forms. Bound fatty acids are generally covalently bonded to other molecules such as proteins whereas free fatty acids are not bound. Brondz (2001) has recently reviewed fatty acid structures, nomenclature and methods of determination. Bound fatty acids in shellfish dominate the lipid content (>70% of total lipid content) (Table 6.1). The major bound fatty acids of some phytoplankton are given in Table 6.2. The main bound fatty acids are found in the phospholipids and the triglycerides of both plankton and shellfish. Environmental conditions, such as irradiance, influence the fatty acid composition of phytoplankton (Thompson et al., 1990 and 1996; Brown et al., 1996). Diet influences the fatty acid composition of shellfish and zooplankton (Lytle et al., 1990 for Crassostrea virginica; Frolov and Pankov, 1992 for Ostrea edulis; Samain et al., 2000 for Pecten maximus and Crassostrea gigas) and in general shellfish fatty acid composition reflects the fatty acid composition in the phytoplankton.

TABLE 6.1 Shellfish lipid and bound fatty acid content (adapted from Exler and Weihrauch, 1977)

Species Total lipids (% wet weight) Total bound fatty acids

(% total lipids) Mytilus edulis 2.0 82.0 Modiolus barbatus 2.0 81.5 Crassostrea gigas 2.3 82.2 Ostrea edulis 1.7 71.2 Pecten sp. 1.5 75.3

Dominant marine fatty acid example structures are given in Figure 6.1.


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TABLE 6.2 Phytoplankton (>5% w/w of total bound fatty acids, bold indicates main acid found in phytoplankton species)

Unsaturated Species Saturated Mono- Poly- Reference

Pavlova pinguis 18:4n-3, 20:5n-3 and 22:6n-3

Isochrysis sp. 18:2n-6, 18:3n-3, 18:4n-3 and 22:6n-3

Dunaliella tertiolecta

Data not available

16:4n-3, 18:2n-6, and 18:3n-3

McCausland et al., 1999

Thalassiosira weissflogii H1

14:0 and 16:0 16:1 20:5n-3 and 22:6n-3

(<5%) Ishida et al., 2000

Phaeodactylum tricornutum

14:0 and 16:0 16:1 20:5n-3 and 22:6n-3

Chaetoceros sp. 14:0 and 16:0 16:1 16:3, 20:4n-6,

20:5n-3 and 22:6n-3Isochrysis galbana

14:0 and 16:0 18:1 18:2n-6, 20:5n-3

and 22:6n-3

Pavlova lutheri 14:0 and 16:0 16:1 18:4n-3, 20:5n-3

and 22:6n-3

Tetraselmis sp. 16:0 18:1 16:4, 18:2n-6,

18:3n-3, 18:4n-3 and 20:5n-3

Nannochloris atomus

14:0 and 16:0

16:1 and 18:1

20:4n-6, 20:5n-3 and 22:6n-3

Gymnodinium sp.

14:0 and 16:0

16:1 and 20:11

18:4n-3, 20:5n-3 and 22:6n-3

Reitan et al., 1997

Tetraselmis suecica 16:0 18:1n-9 18:3n-3, 18:4n-3

and 20:5n-3 Caers et al., 2000

Figure 6.1. Structures of dominant fatty acids in phytoplankton and shellfish

Hexadecanoic acid (palmitic acid)

cis-5,8,11,14,17-Eicosapentaenoic acid (EPA, 20:5n-3)

cis-4,7,10,13,16,19-Docosahexaenoic acid (DHA, 22:6n-3)


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Occurrence Free fatty acids, from red tides, were responsible for fish kills in Japan in 1977, 1978, and 1982 (Okaichi, 1985). It was suggested that the free fatty acids destroyed surface cells of the fish gills; there was no evidence of other toxic substances. Frolov and Pankov (1992) found free fatty acids to be present at low levels in some phytoplankton (2.2-6.1% w/w of neutral lipids) and shellfish (0.1-0.2 % w/w of neutral lipids), the majority of fatty acid being in the bound form. Dunstan et al. (1993) found free fatty acids to account for 0-29.2% w/w of the total lipids in 14 species of diatoms, highlighting the high interspecies variability of the free fatty acid content. However increased levels of free fatty acids have been linked with the degradation of sample integrity in both phytoplankton (Berge et al., 1995; Baldi et al., 1997) and shellfish (Balasundari et al., 1997; Hatate et al., 1992). Upon treatment with boiling water, prior to lipid extraction, Berge et al. (1995) and Budge with Parrish (1999) illustrated phytoplankton had no free fatty acids present. However, non-treated phytoplankton was found to contain significant concentrations of free fatty acids. An increase in the concentration of free fatty acid was observed in Crassostrea madrasensis upon storage at -18ºC (Balasundari et al., 1997). This degradation effect was also observed in C. gigas, especially in the phospholipid, between 4 to -15ºC (Hatate et al., 1992). The suggested cause was the hydrolytic action of enzymes, such as lipases, to produce free fatty acids from bound fatty acids (Saijiki, 1996; Hatate et al., 1992). However, at the lower temperature of –25ºC the enzymatic hydrolysis of free fatty acids was substantially inhibited (Hatate et al., 1992) although oxidation still occurred. Therefore storage below this temperature (-25ºC) would prevent the release of fatty acids from the phospholipid and triglyceride layers. Toxicity in Mice The toxicity of free fatty acids towards mice, in particular DHA and EPA, has been discussed in a number of published papers. The lethal effects in mice were found in the lipid fraction extracted from oysters (Mori et al., 1995; Sajiki et al., 1995), scallops (Takagi et al., 1982), anchovies (Sajiki and Takahashi, 1992) and mussels (Lawrence et al., 1994; Suzuki et al., 1996). Details of the toxicology of free fatty acids were not reported in any of these publications although Sajiki and Takahashi (1992) and Lawrence et al. (1994) gave information on the symptoms of the mouse reaction to a free fatty acid toxic shellfish extract. Sajiki and Takahashi (1992) described the symptoms as ‘immobilisation and crouching down, but not diarrhoea’. Lawrence et al. (1994) described the symptoms ‘abdominal stretching, lying with abdomen flat on the cage floor, hunched back, walking with side to side motion (waddling), an uneven appearance of the coat, jerky head and/or whole body movements, laboured breathing, partially closed eyes and inactivity. The mice that died slept and/or became semicomatose …’. Takagi et al. (1982) documented one of the first incidents of toxicity of free fatty acids in mice. The fat-soluble fraction of the scallop, Patinopecten yessoensis, was found to contain ~400 mg g-1 (calculated from reference Takagi et al., 1982) of free fatty acids. A number of free polyunsaturated fatty acids were reported to be toxic to mice with the two major toxic components being EPA and DHA. However no concentrations of EPA and DHA were given. In the hepatopancreas of mussels (Mytlius edulis) from Nova Scotia, high concentrations of free fatty acids (1.2-2.9 mg g-1) were found in mussels which had given a positive mouse bioassay result (Lawrence et al., 1994, Yasumoto et al., 1984). In comparison, control mussels (negative mouse bioassay result) contained 0.15 mg g-1 of free fatty acids. HPLC and immunoassay techniques showed no significant presence of DSP toxins in the samples (Lawrence et al., 1994). The potential source of this lethality was investigated by assessing


29

the lipid composition of the toxic samples and comparing the value to that of a control mussel. Both the control and toxic samples were compared using both the DSP bioassay extraction with a additional lipid extraction technique (Bligh and Dyer, 1959) and using the Bligh and Dyer method alone. A major difference in the compositions was in the free fatty acid content, the control having a much lower content of free fatty acids than the toxic extracts. Another difference was shown to be in the extraction techniques. The DSP bioassay extraction extracted less free fatty acids than the Bligh and Dyer method, as would be expected, as the DSP bioassay purpose is not the extraction of free fatty acids. Once the composition of the control and toxic extracts had been ascertained, this was duplicated and injected into the mice. The predominant fatty acids were found to be polyunsaturated (18:2n-6, 18:3n-3, 18:4n-3 and EPA). The injection of the duplicate sample resulted in the mice dying however when the sample mass equivalent value was lowered by 40% the mice did not die. Saturated fatty acids, 14:0 and 16:0, were established as being non-contributory to the toxicity of the extracts (Lawrence et al., 1994). Mouse lethal extracts have also been extracted from Japanese anchovies (Engraulis japonica) (Sajiki and Takahashi, 1992). The predominant fatty acids were found to be polyunsaturated (18:2, 18:3, 18:4, 20:4, DHA and EPA). EPA, linolenic (18:3) and arachidonic (20:4) acids were found to have equivalent toxicity whereas linoleic (18:2) and DHA were found to be less toxic. This paper reports that EPA was injected as a pure compound into mice and found to have a lethal effect. Mori et al. (1995) found DHA, EPA and their oxides to inhibit the growth of Escherichia coli and to cause mouse deaths upon intraperitoneal injection after five hours (Table 6.3). High mouse toxicity of crude lipid fractions from C. gigas digestive glands, preserved in 4% acetic acid at 37ºC for 3 hours, showed increases in the molar concentrations of saturated, monounsaturated and polyunsaturated fatty acids. These increases were 12.6, 23.7 and 37.8 fold, respectively, in the free fatty acid fraction when compared to non-treated glands (Sajiki et al., 1995). Changes in the profiles of the triglyceride, phospholipid and free fatty acid fractions were reported and large increases in free polyunsaturated fatty acids (18:3n-3, 18:4n-3, 20:4n-6, 20:5n-3 and 22:6n-3) for the treated samples. Further work suggested that this effect was as a result of cellular degradation through activation of endogenous enzymes, under acidic conditions (Sajiki, 1996). Suzuki et al. (1996) conducted research on the free fatty acid content of mussel (Mytilus coruscus) hepatopancreas on a weekly basis over a period of one month. Samples were extracted using acetone, evaporated, reconstituted in 80% methanol and re-extracted with hexane. This hexane extract was subsequently derivatised with ADAM (9-anthryldiazomethane) prior to analysis by HPLC-FLD. The major free fatty acids were 16:0, 16:1n-7, EPA and DHA. The proportion of EPA and DHA increased notably, over the month, while 16:0 and 16:1n-7 was relatively stable. It was noted that one of the samples could “give rise to a false-positive in the official mouse bioassay for DSP toxins”. The above reports are summarised in Table 6.3.


30

TABLE 6.3 Summary of free fatty acids, extracted from shellfish, which have been associated with mouse mortality

Reference Species Fatty acid associated with toxicity in mice

Takagi et al., 1982 Patinopecten yessoensis EPA and DHA Sajiki and Takahashi, 1992 Engraulis japonica 18:2, 18:3, 18:4, 20:4,

DHA and EPA Lawrence et al., 1994 Mytilus edulis 18:2, 18:3, 18:4 and EPA

Sajiki et al., 1995 Crassostrea gigas 18:3, 18:4, 20:4, DHA and EPA

Mori et al., 1995 Crassostrea gigas EPA, DHA and oxides Suzuki et al., 1996 Mytilus coruscus EPA and DHA

Isolation Published papers on the isolation of free fatty acid compounds from phytoplankton and shellfish were not found. There seems to be a limited number of papers detailing the isolation of bound polyunsaturated fatty acids from phytoplankton (Robles Medina et al., 1998; Belarbi et al., 2000), with the majority of papers on other sources such as fish oil (Belarbi et al., 2000). Methods of Determination The extraction of lipids, in general, is carried out by one of two methods, either Folch et al. (1957) or Bligh and Dyer (1959). These extractions both utilise an organic solvent mixture of 2:1 chloroform-methanol to extract the lipids. Analysis is usually facilitated by the derivatisation of fatty acids to fluorogenic or more volatile compounds such as fatty acid methyl esters (FAMEs). The use of chromatographic methods, such as gas chromatography (GC) and high performance liquid chromatography (HPLC), is widespread, GC being the most popular. The sensitivities of the detectors used in fatty acid analyses are given in Table 6.4. Two excellent reviews have been published on this matter (Gutnikov, 1995; Brondz, 2001). Investigations into the fatty acids in shellfish and phytoplankton have been carried out by methods detailed in these reviews, however little is published, in general, on the analysis of free fatty acids in these two matrices. The analysis of polyunsaturated fatty acids by LC-MS is possible (Yamane, 2001) and papers exist for matrices such as crustaceans (Rezanka, 2000) but give no validation data or limits of determination. Therefore there is need for more research in this area.


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TABLE 6.4 Common detection techniques used for the fatty acid analysis (adapted from Brondz, 2001 and Gutnikov, 1995)

Technique Common Detection Techniques Sensitivity

(on column) Thermal conductivity detector (TCD) Nanograms

Flame ionisation detector (FID) Picograms Electron capture detector (ECD) Femtograms GC

Nitrogen phosphorus detector (NPD) Picograms Ultraviolet detection (UV) Nano- to picograms HPLC Fluorescence (FLD) Picograms

7. CONCLUSIONS From the information presented in this review, it is clear that AZAs, YTXs, and PTXs have been detected in European shellfish and phytoplankton. The establishment of accurate and reliable methods for the detection of these toxins, within monitoring programmes across Europe, is essential for the protection of human health. This will become a formal requirement of EU Member States if the draft Decision SANCO/2227/2001 Rev 3 is adopted. The draft Decision states that the animal test known commonly as the DSP mouse bioassay (Yasumoto et al., 1978) should be used in monitoring and details various extraction techniques for sample preparation. It also allows for the introduction of non-animal methods providing they have been validated. Curiously, the mouse bioassay in its various formats has not, to the authors’ knowledge, undergone any formal validation trials. The DSP mouse bioassay has various shortcomings, some of which are summarised in Table 7.1. One of these is potential interference from free fatty acids, details of which are given in Section 6.


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TABLE 7.1 Summary of DSP mouse bioassay shortcomings regarding new toxins

Toxin Group Known mouse bioassay

shortcomings Reference(s)

Azaspiracids Poor extraction in acetone *+ James et al., 2000

Yessotoxins Poor extraction in diethyl ether + Draisci et al., 1998 Ramstad et al., 2001

Pectenotoxins Unknown, no data published Spirolides Unknown, no data published

Free fatty acids Known to cause false positives due to toxicity in mice* Lawrence et al., 1994

*(Yasumoto et al., 1978) +(Yasumoto et al., 1984) Also covered in this review is information on spirolides (Section 5). The potential hazard to humans from exposure to these toxins is currently unknown and at this stage spirolides have not been reported in shellfish from Europe. The causative agent, A. ostenfeldii was detected in waters around the arctic north-west coast of Norway (Okolodkov and Dodge, 1996), Denmark (Ruhl et al., 2001) and Spain (Taylor et al., 1995). Ruhl et al. (2001) has also found low concentrations of spirolides in phytoplankton samples collected from the east coast of Scotland and the North Atlantic. It is therefore beneficial to be pro-active in developing detection methods for this toxin should the need arise to investigate shellfish samples. There is an obvious requirement to introduce non-animal methods in monitoring for these lipophilic compounds in shellfish in terms of both a) ethical consideration and b) the provision of accurate data specific to the individual toxins. As detailed throughout this literature review LC-MS seems to be the most promising alternative technique for toxins. A number of LC-MS methods for toxin detection have been published however, they currently suffer a number of shortcomings: • Qualifier ions for LC-MS require investigation for all toxins and more distinctive LC-

MS/MS qualifier ions are required for AZAs and PTXs. • Development of validated extraction and clean up techniques are required

particularly for AZAs, spirolides and some of the YTXs and PTXs. • Complete validation data for analysis is required, this includes spiked recoveries for

all compounds and matrices. Qualifier ions are important for LC-MS as their use improves the certainty of the identity of the shellfish toxin. All of the published LC-MS methods (Tables 2.3, 3.4, 4.5 and 5.2) have used a single ion for identification of the toxins and do not analyse for distinct fragment ions (qualifier ions). LC-MS methods for AZAs, YTXs, PTXs and spirolides have, to date, been unable to produce these qualifier ions and generally monitor for the molecular ion, sodium adduct or ammonium adduct. Successive losses of water from the molecular ion has often been used as qualifier ions, particularly for PTX-2 (Draisci et al., 1996) and the AZAs (James et al., 2000; Draisci et al., 2000), using LC-MS/MS. However, this loss is not specific, and is common in most organic molecules. Therefore efforts should be made for the detection of more distinctive qualifier ions (i.e. the loss of carboxylic groups). However, using LC-MS/MS distinctive qualifier ions were produced upon fragmentation for some of the


33

spirolides and yessotoxins (Tables 4.5 and 5.2). However, work may be required for the other toxins in these groups. In the published methods available validation data is minimal, particularly with respect to spiked recovery data for a range of shellfish matrices (Table 7.2). Often spiking is carried out following sample extraction and therefore only gives recoveries for the clean-up steps. A number of methods use lengthy and laborious extraction and clean-up procedures, which cannot easily be applied to routine monitoring situations. Within the new toxin groups not all compounds have been investigated and validation data is only available for some of these. Therefore research is required to develop fully validated, high throughput extraction and clean-up techniques capable of coping with monitoring programme demands. Unfortunately progress is somewhat hindered by the lack of commercially available standards. This is problematic due to: • Expense and need for high level of expertise to produce certified standards. • In most cases, lack of easy availability of highly contaminated material from which to

isolate the desired toxin, (e.g. phytoplankton sources can be difficult to culture in laboratory or insufficient source of contaminated shellfish).

Standards which are available are produced by the laboratory who wish to investigate the compound for a specific purpose (i.e. structural elucidation) or they have been received as a gift from a laboratory that has isolated the compound of interest. These standards are generally uncertified and have not been assessed for purity or stability as would be required for the certification process. R&D project B04004 has attempted to address this issue by working with scientists from Canada (Institute of Marine Biology, National Research Council, Canada) who are experienced in the production and certification of toxin standards. Standards produced will be used to validate the methods developed. Overall, development of LC-MS for toxin analysis is moving into an exciting phase with the availability of cheaper instrumentation and increased efforts to produce toxin standards. It has the ability to detect and quantify individual toxin compounds as well as offering the potential for conducting a general screen for a range of substances. This literature review summarises details on the toxins of interest and the LC-MS methods published up to January 2002. It highlights shortcomings in the LC-MS methods and areas requiring further development. The information will be kept updated and used to enhance the information available to the R&D project B04004.

8. REFERENCES Aune, T., Stabell O.B., Nordstoga K. and Tjota K. 1998. Oral toxicity in mice of algal toxins

from the diarrhetic shellfish toxin (DST) complex and associated toxins. J. Natural Toxins, 7, 141-158.

Aune, T., Sorby, R., Yasumoto, T., Ramstad, H. and Landsverk, T. 2002. Comparison of

oral and intraperitoneal toxicity of yessotoxin towards mice. Toxicon., 40, 77-82.

Balasundari, S., Shanmugam, S.A., Jasmine, G.L. 1997. Frozen Storage Performance of Edible Oyster Crassostrea madrasensis (Preston). J. Food Sci. Tech., 34, 434-436.

Baldi, F., Minacci, A. and Malej, A. 1997. Cell lysis and release of polysaccharides in

extensive marine mucilage assessed by lipid biomarkers and molecular probes. Marine ecology progress series, 1531/3, 45.


34

Belarbi, E., Molina, E. and Chisti, Y. 2000. A process for high yield and scaleable recovery of high purity eicosapentaenoic acid esters from microalgae and fish oil. Process Biochemistry, 35, 951-969.

Berge, J.P., Gouygou, J.P., Durand, P. and Dubacq, J.P. 1995. Reassessment of lipid

composition of the diatom, Skeletonema costatum. Phytochemistry, 39, 1017-1021. Bligh, E.G. and Dyer, W.J. 1959. J. Biochem. Physiol., 37, 991. Brondz, I. Development of fatty acid analysis by high-performance liquid chromatography,

gas chromatography, and related techniques. 2002. Analytica Chimica Acta, In Press, Uncorrected Proof.

Brown, M.R., Dunstan, G.A., Norwood, S.J. and Miller, K.A. 1996. Effects of harvest stage

and light on the biochemical composition of the diatom Thalassiosira pseudonana. Journal of Phycology, 32, 64-73.

Budge, S. and Parrish, C. 1999. Lipid class and fatty acid composition of Pseudo-nitzschia

multiseries and Pseudo-nitzschia pungens and effects of lipolytic enzyme deactivation. Phytochemistry, 52, 561-566.

Burgess, V., Seawright, A., Shaw, G. and Moore, M.R. 2001. Acute oral toxicity of

pectenotoxin-2 Seco acid and 7 epi-pectenotoxin-2 seco acid in mice. Toxicology Abstracts, 164, 204.

Burgess, V. and Shaw, G. 2001. Pectenotoxins-an issue for public health. A review of their

comparative toxicology and metabolism. Environment International, 27, 275-283. Caers, M., Coutteau, P. and Sorgeloos, P. 2000. Incorporation of different fatty acids,

supplied as emulsions or liposomes, in the polar and neutral lipids of Crassostrea gigas spat. Aquaculture, 186, 157-171.

Cembella, A.D., Lewis, N.I. and Quilliam, M.A. 1999. Spirolide composition of micro-

extracted pooled cells isolated from natural plankton assemblages and from cultures of the dinoflagellates Alexandrium ostenfeldii. Natural Toxins, 7, 197-206.

Community Reference Laboratory, 2001. Report on the EU-NRLs intercalibration exercise

on DSP determination, April 2001. Ciminiello, P., Fattorusso E., Forino M., Magno S., Poletti, R., Satake, M., Viviani, R. and

Yasumoto, T. 1997. Yessotoxin in mussels of the northern Adriatic Sea. Toxicon, 35, 177-183.

Ciminiello, P., Fattorusso, E., Forino, M., Magno, S., Poletti, R., and Viviani R. 1998.

Isolation of adriatoxin, a new analogue of yessotoxin, from mussels of the Adriatic Sea. Tetrahedron Letters, 39, 8897-8900.

Ciminiello, P., Fattorusso E., Forino M., Magno S., Poletti R., and Viviani R. 2000. A new

analogue of yessotoxin, carboxyyessotoxin, isolated from Adriatic sea mussels. Eur. J Org. Chem., 291-295.

Daiguji, M., Satake, M., James, K.J., Bishop, A., MacKenzie, L., Naoki, H. and Yasumoto T.

1998a. Structures of new pectenotoxin analogs, pectenotoxin-2 seco acid and 7-epi-pectenotoxin-2 seco acid, isolated from a dinoflagellate and greenshell mussels. Chemistry Letters, 653-654.


35

Daiguji, M., Satake, M., Ramstad, H., Aune, T., Naoki, H. and Yasumoto, T. 1998b. Structure and fluorometric HPLC determination of 1-desulfoyessotoxin, a new yessotoxin analog isolated from mussels from Norway. Natural Toxins, 6, 235-239.

Daranas, A.H., Norte, M. and Fernandez, J.J. Toxic marine microalgae. Toxicon., 39, 1101-

1132. 2001. de la Rosa, L.A., Alfonso, A., Vilarino, N., Vieytes, M.R. and Botana, L.M. 2001. Modulation

of cytosolic calcium levels of human lymphocytes by yessotoxin, a novel marine phycotoxin. Biochem. Pharmacol., 61, 827-33.

Draisci, R., Lucentini, L., Giannetti, L., Boria, P. and Poletti, R. 1996. First report of

pectenotoxin-2 (PTX-2) in algae (Dinophysis fortii) related to seafood poisoning in Europe. Toxicon, 34, 923-935.

Draisci, R., Giannetti, L., Lucentini, L., Feretti, E., Palleschi, L. and Marchiafava, C. 1998.

Direct identification of yessotoxin in shellfish by liquid chromatography coupled with mass spectrometry and tandem mass spectrometry. Rapid Commun. Mass Spectrom., 12, 1291-1296.

Draisci, R., Palleschi, L., Giannetti, L., Lucentini, L., James, K.J., Bishop, A.G., Satake, M.,

and Yasumoto, T. 1999. New approach to the direct detection of known and new diarrhoeic shellfish toxins in mussels and phytoplankton by liquid chromatography-mass spectrometry. J. Chromatogr. A, 847, 213-221.

Draisci, R., Ferreti, E., Palleschi, L., Marchiafava, C., Poletti, R., Milandri, A., Ceredi, A. and

Pompei, M. 1999. High levels of yessotoxin in mussels and presence of yessotoxin in dinoflagellates of the Adriatic sea. Toxicon, 37, 1187-1193.

Draisci, R., Palleschi, L., Ferretti, E., Furey, A., James, K.J., Satake, M. and Yasumoto, T.

2000. Development of a method for the identification of azaspiracid in shellfish by liquid chromatography-tandem mass spectrometry. J. Chromatogr. A, 871, 13-21.

Exler, J. and Weihrauch J.L. 1977. Comprehensive evaluation of fatty acids in foods. J.

Am. Diet. Assoc., 71, 518-521. Falk, M., Burton, I.W., Hu, T., Walter, J.A. and Wright, J.L.C. 2001. Assignment of the

relative stereochemistry of the spirolides, macrocyclic toxins isolated from shellfish and from the cultured dinoflagellate Alexandrium ostenfeldii. Tetrahedron, 57, 8659-8665.

Fladmark, K.E., Serres, M.H., Larsen, N.L., Yasumoto, T., Aune, T. and Doskeland, S.O.

1998. Sensitive detection of apoptogenic toxins in suspension cultures of rat and salmon hepatocytes. Toxicon, 36, 1101-1114.

Flanagan, A.F., Kane, M., Donlon, J. and Palmer, R. 1999. Azaspiracid; detection of a

newly discoverd phycotoxin in vitro. J. Shell. Res., 18, 716. Flanagan, A.F., Callanan, K.R., Donlon, J., Palmer, R., Forde, A. and Kane M. 2001. A

cytotoxicity assay for the detection and differentiation of two families of shellfish toxins. Toxicon., 39, 1021-1027.

Folch, J., Lees, M. and Stanley, G.H.S. 1957. J. Biol. Chem., 226, 497. Frolov, A.V. and Pankov, S.L. 1992. The reproduction strategy of oyster Ostrea Edulis L.

from the biochemical point of view. Comp. Biochem. Physiol., 103, 161-182.


36

Goto, H., Igarashi, T., Yamamoto, M., Yasuda, M., Sekiguchi, R., Watai, M., Tanno, K. and Yasumoto. 2001. Quantitative determination of marine toxins associated with diarrhetic shellfish poisoning by liquid chromatography coupled with mass spectrometry. J. Chromatogr. A, 907, 181-9.

Gutnikov, G. 1995. Fatty acid profiles of lipid samples. J. Chromatogr. B, 671, 71-89. Hallegraeff, G.M. 1995. Harmful algal blooms: a global overview. In: Hallegraeff, G.M.;

Anderson, D.M., and Cembella, A.D., Eds. Manual on Harmful Marine Microalgae. Paris: IOC-UNESCO; pp. 1-24. (IOC Manual and Guides; v. 33).

Hatate, H., Hukada, S. and Imura, H. 1992. Lipid oxidation and hydrolysis in homogenates

of Japanese oyster during cold storage. Nippon Suisan Gakkaishi, 58, 495. Hess, P., McMahon, T., Slattery, D. 2001. Ongoing research on toxic algae. Proceedings

of the 2nd Irish Marine Biotoxin Science Workshop, 2001. Hu, T., Curtis, J.M., Oshima, Y., Quilliam, M.A., Walter, J.A., Watson-Wright, W.M. and

Wright, J.L.C. 1995. Spirolides B and D, two novel macrocycles isolated from the digestive glands of shellfish. J. Chem. Soc. Chem. Commun., 2159-2161.

Hu, T., Curtis, J.M., Walter, J.A. and Wright, J.L.C. 1996. Characterisation of biologically

inactive spirolides E and F: identification of the spirolide pharmacophore. Tetrahedron Lett., 37, 7671-7674.

Hu, T., Burton, I.W., Cembella, A.D., Curtis, J.M., Quilliam, M.A. and Wright, J.A. 2001

Characterisation of spirolides A,C and 13-Desmethyl C, new marine toxins isolated from toxic plankton and contaminated shellfish. J. Nat. Prod., 64, 308-312.

Ishige, M., Satoh, N. and Yasumoto, T. 1988. Pathological studies on mice administered

with the causative agent of diarrhetic shellfish poisoning (okadaic acid and pectenotoxin-2). Hokkaidoritsu Eisei Kenkyushoho, 38, 15-18.

Ito, E., Satake, M., Ofuji, K., Kurita, N., McMahon, T., James, K. and Yasumoto, T. 2000.

Multiple organ damage caused by a new toxin azaspiracid, isolated from mussels produced in Ireland. Toxicon, 38, 917-30.

Ito, E., Satake, M., Ofuji, K., Higashi, M., Harigaya, K., McMahon, T. and Yasumoto, T.

2002. Chronic effects in mice caused by oral administration of sublethal doses of azaspiracid, a new marine toxin isolated from mussels. Toxicon., 40, 193-203.

Ito, S. and Tsukada, K. 2002. Matrix effect and correction by standard addition in

quantitative liquid chromatographic-mass spectrometric analysis of diarrhetic shellfish poisoning toxins. J. Chromatogr. A, 943, 39-46.

James, K.J., Bishop, A.G., Draisci, R., Palleschi, L., Marchiafava, C., Ferretti, E., Satake, M.,

and Yasumoto, T. 1999. Liquid chromatographic methods for the isolation and identification of new pectenotoxin-2 analogues from marine phytoplankton and shellfish. J. Chromatogr. A, 844, 53-65.

James, K.J., Furey, A., Satake, M. and Yasumoto, T. 2000a. Abstract: Azaspiracid

poisoning (AZP): a new shellfish toxic syndrome in europe. International conference on Harmful Algal Blooms. Ninth conference Tasmania 2000.


37

James, K.J., Furey, A., Lehane, M., Moroney, C., Satake, M. and Yasumoto, T. 2001b. LC-MS methods for the investigation of a new shellfish toxic syndrome-azaspiracid poisoning (AZP). In: Mycotoxins and Phycotoxins in perspective at the turn of the millenium, eds. De Koe W. J. et al. 401-408.

Jung, J.H., Sim, C.J. and Lee, C.O. 1995. Cytotoxic compounds from a two-sponge

association. J. Natural Products, 58, 1722-1726. Lawrence, J.F., Chadha, R.K., Ratnayake, W.M.N. and Truelove, J.F. 1994. An incident of

elevated levels of unsaturated free fatty acids in mussels from Nova Scotia and their toxic effect in mice after intraperitoneal injection. Nat. Toxins, 2, 318-321.

Lee, J.S., Yanagi, T., Kenma, R. and Yasumoto, T. 1987. Fluorimetric determination of

diarrhetic shellfish toxins by high-performance liquid chromatography. Agric. Biol. Chem., 51, 877-881.

Lee, J.S., Murata, M. and Yasumoto, T. 1989. Analytical methods for determination of

diarrhetic shellfish toxins. Bioact. Mol., 10, 327-34. Lytle, J.S, Lytle, T.F and Ogle, J.T. 1990. Polyunsaturated fatty acid profiles as a

comparative tool in assessing maturation diets in enaeus vannmei. Aquaculture, 89, 287-299.

Mori, K., Saijiki, J. and Takahashi, H. 1995. Toxicity of lipophilic fractions extracted from

summer oysters. Jap. J. of Toxicology and Environmental Health, 41, 386-391 (in japanese with english abstract).

Murata, M., Sano, M., Iwashita, T., Naoki, H. and Yasumoto, T. 1986.The structure of

pectenotoxin-3, a new constitutent of diarrhetic shellfish toxins. Agric. Biol. Chem., 50, 2693-5.

Murata, M., Kumagai, M., Lee, J.S. and Yasumoto, T. 1987. Isolation and structure of

yessotoxin, a novel polyether compound implicated in diarrhetic shellfish poisoning. Tetrahedron Lett., 28, 5869-72.

Naoki, H., Murata M. and Yasumoto T. 1993. Negative-ion fast-atom bombardment tandem

mass spectrometry for the structural study of polyether compounds: structural verification of yessotoxin. Rapid Commun. Mass Spectrom., 7, 179-182.

Ofuji, K., Satake, M., McMahon, T., Silke, J., James, K.J., Naoki, H., Oshima, Y. and

Yasumoto, T. 1999a. Two analogs of azaspiracid isolated from mussels, Mytilus edulis, involved in human intoxication in Ireland. Nat Toxins, 7, 99-102.

Ofuji, K., Satake M., Oshima Y., McMahon T., James K.J. and Yasumoto T. 1999b. A

sensitive and specific determination method for azaspiracids by liquid chromatography mass spectrometry. Nat. Toxins, 7, 247-250.

Ofuji, K., Satake M., McMahon T., James K.J., Naoki H., Oshima Y. and Yasumoto T. 2001.

Structures of azaspiracid analogs, azaspiracid-4 and azaspiracid-5, causative toxins of azaspiracid poisoning in Europe. Biosci. Biotechnol. Biochem., 65, 740-2.

Ogino, H., Kumagai, M. and Yasumoto, T. 1997. Toxicologic evaluation of yessotoxin. Nat.

Toxins, 5, 255-259. Okaichi, T. 1985. Fish kills due to the red tides of Chattonella. Bull. Mar. Sci., 37, 772.


38

Otofuji, T., Ogo A., Koishi J., Matsuo K., Tokiwa H., Yasumoto T., Nishihara K., Yamamoto E., Saisho M., Kurihara Y. and Hayashida K. 1981. Food Sanit. Res., 31, 76.

Pavela-Vrancic, M., Mestrovic, V., Marasovic, I., Gillman, M., Furey, A. and James, K.J.

2001. The occurrence of 7-epi-pectenotoxin-2 seco acid in the coastal waters of the central Adriatic (Kastela Bay). Toxicon., 39, 771-9.

Quilliam, M.A. and Wright, J.L. 1989. The amnesic shellfish poisoning mystery. Anal.

Chem., 61, 1053-60. Quilliam, M., Eaglesham, G., Hallegraeff, G., Quaine, J., Curtis, J., Richard, D. and

Nunez, P. 2000. Abstract: Detection and identification of toxins associated with a shellfish poisoning incident in new south wales, Australia. International conference on Harmful Algal Blooms. Ninth conference Tasmania 2000.

Quilliam, M.A., Hess, P. and Dell'Aversano, C. 2001a. Recent developments in the analysis

of phycotoxins by liquid chromatography-mass spectrometry. In: Mycotoxins and Phycotoxins in perspective at the turn of the millenium, eds. De Koe W.J. 383-391.

Quilliam, M.A., Hardstaff, W.R., Richard, D.R., Cembella, A. and Hancock, S. 2001b. Liquid

chromatography-mass spectrometry analysis of spirolide toxins. 2nd Int. Conf. on Harmful Algae Management and Mitigation, Qingdao, China poster presentation.

Quilliam, M.A. 2002. Chemical methods for lipophilic shellfish toxins, In press. Ramstad, H., Hovgaard, P., Yasumoto, T., Larsen, S. and Aune, T. 2001. Monthly

variations in diarrhetic toxins and yessotoxin in shellfish from coast to the inner part of the Sognefjord, Norway. Toxicon., 39, 1035-43.

Ramstad, H., Larsen, S. and Aune, T. 2001. Repeatability and validity of a fluorimetric

HPLC method in the quantification of yessotoxin in blue mussels (Mytilus edulis) related to the mouse bioassay. Toxicon., 39, 1393-7.

Rezanka, T. 2000. Analysis of very long chain polyunsaturated fatty acids using high-

performance liquid chromatography - atmospheric pressure chemical ionization mass spectrometry. Biochemical Systematics and Ecology, 28, 847-856.

Roden, C. 2001. Ongoing research on toxic algae. Proceedings of the 2nd Irish Marine

Biotoxin Science Workshop, 2001. Ruhl, A., Hummert, C., Gerdts, G. and Luckas, B. 2001. Determination of new algal toxins

(spirolides) near the Scottish east coast. http://www.ices.dk/asc/2001/callforpapers/slist.htm

Saijiki, J. and Takahashi K. 1992. Free fatty acids inducing mouse lethal toxicity in lipid

extracts of Engraulis japonica, the Japanese anchovy. Lipids, 27, 988-992. Sajiki, J., Takahashi, H. and Takahashi, K. 1995. Impact of Vinegar Acetic Acid on

Hydrolysis and Oxidation of Lipids in Tissues of the Oyster, Crassostrea gigas, at 37 degrees C. J. Agric. Food Chem., 43, 1467.

Sajiki, J. 1996. Is phospholipase in vinegared oysters a casual agent for human poisoning?

J. Toxicology and Environmental Health, 47, 221-232.


39

Samain, J.F., Soudant, P., Marty, Y., Moal, J. and Van Ryckeghem, K. 2000. Fatty acid specifity of polar lipid classes in two bivalves molluscs. Changes in relation to diet, reproduction and larval development. Proceedings of the Symposium held in Brest, IFREMER. 2000.

Sasaki, K., Satake, M. and Yasumoto, T. 1997. Identification of the absolute configuration

of pectenotoxin-6, a polyether macrolide compound, by NMR spectroscopic method using a chiral anisotropic reagent, phenylglycine methyl ester. Biosci. Biotechnol. Biochem., 61, 1783-1785.

Sasaki, K., Wright, J.L.C. and Yasumoto, T. 1998. The identification and characterization of

pectenotoxin (PTX) 4 and PTX7 as spiroketal stereoisomers of two previously reported pectenotoxins. J. Org. Chem., 63, 2475-2480.

Sasaki, K., Takizawa, A., Tubaro, A., Sidari, L., Della Loggia, R. and Yasumoto, T. 1999.

Fluorometric analysis of Pectenotoxin-2 in microalgal samples by High Performance Liquid Chromatography. Nat. Toxins, 7, 241-246.

Satake, M., Terasawa, K., Kadowaki, Y. and Yasumoto, T. 1996. Relative configuration of

yessotoxin and isolation of two new analogs from toxic scallops. Tetrahedron Lett., 37, 5955-5958.

Satake, M., Tubaro, A., Lee, J.S. and Yasumoto, T. 1997a. Two new analogs of yessotoxin,

homoyessotoxin and 45-hydroxyhomoyessotoxin, isolated from mussels of the Adriatic Sea. Nat. Toxins, 5, 107-110.

Satake, M., MacKenzie, L. and Yasumoto, T. 1997b. Identification of Protoceratium

reticulatum as the biogenetic origin of yessotoxin. Nat. Toxins, 5, 164-167. Satake, M., Ofuji, K., Naoki, H., James, K.J., Furey, A., McMahon, T., Silke, J. and

Yasumoto, T. 1998a. Azaspiracid, a new marine toxin having unique spiro ring assemblies, isolated from Irish mussels, Mytilus edulis. J. Amer. Chem. Soc., 120, 9967-9968.

Satake, M., Ofuji, K., James, K., Furey, A. and Yasumoto, T. 1998b. New toxic event

caused by Irish mussels. In: Reguera, B., Blanco, J., Fernandez, M.L. and Wyatt, T. Harmful Algae. 468-469. 1998. Vigo, Xunta de Galicia and IOC/UNESCO.

Satake, M., Ichimura, T., Sekiguchi, K., Yoshimatsu, S. and Oshima, Y. 1999. Confirmation

of yessotoxin and 45,46,47-trinoryessotoxin production by Protoceratium reticulatum collected in Japan. Nat. Toxins, 7, 147-150.

Shimada, Y., Komiyama, M., Terao, K. and Zhou, Z.H. 1994. Effects of pectenotoxin-1 on

liver cells in vitro. Nat. Toxins, 2, 132-5. Suzuki, T., Yoshizawa, R., Yamasaki, M. 1996. Interference of Free Fatty Acids from the

Hepatopancreas of Mussels with the Mouse Bioassay for Shellfish Toxins. Lipids, 31, 641-645.

Suzuki, T., Mitsuya, T., Matsubara, H. and Yamasaki, M. 1998. Determination of

pectentoxin-2 after solid phase extraction from seawater and from the dinoflagellate Dinophysis fortii by liquid chromatography with electrospray mass spectrometry and ultraviolet detection. Evidence of oxidation of pectenotoxin-2 to pectenotoxin-6 in scallops. J. Chromatogr. A, 815, 155-160.


40

Suzuki, T. and Yasumoto, T. 2000. Liquid chromatography-electrospray ionization mass spectrometry of the diarrhetic shellfish-poisoning toxins okadaic acid, dinophysistoxin-1 and pectenotoxin-6 in bivalves. J. Chromatogr A, 874, 199-206.

Suzuki, T., Mackenzie L., Stirling D. and Adamson J. 2001a. Pectenotoxin-2 seco acid: a

toxin converted from pectenotoxin-2 by the New Zealand Greenshell mussel, Perna canaliculus. Toxicon., 39, 507-14.

Suzuki, T., Mackenzie, L., Stirling, D. and Adamson, J. 2001b. Conversion of pectenotoxin-

2 to pectenotoxin-2 seco acid in the New Zealand scallop, Pecten novaezelandiae. Fisheries Science, 67, 506-510.

Takagi, T., Hayashi, K. and Itabashi, Y. 1982. Toxic effect of free polyenoic acids:a fat

soluble marine toxin. Bull. Fac. Fish. Hokkaido Univ., 33, 255-262. Takahashi, H., Kusumi, T., Kan, Y., Satake, M. and Yasumoto, T. 1996. Determination of

the absolute configuration of yessotoxin, a polyether compound implicated in diarrhetic shellfish poisoning, by NMR spectroscopic method using a chiral anisotropic reagent, methoxy-(2-naphthyl)acetic acid. Tetrahedron Lett., 37, 7087-7090.

Taylor, F.J.R., Fukuyo, Y. and Larsen, J. 1995. Taxonomy of Harmful Dinoflagellates. In:

Hallegraeff, G.M., Anderson, D.M. and Cembella, A.D. Eds. Manual on Harmful Marine Microalgae. Paris: IOC-UNESCO; pp. 1-24. (IOC Manual and Guides; v 33).

Terao, K., Ito, E., Yanagi, T. and Yasumoto, T. 1986. Histopathological studies on

experimental marine toxin poisoning. I. Ultrastructural changes in the small intestine and liver of suckling mice induced by dinophysistoxin 1 and pectenotoxin 1. Toxicon., 24, 1141-51.

Terao, K., Ito, E., Oarada, M., Murata, M. and Yasumoto, T. 1990. Histopathological studies

on experimental marine toxin poisoning--5. The effects in mice of yessotoxin isolated from Patinopecten yessoensis and of a desulfated derivative. Toxicon., 28, 1095-1104.

Thompson, P. A., Harrison, P.J. and Whyte, J.N.C. 1990. Influence of irradiance on the

fatty acid composition of phytoplankton. Journal of Phycology, 26, 278-288. Thompson, P.A., Ming-xin, G. and Harrison, P.J. 1996. Nutritional value of diets that vary in

fatty acid composition for larva oysters (Crassostrea gigas). Aquaculture, 143, 379-391.

Tubaro, A., Sidari, L., Della Loggia, R. and Yasumoto, T. 1998. Occurrence of yessotoxin-

like toxins in phytoplankton and mussels from northern Adriatic Sea. In: Reguera, B., Blanco, J., Fernandez, M. L., and Wyatt, T. Harmful Algae. 470-472. 1998. Vigo, Xunta de Galicia and IOC/UNESCO.

Wright, J.L.C. 1995. Dealing with seafood toxins: present approaches and future options.

Food Res. Int., 28, 347-358. Yamane, M. 2001. High-performance liquid chromatography-thermospray ionization-mass

spectrometry of the oxidation products of polyunsaturated-fatty acids. Analytica Chimica Acta, In Press, Uncorrected Proof.

Yasumoto T., Oshima. and Yamaguchi M. 1978. Occurrence of a new type of shellfish

poisoning in the Tohoku district. Bull. Jpn. Soc. Sci. Fish., 44, 1249-1255.


41

Yasumoto, T., Murata, M., Oshima, Y., Matsumoto, G.L. and Clardy, J. 1984. Diarrhetic

shellfish poisoning. In: Seafood Toxins (Am. Chem. Soc. Symp. Ser.) ed: Ragelis, E.P. 207-214.

Yasumoto, T., Murata, M., Oshima, Y., Sano, M., Matsumoto, G.K. and Clardy, J. 1985.

Diarrhetic shellfish toxins. Tetrahedron, 41, 1019-1025. Yasumoto, T., Murata, M., Lee, J.-S. and Torigoe, K. 1989. Polyether toxins produced by

dinoflagellates. in: "Mycotoxins and Phycotoxins '88", eds.: Natori S., Hashimoto K., and Ueno Y.375-382.

Yasumoto, T. and Takizawa, A. 1997. Fluorometric measurement of yessotoxins in shellfish

by high-pressure liquid chromatography. Biosci. Biotech. Biochem., 61, 1775-1777.

occurrence of azaspiracids, spirolides, yessotoxins, pectenotoxins

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