Determination of paralytic shellfish poisoning toxins by high-performance ion-exchange chromatography

An efficient LC method has been developed for the determination of paralytic shellfish poisoning (PSP) toxins based on ion-exchange chromatographic separation of the toxins followed by electrochemical post-column oxidation and fluorescence detection as well as mass spectrometric (MS) detection. The method can be applied to the determination of PSP toxins in phytoplankton and to control seafood for PSP content.     

Application of a new zwitterionic hydrophilic interaction chromatography column for determination of paralytic shellfish poisoning toxins

A novel method for paralytic shellfish poisoning (PSP) toxins which is based on the chromatographic separation of the toxins using a zwitterionic (ZIC) hydrophilic interaction chromatography (HILIC) column is presented. Efficient retention of the polar PSP toxins on the ZIC-HILIC column allowed their selective and sensitive determination by the application of mass spectrometric (MS/MS) detection or as derivatives after oxidation prior to fluorescence detection (FD). Low buffer concentrations and the omission of ion-pair reagents decreased the limits of detection (LODs) by MS/MS analysis and showed a good linearity for both methods of detection. This method can be applied for the qualitative and quantitative determination of PSP toxins in various types of phytoplankton, and for the routine analysis of seafood.     

Determination of marine biotoxins relevant for regulations: from the mouse bioassay to coupled LC-MS methods

The frequency of occurrence and intensity of harmful algal blooms (HABs) appear to be increasing on a global scale. Consequently, methods were established for the evaluation of possible hazards caused by the enrichment of algal toxins in the marine food chain. Different clinical types of algae-related poisoning have attracted scientific attention: paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), and amnesic shellfish poisoning (ASP). In several countries fish specialties are consumed which may be contaminated with algal toxins typical for the respective region (e.g., ciguatera and tetrodotoxins). Bioassays are common methods for the determination of marine biotoxins. However, biological tests are not completely satisfactory, due to the low sensitivity and the absence of specialized variations. Moreover, there is growing resistance against the use of animal experiments. Therefore, many efforts have been made to determine algal toxins with chemical methods. In this context LC-MS methods replaced HPLC methods with optical detectors, allowing both effective seafood control and monitoring of phytoplankton in terms of the different groups of marine biotoxins.     

Analysis of paralytic shellfish poisons by capillary electrophoresis

A capillary electrophoresis (CE) method with UV detection is described for the separation and determination of underivatized toxins associated with paralytic shellfish poisoning (PSP). Confirmation of the electrophoretic peaks was facilitated by mass spectrometric (MS) detection using an ionspray CE-MS interface and by high-performance liquid chromatography with fluorescence detection. The determination of PSP toxins, such as saxitoxin and neosaxitoxin, in toxic dinoflagellates and scallops is demonstrated and comparisons are made with existing techniques.     

Analysis of the characteristic PSP profiles ofPyrodinium bahamense and several strains ofAlexandrium by HPLC based on ion-pair chromatographic separation, post-column oxidation, and fluorescence detection

Summary A sensitive HPLC method for determination of paralytic shellfish poisoning (PSP) based on ion-pair chromatographic separation of PSP toxins, post-column oxidation with periodic acid, and fluorescence detection has been used to determine toxin profiles ofPyrodinium bahamense and several strains ofAlexandrium. The HPLC chromatograms revealed clear differences betweenPyrodinium bahamense andAlexandrium strains. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996.     

Evolving to the optoelectronic mouse for phycotoxin analysis in shellfish

Despite ethical and technical concerns, the in vivo method, or more commonly referred to mouse bioassay (MBA), is employed globally as a reference method for phycotoxin analysis in shellfish. This is particularly the case for paralytic shellfish poisoning (PSP) and emerging toxin monitoring. A high-performance liquid chromatography method (HPLC-FLD) has been developed for PSP toxin analysis, but due to difficulties and limitations in the method, this procedure has not been fully implemented as a replacement. Detection of the diarrhetic shellfish poisoning (DSP) toxins has moved towards LC-mass spectrometry (MS) analysis, whereas the analysis of the amnesic shellfish poisoning (ASP) toxin domoic acid is performed by HPLC. Although alternative methods of detection to the MBA have been described, each procedure is specific for a particular toxin and its analogues, with each group of toxins requiring separate analysis utilising different extraction procedures and analytical equipment. In addition, consideration towards the detection of unregulated and emerging toxins on the replacement of the MBA must be given. The ideal scenario for the monitoring of phycotoxins in shellfish and seafood would be to evolve to multiple toxin detection on a single bioanalytical sensing platform, i.e. ‘an artificial mouse’. Immunologically based techniques and in particular surface plasmon resonance technology have been shown as a highly promising bioanalytical tool offering rapid, real-time detection requiring minimal quantities of toxin standards. A Biacore Q and a prototype multiplex SPR biosensor have been evaluated for their ability to be fit for purpose for the simultaneous detection of key regulated phycotoxin groups and the emerging toxin palytoxin. Deemed more applicable due to the separate flow channels, the prototype performance for domoic acid, okadaic acid, saxitoxin, and palytoxin calibration curves in shellfish achieved detection limits (IC₂₀) of 4,000, 36, 144 and 46 μg/kg of mussel, respectively. A one-step extraction procedure demonstrated recoveries greater than 80 % for all toxins. For validation of the method at the 95 % confidence limit, the decision limits (CCα) determined from an extracted matrix curve were calculated to be 450, 36 and 24 μg/kg, and the detection capability (CCβ) as a screening method is ≤10 mg/kg, ≤160 μg/kg and ≤400 μg/kg for domoic acid, okadaic acid and saxitoxin, respectively.     

Sensitive HPLC-fluorometric and HPLC-MS determination of diarrhetic shellfish poisoning (SP)-toxins as 4-bromomethyl-7-methoxycoumarin esters

 Extracts containing the diarrhetic shellfish poisoning (DSP) toxins okadaic acid (OA), dinophysistoxin-2 (DTX₂), and dinophysistoxin-1 (DTX₁) were purified on a silica gel cartridge and derivatized with 4-bromomethyl-7 methoxycoumarin (BrMmc). After pre-column derivatization the BrMmc derivatives of the DSP toxins were directly injected into an HPLC system, isocratically eluted, and quantified by fluorescence detection. The signals of the esters showed good linearity in the fluorescence detector within the examined contamination range of 0.03 mg DSP/kg to 2.5 mg DSP/kg. The detection limits for the DSP toxins as 7-Mmc esters were 0.04 ng (corresponding to 0.05 mg DSP/kg). The chromatographic conditions allow to couple the HPLC device with mass spectrometry. The method was tested with various mussel tissue samples. Received: 14 December 1995/Revised: 26 January 1996/Accepted: 30 January 1996     

Sensitive HPLC-fluorometric and HPLC-MS determination of diarrhetic shellfish poisoning (SP)-toxins as 4-bromomethyl-7-methoxycoumarin esters

 Extracts containing the diarrhetic shellfish poisoning (DSP) toxins okadaic acid (OA), dinophysistoxin-2 (DTX₂), and dinophysistoxin-1 (DTX₁) were purified on a silica gel cartridge and derivatized with 4-bromomethyl-7 methoxycoumarin (BrMmc). After pre-column derivatization the BrMmc derivatives of the DSP toxins were directly injected into an HPLC system, isocratically eluted, and quantified by fluorescence detection. The signals of the esters showed good linearity in the fluorescence detector within the examined contamination range of 0.03 mg DSP/kg to 2.5 mg DSP/kg. The detection limits for the DSP toxins as 7-Mmc esters were 0.04 ng (corresponding to 0.05 mg DSP/kg). The chromatographic conditions allow to couple the HPLC device with mass spectrometry. The method was tested with various mussel tissue samples. Received: 14 December 1995/Revised: 26 January 1996/Accepted: 30 January 1996     

New strategy for the determination of microcystins and diarrhetic shellfish poisoning (DSP) toxins, two potent phosphatases 1 and 2A inhibitors and tumor promoters

A new analytical strategy was established to improve the determination and identification performance during analyses of microcystins and diarrhetic shellfish poisoning (DSP) toxins in different matrices. Automated high performance size exclusion chromatography (gel permeation chromatography, SEC) was applied for the clean-up of raw extracts from algae and mussel tissue containing either microcystins or DSP toxins. The cleaned raw extracts are well suited for the direct determination of microcystins and DSP toxins by HPLC/MS. The analyses of cleaned raw extracts containing microcystin by HPLC and UV/diode array detection (DAD) revealed chromatograms without interfering peaks. Additionally, methods for the identification of unknown microcystins and those not available as standards were developed and established. The proposed strategy is exemplarily demonstrated for the analyses of a natural algae community from a lake in Slowakia and a naturally contaminated mussel from Portugal.     

Biosensors to detect marine toxins: Assessing seafood safety

This article describes the different types of marine toxins and their toxic effects, and reviews the bio/analytical techniques for their detection, putting special emphasis to biosensors. Important health concerns have recently appeared around shellfish (diarrheic, paralytic, amnesic, neurologic and azaspiracid) and fish (ciguatera and puffer) poisonings produced by different types of phycotoxins, making evident the urgent necessity of counting on appropriate detection technologies. With this purpose, several analysis methods (bioassays, chromatographic techniques, immunoassays and enzyme inhibition-based assays) have been developed. However, easy-to-use, fast and low-cost devices, able to deal with complicated matrices, are still required. Biosensors offer themselves as promising biotools, alternative and/or complementary to conventional analysis techniques, for fast, simple, cheap and reliable toxicity screening. Nevertheless, despite the wide range of seafood toxins and the already rooted biosensing systems, the literature on biosensors for phycotoxins is scarce. This article discusses the existing biosensor-based strategies and their advantages and limitations. Finally, the article gives a general overlook about the regulation toxin levels and monitoring programmes currently established around the world concerning seafood safety.     

Ionspray mass spectrometry of marine toxins. III. Analysis of paralytic shellfish poisoning toxins by flow-injection analysis, liquid chromatography/mass spectrometry and capillary electrophoresis/mass spectrometry.

Ionspray mass spectrometry has been used to monitor the purification of saxitoxin, the parent compound in the family of toxins responsible for paralytic shellfish poisoning (PSP), from a strain of the dinoflagellate Alexandrium excavatum. Quantitative results obtained by flow-injection analysis are compared to those obtained by high-performance liquid chromatography with post-column oxidation and fluorescence detection. The coupling of liquid chromatography and capillary electrophoresis with ionspray mass spectrometry is described for the separation of mixtures of PSP toxins and the highly potent pufferfish toxin tetrodotoxin. Tandem mass spectrometry is used to provide the structural information, and the ability to distinguish isomeric PSP toxins both chromatographically and mass spectrometrically is demonstrated.     

New strategy for the determination of microcystins and diarrhetic shellfish poisoning (DSP) toxins, two potent phosphatases 1 and 2A inhibitors and tumor promoters

A new analytical strategy was established to improve the determination and identification performance during analyses of microcystins and diarrhetic shellfish poisoning (DSP) toxins in different matrices. Automated high performance size exclusion chromatography (gel permeation chromatography, SEC) was applied for the clean-up of raw extracts from algae and mussel tissue containing either microcystins or DSP toxins. The cleaned raw extracts are well suited for the direct determination of microcystins and DSP toxins by HPLC/MS. The analyses of cleaned raw extracts containing microcystin by HPLC and UV/diode array detection (DAD) revealed chromatograms without interfering peaks. Additionally, methods for the identification of unknown microcystins and those not available as standards were developed and established. The proposed strategy is exemplarily demonstrated for the analyses of a natural algae community from a lake in Slowakia and a naturally contaminated mussel from Portugal. Received: 23 July 1999 / Revised: 9 September 1999 / Accepted: 16 September 1999     

Quantitative determination of paralytic shellfish poisoning toxins in shellfish using prechromatographic oxidation and liquid chromatography with fluorescence detection: interlaboratory study

An interlaboratory study was conducted for the determination of paralytic shellfish poisoning (PSP) toxins in shellfish. The method used liquid chromatography with fluorescence detection after prechromatographic oxidation of the toxins with hydrogen peroxide and periodate. The PSP toxins studied were saxitoxin (STX), neosaxitoxin (NEO), gonyautoxins 2 and 3 (GTX2,3 together), gonyautoxins 1 and 4 (GTX1,4 together), decarbamoyl saxitoxin (dcSTX), B-1 (GTX5), C-1 and C-2 (C1,2 together), and C-3 and C-4 (C3,4 together). B-2 (GTX6) toxin was also included, but for qualitative identification only. Samples of mussels, both blank and naturally contaminated, were mixed and homogenized to provide a variety of PSP toxin mixtures and concentration levels. The same procedure was followed with samples of clams, oysters, and scallops. Twenty-one samples in total were sent to 21 collaborators who agreed to participate in the study. Results were obtained from 18 laboratories representing 14 different countries.     

Advanced studies for the application of high-performance capillary electrophoresis for the analysis of yessotoxin and 45-hydroxyyessotoxin

Yessotoxins (YTXs) are a group of polyether toxins which have been previously reported as responsible for seafood contamination in several places worldwide. Despite their toxicity, which is not yet fully discussed, YTXs have been reported as an interference in the success of mouse bioassay for the determination of diarrhetic shellfish poisoning (DSP) toxins, and therefore, efficient and reliable analytical methodologies are required to evaluate their presence, avoiding false positives for DSP. High-performance capillary electrophoresis (HPCE) is presented in this work as an alternative to HPLC technique widely used for the analysis of YTXs. Improvements in the applicability of HPCE have been carried out through the development of different CE modes as well as different detection modes. With this aim, micellar electrokinetic chromatography (MEKC) has been considered for an increased selectivity while an increased sensitivity was achieved by using sample stacking. Moreover, the coupling of CE with mass spectrometry allowed the confirmation of YTXs present in the contaminated samples evaluated in this work. The results obtained showed the potential of CE as an alternative to HPLC for the analysis of YTXs present in naturally contaminated samples.     

Quantitative determination of paralytic shellfish poisoning toxins in shellfish using prechromatographic oxidation and liquid chromatography with fluorescence detection: collaborative study

A collaborative study was conducted for the determination of paralytic shellfish poisoning (PSP) toxins in shellfish. The method used liquid chromatography with fluorescence detection after prechromatographic oxidation of the toxins with hydrogen peroxide and periodate. The PSP toxins studied were saxitoxin (STX), neosaxitoxin (NEO), gonyautoxins 2 and 3 (GTX2,3; together), gonyautoxins 1 and 4 (GTX1,4; together), decarbamoyl saxitoxin (dcSTX), B-1 (GTX5), C-1 and C-2 (C1,2; together), and C-3 and C-4 (C3,4; together). B-2 (GTX6) toxin was also included, but for qualitative identification only. Mussels, both blank and naturally contaminated, were mixed and homogenized to provide a variety of PSP toxin mixtures and concentration levels. The same procedure was followed with clams, oysters, and scallops. Twenty-one test samples in total were sent to 21 collaborators who agreed to participate in the study. Results were obtained from 18 laboratories representing 14 different countries. It is recommended that the method be adopted First Action by AOAC INTERNATIONAL.     

Integrated enzyme-linked immunosorbent assay screening system for amnesic, neurotoxic, diarrhetic, and paralytic shellfish poisoning toxins found in New Zealand

Enzyme-linked immunosorbent assays (ELISAs) were developed for amnesic, neurotoxic, and diarrhetic shellfish poisoning (ASP, NSP, and DSP) toxins and for yessotoxin. These assays, along with a commercially available paralytic shellfish poisoning (PSP) ELISA, were used to test the feasibility of an ELISA-based screening system. It was concluded that such a system to identify suspect shellfish samples, for subsequent analysis by methods approved by international regulatory authorities, is feasible. The assays had sufficient sensitivity and can be used on simple shellfish extracts. Alcohol extraction gave good recovery of all toxin groups. The ease of ELISAs permits the ready expansion of the system to screen for other toxins, as new ELISAs become available.     

Requirements for screening and confirmatory methods for the detection and quantification of marine biotoxins in end-product and official control

An overview is given of the biological origin of phycotoxins, as well as their chemical characteristics. Major poisoning types are described and examples of poisoning events are given to illustrate the importance of the phenomenon to both shellfish consumers and the shellfish producing industry. The characteristics of phycotoxins as natural products, the lack of predictability of their occurrence, economic drivers and the freshness of shellfish consumed in many countries result in a number of requirements for methods to be used in the efficient detection of these compounds. Subsequently, the performance of mouse bioassays and mass spectrometry as detection tools are compared for examples from Irish and French monitoring programmes to assess the usefulness of qualitative and quantitative tools in official control, and their fitness for purpose compared with the requirements. The final part of the paper critically reviews methods available for the end-product and official control of shellfish toxins and their use in screening and confirmatory approaches in monitoring. Recent expert consultations on the methodology for phycotoxins at European and global level are summarised and recommendations are made for future progress in this area.     

Immunoassay methods for paralytic shellfish poisoning toxins

The current status of immunochemical techniques for analysis of paralytic shellfish poisoning (PSP) toxins is summarized. Important aspects regarding production of the biological reagents necessary for immunochemical methods, the characteristics of polyclonal and monoclonal antibodies against saxitoxin and neosaxitoxin, and the importance of test sensitivity and specificity are discussed. Applications of immunochemical techniques for PSP toxins include microtiter plate enzyme immunoasays and enzyme-linked immunofiltration assays for toxin detection, and immunoaffinity chromatography (IAC) for sample extract cleanup. A major advantage of enzyme immunoassay (EIA) is simplicity and rapidity of the test procedure, and higher sensitivity than other methods. However, quantitative agreement between EIA and mouse bioassay is dependent on antibody specificity and the toxin profile in the shellfish; thus, both over- and underestimation of total toxicity may occur. For screening purposes, however, EIAs offer major advantages over the mouse bioassay, which is criticized in Europe because of animal welfare. A major application of antibodies against PSP toxins is their use for extract cleanup by IAC, which gives highly purified extracts, thereby enhancing determination of PSP toxins by conventional physicochemical methods such as liquid chromatography. IAC can also be used to isolate PSP toxins for preparation of analytical standard solutions.     

Development and single laboratory validation of an optical biosensor assay for tetrodotoxin detection as a tool to combat emerging risks in European seafood

Tetrodotoxin (TTX) is a potent neurotoxin emerging in European waters due to increasing ocean temperatures. Its detection in seafood is currently performed as a consequence of using the Association of Analytical Communities (AOAC) mouse bioassay (MBA) for paralytic shellfish poisoning (PSP) toxins, but TTX is not monitored routinely in Europe. Due to ethical and performance-related issues associated with this bioassay, the European Commission has recently published directives extending procedures that may be used for official PSP control. An AOAC-accredited high-performance liquid chromatography (HPLC) method has now been accepted by the European Union as a first action screening method for PSP toxins to replace the MBA. However, this AOAC HPLC method is not capable of detecting TTX, so this potent toxin would be undetected; thereby, a separate method of analysis is required. Surface plasmon resonance (SPR) optical biosensor technology has been proven as a potential alternative screening method to detect PSP toxins in seafood. The addition of a similar SPR inhibition assay for TTX would complement the PSP assay in removing the MBA. The present report describes the development and single laboratory validation in accordance with AOAC and IUPAC guidelines of an SPR method to be used as a rapid screening tool to detect TTX in the sea snail Charonia lampas lampas, a species which has been implicated in 2008 in the first case of human TTX poisoning in Europe. As no current regulatory limits are set for TTX in Europe, single laboratory validation was undertaken using those for PSP toxins at 800 μg/kg. The decision limit (CCα) was 100 μg/kg, with the detection capability (CCβ) found to be ≤200 μg/kg. Repeatability and reproducibility were assessed at 200, 400, and 800 μg/kg and showed relative standard deviations of 8.3, 3.8, and 5.4 % and 7.8, 8.3, and 3.7 % for both parameters at each level, respectively. At these three respective levels, the recovery of the assay was 112, 98, and 99 %.     

Phycotoxins.

The 1997-1998 period brought many new developments to the phycotoxin field. There were several reviews on phycotoxins in general, on their toxicological evaluation, and on their analysis. The ecophysiology, biosynthesis, and metabolism of polyether toxins and paralytic shellfish poisoning (PSP) toxins were also reviewed. The proceedings of the Eighth International Conference on Harmful Algae (Vigo, Spain, June 25-29, 1997) have been published and provide an excellent source of information on phycotoxins and toxic plankton bloom research. In addition, the much anticipated proceedings of the IX International IUPAC Symposium on Mycotoxins and Phycotoxins (Rome, Italy, May 27-31, 1996) have been published. Further evidence was provided to support the theory that Prorocentrum lima is the source organism for diarrhetic shellfish poisoning (DSP) toxins in Nova Scotian shellfish. In another study, different Prorocentrum species and isolates were analyzed for DSP toxins. In addition to detecting some new compounds, such as a DTX1 isomer, it was found that toxins were produced by both axenic and nonaxenic batch cultures, indicating that bacteria are probably not involved in the biosynthesis. The source organism for the spirolides, a family of fast-acting toxins reported from Nova Scotia, Canada, was determined to be Alexandrium ostenfeldii, a species that is found worldwide. The biogenetic origin of yessotoxin was reported to be Protoceratium reticulatum, another widely occurring organism. A great deal of attention and research funding has been directed at the serious problems associated with Pfiesteria piscicida. Analysts are eagerly awaiting publication of toxin structures, which will then allow the development of analytical methods. An incident of the mass mortality of California sea lions was reported in the Monterey area in May 1998. Analyses of tissue and urine samples revealed the presence of domoic acid. High levels of domoic acid were also found in anchovies and sardines, a common food source of sea lions. This is reminiscent of an incident of mass bird mortality in 1992 in the same region. Toxicological studies of domoic acid continue with one investigation on the effect of pH on toxicity in the mouse assay and others examining toxic effects in rats and cynomolgus monkeys. A study on the uptake and depuration of domoic acid in the Dungeness crab was reported. On October 20, 1997, EU (European Union) directive CE97/61 established a regulatory limit of 20 ppm for domoic acid in European shellfish, the same level as in North America. A detailed study on the oral toxicity of DSP toxins in mice was reported. Recent work by several researchers has revealed the genotoxic potential of okadaic acid and other DSP toxins. Previous work had clearly demonstrated the tumor-promoting potential of DSP toxins, but this recent evidence, which shows mutations in the progeny of okadaic acid-treated cells and the formation of DNA-adducts, increases concerns over the hazards associated with DSP-contaminated shellfish. The toxicology of yessotoxin was evaluated by Ogino et al. The toxin showed weak cytotoxicity, but was not orally lethal to mice at 10 mg/kg, and did not cause intestinal fluid accumulation, inhibition of protein phosphatase 2A (PP2A), or hemolytic effects. Similarly, Tubaro et al. saw no evidence for diarrheogenicity of homoyessotoxin isolated from mussels and from the proposed planktonic producer, Lingulodinium polyedrum. All this provides further evidence that yessotoxin should not be classed as a DSP toxin. A number of new toxins have been detected and identified. Two analogues of yessotoxin, homoyessotoxin, and 45-hydroxyhomoyessotoxin were isolated from mussels of the Adriatic Sea and identified by Satake et al. A recent DSP event in Ireland associated with cultured mussels led to the identification of azaspiracid, a unique marine toxin with spiro ring assemblies. (ABSTRACT TRUNCATED)