Assessment of MIST Alert,a commercial qualitative assay for detection of paralytic shellfish poisoning toxins in bivalve molluscs

A recently developed commercial rapid test kit (MIST Alert) was assessed for determination of the presence of paralytic shellfish poisoning (PSP) toxins in shellfish. Several commercially important shellfish species obtained from the UK shellfish toxin monitoring program, containing a range of total PSP toxicities as determined by the mouse bioassay (MBA), were tested. The kit detected toxin in all samples containing the European Community tolerance level of 80 microg saxitoxin (STX) equivalents/100 g shellfish flesh as determined by the MBA. With one exception, the kit detected toxin in all samples that contained >40 microg STX equivalents/100 g according to the MBA. Among samples in which the MBA did not detect toxin, the kit disagreed in 25% of the tests, although further analysis by liquid chromatography (LC) and MBA of some samples confirmed the presence of toxins. These results suggest that MIST Alert may be suitable as an initial screen for PSP toxins as part of routine monitoring programs, thereby greatly reducing the number of MBAs. Trials were also performed by nonscientific personnel to evaluate the ease of use and interpretation of results obtained by MIST Alert. The results indicated that the kits could be readily used and accurately interpreted by individuals with no technical or scientific background.     

Results of a European interlaboratory method validation study for the quantitative determination of lipophilic marine biotoxins in raw and cooked shellfish based on high-performance liquid chromatography–tandem mass spectrometry. Part I: collaborative study

A European interlaboratory collaborative study was conducted to validate a method for the quantitative determination of lipophilic marine biotoxins based on high-performance liquid chromatography–tandem mass spectrometry. During this study, the diarrhetic shellfish poisoning toxins okadaic acid, dinophysis toxin1 and 2 including their esters, the azaspiracids 1-3, pectenotoxin2, and the yessotoxins were investigated at concentration levels near the limit of quantification and near the legal limit. Naturally contaminated blue mussels, both raw and cooked and spiked extracts of clams and oysters were studied and results were obtained for 16 test samples from 16 laboratories representing eight different countries. This article summarizes the study outcome concerning validation key parameters like specificity, linearity, limit of detection, accuracy/recovery, and precision. Further, influences of cooking of mussels before homogenization or hydrolysis on method robustness have been evaluated.     

The development of a rapid method for the isolation of four azaspiracids for use as reference materials for quantitative LC–MS–MS methods

The azaspiracids are a family of lipophilic polyether marine biotoxins that have caused a number of human intoxication incidents in Europe since 1995 after consumption of contaminated shellfish (Mytilus edulis). Levels of azaspiracids in shellfish for human consumption are monitored in accordance with EU guidelines: only shellfish with less than 160 μg kg⁻¹ are deemed safe. The limited availability of commercially available standards for azaspiracids is a serious problem, because validated LC–MS methods are required for routine analysis of these toxins in shellfish tissues. The procedure described herein has been used for the separation and the isolation of four azaspiracid (AZA) toxins from shellfish, for use as LC–MS–MS reference materials. Five separation steps have been used to isolate azaspiracids 1, 2, 3, and 6. The purity of the toxins obtained has been confirmed by multiple mass spectrometric methods using authentic azaspiracid standards. The same techniques have been used for quantification of the toxins extracted. The isolation procedure involves several chromatographic purification techniques: solid-phase extraction (diol sorbent, 90% mass reduction, and 95 ± 1% toxin recovery); Sephadex size-exclusion chromatography (87% mass reduction and up to 95 ± 2% toxin recovery), Toyopearl HW size-exclusion chromatography (90% mass reduction and up to 92.5 ± 2.5% toxin recovery), and semi-preparative LC (78 ± 3% toxin recovery). The procedure effectively separates the toxins from the sample matrix and furnishes azaspiracid toxins (AZA1, AZA2, AZA3 and AZA6) of sufficient purity with an average yield of 65% (n = 5). Triple-quadrupole mass spectrometry was used for qualitative and quantitative monitoring of the isolation efficiency after each stage of the process. High-resolution mass spectrometric evaluation of the toxic isolated material in both positive and negative modes suggests high purity.     

Potential use of gamma irradiation in the production of mussel and oyster reference materials for paralytic shellfish poisoning toxins

Bivalve shellfish samples containing paralytic shellfish poisoning toxins were subjected to gamma irradiation dosage trials in order to assess the potential suitability of the technique in the production of toxin reference materials. Two candidate reference materials of tissue homogenates, mussels (Mytilus sp.) and native oysters (Ostrea edulis), were prepared in-house. Both were subjected to gamma irradiation at four different dose levels, 3.0, 6.0, 13.0 and 18.1 kGy. Bacterial levels were shown to be eliminated in the mussels and significantly reduced in the oysters following irradiation at all four dose levels. Paralytic shellfish poisoning (PSP) toxin concentrations were not significantly reduced in any of the samples indicating the treatment had no adverse affect on the initial stability of any of the PSP toxins monitored. Chromatographic results showed near-identical profiles for treated and non-treated samples inferring that no fluorescent toxin degradation products or matrix interferences were produced during the irradiation process. Results therefore proved that gamma irradiation treatment reduced bacterial levels within paralytic shellfish poisoning reference materials without compromising analyte content, with the subsequent potential to enhance the stability of future candidate reference materials treated in this manner.     

Feasibility of gamma irradiation as a stabilisation technique in the preparation of tissue reference materials for a range of shellfish toxins

The effect of γ-irradiation on concentrations of hydrophilic and lipophilic phycotoxins has been investigated by use of HPLC–UV and LC–MS. Pure toxins in organic solvents and toxins in mussel (Mytilus edulis) tissues were irradiated at three different doses. In solution all toxin concentrations were reduced to some extent. Most severe decreases were observed for domoic acid and yessotoxin, for which the smallest dose of irradiation led to almost complete destruction. For pectenotoxin-2 the decrease in concentration was less severe but still continuous with increasing dose. Azaspiracid-1 and okadaic acid were the least affected in solution. In shellfish tissue the decrease in toxin concentrations was much reduced compared with the effect in solution. After irradiation at the highest dose reductions in concentrations were between ca. 5 and 20% for the lipophilic toxins and there was no statistical difference between control and irradiated samples for azaspiracids in tissue. Irradiation of shellfish tissues contaminated with domoic acid led to a more continuous decrease in the amount of the toxin with increasing dose. The effect of irradiation on the viability of microbial activity in shellfish tissues was assessed by using total viable counting techniques. Microbial activity depended on the type of shellfish and on the pretreatment of the shellfish tissues (with or without heat treatment). As far as we are aware this is the first investigation of the effectiveness of irradiation as a technique for stabilising tissue reference materials for determination of phycotoxins. Our results suggest that this technique is not effective for materials containing domoic acid. It does, however, merit further investigation as a stabilisation procedure for preparation of shellfish tissue materials for some lipophilic toxins, in particular azaspiracids. Chemical structures of the toxins investigated in the study     

Screening of lipophilic marine toxins in shellfish and algae: development of a library using liquid chromatography coupled to orbitrap mass spectrometry

Most liquid chromatography (LC) mass spectrometric (MS) methods used for routine monitoring of lipophilic marine toxins focus on the analysis of the 13 toxins that are stated in European Union legislation. However, to date over 200 lipophilic marine toxins have been described in the literature. To fill this gap, a screening method using LC coupled to high resolution (HR) orbitrap MS (resolution 100 000) for marine lipophilic toxins has been developed. The method can detect a wide variety of okadaic acid (OA), yessotoxin (YTX), azaspiracid (AZA) and pectenotoxin (PTX) group toxins. To build a library of toxins, shellfish and algae samples with various toxin profiles were obtained from Norway, Ireland, United Kingdom, Portugal and Italy. Each sample extract was analyzed with and without collision induced dissociation fragmentation. Based on their mass and specific fragmentation pattern, 85 different toxins were identified comprising 33 OA, 26 YTX, 18 AZA and 8 PTX group toxins. A major complication of full scan HRMS is the huge amount of data generated (file size), which restricts the possibility of a fast search. A software program called metAlign was used to reduce the orbitrap MS data files. The 200-fold reduced data files were screened using an additional software tool for metAlign: ‘Search_LCMS’. A search library was constructed for the 85 identified toxins. The library contains information about compound name, accurate mass, mass deviation (<5 ppm), retention time (min) and retention time deviation (<0.2 min). An important feature is that the library can easily be exchanged with other instruments as the generated metAlign files are not brand-specific. The developed screening procedure was tested by analyzing a set of known positive and blank samples, processing them with metAlign and searching with Search_LCMS. A toxin profile was determined for each of the contaminated samples. No toxins were found in the blank sample, which is in line with the results obtained for this sample in the routine monitoring program (rat bioassay and tandem LC–MS).     

超高效液相色谱-串联质谱法同时测定血浆与尿液中12种脂溶性贝类毒素

生物样品中脂溶性贝类毒素的检测,可为食物中毒等突发公共卫生事件的流行病学调查以及中毒者的临床救治提供技术支持.目前的研究存在目标化合物少,以及方法前处理复杂、灵敏度低等问题.该研究通过优化前处理和色谱分离技术,建立了超高效液相色谱-串联质谱法测定血浆、尿液中12种脂溶性贝类毒素的方法.实验对提取试剂以及流动相的选择进行...     

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.     

l‐Cysteine‐modified magnetic microspheres for extraction and quantification of saxitoxin in rat plasma with liquid chromatography and tandem mass spectrometry

Saxitoxin, which is one of the most typical paralytic shellfish poisoning toxins, ranks the highest intoxication rate of marine biological poisoning cases globally. Efficient clean‐up and extraction of saxitoxin from complex biological matrices are imperative for the analysis and concentration monitoring of the toxin when correlative poisoning cases happen. Herein, l ‐cysteine‐modified magnetic microspheres based on metal‐organic coordination were synthesized by a facile approach and applied for magnetic solid‐phase extraction of saxitoxin from rat plasma samples before liquid chromatography–tandem mass spectrometry detection. Parameters, including adsorbent amount, extraction time, desorption solution, and desorption time that could affect the extraction efficiency, were respectively investigated. The developed method demonstrated good linearity in the range of 5–300 ng/mL (R² = 0.9985) with a limit of quantification of 5 ng/mL and a limit of detection of 0.5 ng/mL, acceptable accuracy. and precision of within‐run and between‐run.     

Comparison of biosensor platforms for surface plasmon resonance based detection of paralytic shellfish toxins

Paralytic shellfish poisoning (PSP) toxins are produced by certain marine dinoflagellates and may accumulate in bivalve molluscs through filter feeding. The Mouse Bioassay (MBA) is the internationally recognised reference method of analysis, but it is prone to technical difficulties and regarded with increasing disapproval due to ethical reasons. As such, alternative methods are required. A rapid surface plasmon resonance (SPR) biosensor inhibition assay was developed to detect PSP toxins in shellfish by employing a saxitoxin polyclonal antibody (R895). Using an assay developed for and validated on the Biacore Q biosensor system, this project focused on transferring the assay to a high-throughput, Biacore T100 biosensor in another laboratory. This was achieved using a prototype PSP toxin kit and recommended assay parameters based on the Biacore Q method. A monoclonal antibody (GT13A) was also assessed. Even though these two instruments are based on SPR principles, they vary widely in their mode of operation including differences in the integrated μ-fluidic cartridges, autosampler system, and sensor chip compatibilities. Shellfish samples (n = 60), extracted using a simple, rapid procedure, were analysed using each platform, and results were compared to AOAC high performance liquid chromatography (HPLC) and MBA methods. The overall agreement, based on statistical 2 × 2 comparison tables, between each method ranged from 85% to 94.4% using R895 and 77.8% to 100% using GT13A. The results demonstrated that the antibody based assays with high sensitivity and broad specificity to PSP toxins can be applied to different biosensor platforms.     

石墨烯-管尖固相萃取-液相色谱-串联质谱法测定贝类中10种脂溶性贝类毒素

以石墨烯为吸附剂，制作了石墨烯-管尖固相萃取装置，结合液相色谱-串联质谱，建立了一种同时测定贝类中10种脂溶性贝类毒素的方法。实验对提取剂、石墨烯的用量、淋洗剂的种类和用量、洗脱剂的种类和用量等实验参数进行了详细优化。在最优的实验条件下，10种脂溶性贝类毒素在各自相应浓度范围内线性良好，相关系数均大于0.99，方法检出限（LOD）和定量限（LOQ）分别在0.1~1.1 μg/kg和0.3~3.2 μg/kg之间；对阴性牡蛎样品进行3个水平的加标回收实验，10种脂溶性贝类毒素的回收率在72.0%~101.2%之间，相对标准偏差小于15%。结果表明，该方法灵敏度高，操作简单高效，适用于贝类水产品中脂溶性贝类毒素的检测分析。     

Multiresidue method for determination of algal toxins in shellfish: single-laboratory validation and interlaboratory study

A method that uses liquid chromatography with tandem mass spectrometry (LC/MS/MS) has been developed for the highly sensitive and specific determination of amnesic shellfish poisoning toxins, diarrhetic shellfish poisoning toxins, and other lipophilic algal toxins and metabolites in shellfish. The method was subjected to a full single-laboratory validation and a limited interlaboratory study. Tissue homogenates are blended with methanol-water (9 + 1), and the centrifuged extract is cleaned up with a hexane wash. LC/MS/MS (triple quadrupole) is used for quantitative analysis with reversed-phase gradient elution (acidic buffer), electrospray ionization (positive and negative ion switching), and multiple-reaction monitoring. Ester forms of dinophysis toxins are detected as the parent toxins after hydrolysis of the methanolic extract. The method is quantitative for 6 key toxins when reference standards are available: azaspiracid-1 (AZA1), domoic acid (DA), gymnodimine (GYM), okadaic acid (OA), pectenotoxin-2 (PTX2), and yessotoxin (YTX). Relative response factors are used to estimate the concentrations of other toxins: azaspiracid-2 and -3 (AZA2 and AZA3), dinophysis toxin-1 and -2 (DTX1 and DTX2), other pectenotoxins (PTX1, PTX6, and PTX11), pectenotoxin secoacid metabolites (PTX2-SA and PTX11-SA) and their 7-epimers, spirolides, and homoYTX and YTX metabolites (45-OHYTX and carboxyYTX). Validation data have been gathered for Greenshell mussel, Pacific oyster, cockle, and scallop roe via fortification and natural contamination. For the 6 key toxins at fortification levels of 0.05-0.20 mg/kg, recoveries were 71-99% and single laboratory reproducibilities, relative standard deviations (RSDs), were 10-24%. Limits of detection were <0.02 mg/kg. Extractability data were also obtained for several toxins by using successive extractions of naturally contaminated mussel samples. A preliminary interlaboratory study was conducted with a set of toxin standards and 4 mussel extracts. The data sets from 8 laboratories for the 6 key toxins plus DTX1 and DTX2 gave within-laboratories repeatability (RSD(R)) of 8-12%, except for PTX-2. Between-laboratories reproducibility (RSDR) values were compared with the Horwitz criterion and ranged from good to adequate for 7 key toxins (HorRat values of 0.8-2.0).     

生物样品中大田软海绵酸类毒素代谢规律及检测方法的研究进展

近年来,中国赤潮污染日趋严重,因此引发多起贝类毒素中毒事件,威胁消费者的食用安全。大田软海绵酸（okadaic acid,OA）及其衍生物鳍藻毒素（dinophysistoxins,DTXs）是分布最广、危害最大的一类腹泻性贝类毒,具有急性腹泻毒性及多种慢性毒性。建立生物体液样品中OA类毒素残留的检测方法对辅助诊断患者的中毒情况极为必要。文章简要介绍了OA类毒素的理化性质、中毒事件、毒理作用,并详细总结了生物样品中OA类毒素代谢规律及检测方法的研究进展。     

A feasibility study into the production of a freeze-dried oyster reference material for paralytic shellfish poisoning toxins

Matrix reference materials are an essential component for the validation and quality control of analytical methodologies for the quantitation of marine biotoxins in shellfish. Given the potential advantages of reference materials in powder form, a study was conducted to assess the feasibility for the production of a freeze-dried oyster tissue reference material containing a range of important paralytic shellfish poisoning toxins. One bulk sample of a wet oyster tissue homogenate was generated following mass culturing of toxic Alexandrium and oyster feeding experiments. The bulk tissue was used to prepare untreated wet frozen aliquots with the remainder being freeze-dried and processed into appropriately-sized powder samples. A pre-column oxidation LC-FLD analysis was used to confirm the absence of any chromatographic artefacts resulting from the processing and to confirm acceptable homogeneity of the tissues. Excellent stability over both the short-term (1 month) and long-term (1 year) of the freeze-dried material was demonstrated as compared with the stability of the untreated wet tissue. A post-column oxidation LC-FLD method was used to confirm the absence of toxin epimerisation in freeze-dried tissues which were observed in the wet tissues. Overall the work showed the feasibility of an approach to produce a homogenous freeze-dried oyster matrix material with enhanced stability in comparison to the untreated wet tissue. The potential for use of the process for preparation of large scale production batches of a freeze-dried CRM for paralytic shellfish poisoning toxins has therefore been demonstrated.     

A mussel tissue certified reference material for multiple phycotoxins. Part 1: design and preparation

The development of multi-analyte methods for lipophilic shellfish toxins based on liquid chromatography–mass spectrometry permits rapid screening and analysis of samples for a wide variety of toxins in a single run. Validated methods and appropriate certified reference materials (CRMs) are required to ensure accuracy of results. CRMs are essential for accurate instrument calibration, for assessing the complete analytical method from sample extraction to data analysis and for verifying trueness. However, CRMs have hitherto only been available for single toxin groups. Production of a CRM containing six major toxin groups was achieved through an international collaboration. Preparation of this material, CRM-FDMT1, drew on information from earlier studies as well as improved methods for isolation of toxins, handling bulk tissues and production of reference materials. Previous investigations of stabilisation techniques indicated freeze-drying to be a suitable procedure for preparation of shellfish toxin reference materials and applicable to a wide range of toxins. CRM-FDMT1 was initially prepared as a bulk wet tissue homogenate containing domoic acid, okadaic acid, dinophysistoxins, azaspiracids, pectenotoxin-2, yessotoxin and 13-desmethylspirolide C. The homogenate was then freeze-dried, milled and bottled in aliquots suitable for distribution and analysis. The moisture content and particle size distribution were measured, and determined to be appropriate. A preliminary toxin analysis of the final material showed a comprehensive toxin profile.     

Profiles of paralytic shellfish poisoning toxins in shellfish from Portugal explained by carbamoylase activity

The presence of paralytic shellfish poisoning (PSP) toxins has not been recorded in the Portuguese coast since 1995. A bloom of Gymnodinium catenatum occurred in the NW coast of Portugal in the autumn of 2005, and PSP profiles were determined in several inshore and offshore shellfish species by HPLC after pre-column oxidation. Most of the species studied contained a complex toxin profile, typically representative of contamination by G. catenatum. However, clams such as Spisula solida contained mainly decarbamoyl toxins, while less extensive transformation was found in Scrobicularia plana. In vitro incubation of S. solida digestive glands with PSP standards revealed a rapid transformation of carbamate and N-sulfocarbamoyl toxins into their corresponding decarbamate analogues. After 24 h, less than 5% of the carbamate or N-sulfocarbamoyl toxins tested remained. After a 24 h in vitro incubation of S. plana digestive glands, no decarbamate analogues were detected. Artificial toxification of S. plana with cultures of G. catenatum revealed the conversion into decarbamoyl analogues progressed slowly: initially dcGTX2+3 and dcSTX accounted only for 5% of total non N-1 hydroxilated toxins, after 6 days these toxins accounted for 41% of the toxin composition. In vitro incubations of digestive glands from other commercial bivalves did not reveal production of decarbamoyl analogues over a 24 h period.     

Comparison of liquid-junction and coaxial interfaces for capillary electrophoresis-mass spectrometry with application to compounds of concern to the aquaculture industry.

The application of capillary electrophoresis-mass spectrometry (CE-MS) to the analysis of compounds of concern to the aquaculture industry is reported. Two different approaches to coupling the CE column to an IonSpray atmospheric pressure ionization (API) interface, viz., a liquid-junction and a coaxial arrangement, are describe and compared with regard to ruggedness, ease of use, sensitivity and electrophoretic performance. The different injection modes used in three commercial capillary electrophoresis systems were also evaluated for their applicability to CE-MS. The use of CE-MS for the analysis of a variety of classes of antibiotics used in the fish aquaculture industry, such as the sulfonamides and their potentiators (e.g., trimethoprim), is demonstrated and was used to confirm the presence of these components in shellfish extracts at the low ppm level. CE-MS was also applied to the analysis of marine toxins such as saxitoxin and its analogues which are associated with paralytic shellfish poisoning, and also the toxins responsible for amnesic and diarrheic shellfish poisoning. Tandem mass spectrometry (MS-MS) was used to provide structural information on these analytes, and the ability to distinguish isomeric compounds based on their different migration and fragmentation characteristics using CE-MS-MS is demonstrated.     

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.     

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.