Dinophysistoxin-1 Research Articles

Quantifying Spatio-temporal risk of Harmful Algal Blooms and their impacts on bivalve shellfish mariculture using a data-driven modelling approach

Harmful algal blooms (HABs) intoxicate and asphyxiate marine life, causing devastating environmental and socio-economic impacts, costing at least $8bn/yr globally. Accumulation of phycotoxins from HAB phytoplankton in filter-feeding shellfish can poison human consumers, prompting harvesting closures at shellfish production sites. To quantify long-term intoxication risk from Dinophysis HAB species, we used historical HAB monitoring data (2009–2020) to develop a new modelling approach to predict Dinophysis toxin concentrations in a range of bivalve shellfish species at shellfish sites in Western Scotland, South-West England and Northern France. A spatiotemporal statistical modelling framework was developed within the Generalized Additive Model (GAM) framework to quantify long-term HAB risks for different bivalve shellfish species across each region, capturing seasonal variations, and spatiotemporal interactions. In all regions spatial functions were most important for predicting seasonal HAB risk, offering the potential to inform optimal siting of new shellfish operations and safe harvesting periods for businesses. A 10-fold cross-validation experiment was carried out for each region, to test the models’ ability to predict toxin risk at harvesting locations for which data were withheld from the model. Performance was assessed by comparing ranked predicted and observed mean toxin levels at each site within each region: the correlation of ranks was 0.78 for Northern France, 0.64 for Western Scotland, and 0.34 for South-West England, indicating our approach has promise for predicting unknown HAB risk, depending on the region and suitability of training data.

A simple method to evaluate the toxic effects of Prorocentrum lima extracts to fish (sea bass) kidney cells

The diarrhetic shellfish toxins (DSTs) okadaic acid (OA) and its analogues — the dinophysistoxins (DTXs) — are produced by dinoflagellates such as Prorocentrum lima and can bioaccumulate in filter-feeding organisms as they are transferred through the food web. Although there is no assessment of the harmful effects of these toxins on the fish's immune system, this study developed a primary culture protocol for kidney cells from marine fish Centropomus parallelus and evaluated the immunotoxic effects to P. lima extracts containing DSTs. The cells were obtained by mechanical dissociation, segregated with Percoll gradient, and incubated for 24 h at 28 °C in a Leibovitz culture medium supplemented with 2% fetal bovine serum and antibiotics. The exposed cells were evaluated in flow cytometry using the CD54 PE antibody. We obtained >5.0 × 106 viable cells per 1.0 g of tissue that exhibited no cell differentiation. Exposure to 1.2 or 12 ng DST mL−1 stimulated the immune system activation and increased the proportion of activated macrophages and monocytes in 48 to 52% and in 127 to 146%, respectively. The protocol proved to be an alternative tool to assess the immunotoxic effects of DST exposure on fish's anterior kidney cells.

Rapid enzyme-linked immunosorbent assay and colloidal gold immunoassay for assessing okadaic acid and its derivatives in shellfish

ABSTRACT Okadaic acid (OA) and the analogs of dinophysistoxin (DTX) are important members of diarrhetic shellfish poisoning (DSP) toxins. In this study, five OA-specific mAbs (monoclonal antibodies) were developed from five stable cells of hybridoma. The anti-OA mAb-2D7 showed high sensitivity to OA, the IC50 of the antibody was 0.24 ng/mL, and its cross-reactivity was 91.6% with DTX-1 and 110.5% with DTX-2., In dcELISA, the IC50 was set at 0.182 ng/mL, and the detection limit was set at 0.023 ng/mL by using the anti-OA mAb-2D7. The level of OA recovered from spiked mussel samples was 2–50 ng/g, ranging from 97.6 ± 7.2% to 106.4 ± 9.8%. In contrast, the immunostrip assay with a limit of 5 ng/mL, conducted for detecting OA, was completed in 10 min. The mAb-based dcELISA and immunostrip assay developed were precise and sensitive enough to quickly assess OA, DTX-1, and DTX-2 in shellfish specimens.

In Vitro Interactions between Okadaic Acid and Rat Gut Microbiome.

Okadaic acid (OA) is a marine biotoxin associated with diarrhetic shellfish poisoning (DSP), posing some threat to human beings. The oral toxicity of OA is complex, and the mechanism of toxicity is not clear. The interaction between OA and gut microbiota may provide a reasonable explanation for the complex toxicity of OA. Due to the complex environment in vivo, an in vitro study may be better for the interactions between OA and gut microbiome. Here, we conducted an in vitro fermentation experiment of gut bacteria in the presence of 0–1000 nM OA. The remolding ability of OA on bacterial composition was investigated by 16S rDNA sequencing, and differential metabolites in fermentation system with different concentration of OA was detected by LC-MS/MS. We found that OA inhibited some specific bacterial genera but promoted others. In addition, eight possible metabolites of OA, including dinophysistoxin-2 (DTX-2), were detected in the fermentation system. The abundance of Faecalitalea was strongly correlated with the possible metabolites of OA, suggesting that Faecalitalea may be involved in the metabolism of OA in vitro. Our findings confirmed the direct interaction between OA and gut bacteria, which helps to reveal the metabolic process of OA and provide valuable evidence for elucidating the complex toxicity of OA.

A facile and integrated aptamer-based platform for preliminary and simultaneous screening of group targets

Highly efficient detection of group targets is of great significance. Okadaic acid (OA), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2) are the main diarrhetic shellfish poisons (DSPs) and have similar structures. As essential auxiliary tools, facile methods for simultaneous screening of these three DSPs are urgently needed. In this study, we report an aptamer-based platform which integrates pretreatment and quantitation for preliminary and simultaneous screening of the three DPSs. With computational study-assisted post-selection strategies, a truncated aptamer and a pair of split aptamers were generated with group-specific and high affinity towards the DSPs. The truncated aptamer was adopted to prepare an aptamer affinity column (AAC) for enrichment and purification of DSPs in real samples. The split aptamers were combined with G-quadruplex-hemin nanozyme to fabricate a label-free colorimetric aptasensor for quantitation. The prepared AAC showed good enrichment capacity. The developed colorimetric aptasensor provided high sensitivity, with limit of detection of 0.7011 nM, 14.27 nM, and 5.374 nM towards OA, DTX-1, and DTX-2, respectively, as well as high selectivity and accuracy. UPLC-MS was then only needed to analyze the suspicious positive samples, and highly efficient detection was achieved. This robust platform could be referred for preliminary screening of other group analytes.

Characterization of Diarrhetic Shellfish Toxins in the Mizuhopecten yessoensis (Scallop) Midgut Gland by High-Performance Liquid Chromatography – Tandem Mass Spectrometry (HPLC-MS/MS)

Liquid chromatography – tandem mass spectrometry (LC-MS/MS) is routinely applied for the determination of toxins associated with diarrhetic shellfish poisoning (DSP) as a part of the Japanese official method. For scallops, not only the edible part but also the midgut gland may be used for analysis. In this study, the matrix effects in the LC-MS/MS-based determination of okadaic acid (OA) and dinophysistoxin-1 (DTX1) in the scallop midgut gland were evaluated to assess the impact on the accuracy of the results. The analyzed sample in an interlaboratory comparison study was subjected to the Japanese official method. The solutions obtained with and without a cleanup process (combination of hexane washing and solid-phase extraction using a C18 cartridge) were diluted to varying ratios and used for evaluation. For all prepared solutions, the ionization of OA and DTX1 was enhanced compared to the samples in methanol; the cleanup process did not sufficiently reduce the matrix effects. Although the dilution of the sample solution reduced the matrix effect, the improvement was limited. Another pretreatment technique is necessary for the accurate determination of DSP-associated toxins in this matrix.

Development of a colorimetric aptasensor fabricated with a group-specific aptamer and AuNPs@Fe2+ nanozyme for simultaneous detection of multiple diarrheic shellfish poisons

Okadaic acid (OA), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2) are the main diarrheic shellfish poisons (DSPs). Detection methods to achieve simultaneous detection of the three toxins are urgently needed. In this paper, a terminal fixation design proposed in our previous study was further explored to engineer an aptamer with group-specific recognition ability from an original aptamer binding to one target. Both molecular docking assay and biolayer interferometry assay were applied to reveal the binding mechanisms between three DSPs and the engineered aptamer. Then, a label-free colorimetric aptasensor by fabricating the aptamer with AuNPs@Fe2+ nanozyme was constructed, which provided a wide linear detection range (0.4688–7.5 nM), a very low limit of detection (LOD, 86.28 pM), and good recoveries (96.02–104.9%) when analyzing DSPs in seawater and scallop samples, indicating the engineered aptamer and the developed aptasensor had great potential in recognizing and detecting multiple DSPs in real world.

Simultaneous determination of okadaic acid, dinophysistoxin-1, dinophysistoxin-2, and dinophysistoxin-3 using liquid chromatography-tandem mass spectrometry in raw and cooked food matrices

Diarrheic shellfish poison produced by toxic algae (e.g., Dynophysis sp.) adversely affects humans and marine ecosystems. Contamination occurs mainly from bivalve mollusks like mussels, but contamination from fishes (e.g., flatfish) has also recently been reported. Herein, we validated a liquid chromatography-tandem mass spectrometry-based analytical method for identifying okadaic acid (OA), dinophysistoxin-1 (DTX1), dinophysistoxin-2 (DTX2), and dinophysistoxin-3 (DTX3) in mussels, clams, and flatfishes, and their cooked matrices. The limits of both detection and quantification were 0.2–5.1 μg/kg. The accuracy and precision determined from intra- and inter-day repeated analyses for all matrices were 80.5%–109.8% and 0.9%–20.1%, respectively, satisfying all the performance criteria for the analytical method. The relative measurement uncertainty was estimated to be 17.8–40.8% in all matrices. Trueness was confirmed using mussel matrix certified reference material, the measurement value of which was within the certified value range. OA, DTX1, and DTX3 were detected in four mussels, two clams, and one processed case by applying the validated analytical method to commercially available samples (n = 32). In particular, DTX3 was identified in Korean mussels and clams for the first time. This method can be used to control the safety of raw and cooked bivalves and fishes from contamination with OA group toxin.

2D Carbon Nitride-Based Electrochemical Aptasensor for Label-Free and Highly-Sensitive Detection of Okadaic Acid in Shellfish

Okadaic acid (OA) is a marine toxin accumulated in bivalves causing severe diarrhetic shellfish poisoning, which has become a huge threaten to human health, food safety, and environmental protection. Therefore, it is highly essential to develop cost-effective and convenient approaches for OA detection. Recent advances in the electrochemical transducer and nanomaterials may provide novel approaches to address this issue. Herein, a label-free electrochemical impedance aptasensor was developed for the sensitive detection of OA in shellfish. Two-dimensional nanomaterials (carbon nitride) conjugated with aptamers were employed as the sensitive element for OA detection, which have been well characterized by Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM), Fourier Transform Infrared Spectrometer (FTIR) and X-ray diffraction (XRD). The obtained aptasensor exhibited a good performance for the OA detection with a wide linearity ranging from 1 × 10−14 mol l−1 to 1 × 10−8 mol l−1. The limit of detection was down to 1 × 10−14 mol l−1. Besides, this aptasensor had a good selectivity towards OA in the presence of other toxins, such as dinophysistoxins (DTX), pectenotoxins (PTX), and yessotoxin (YTX). Meanwhile, it also showed a good reproducibility and stability. The real mussel samples results showed a good recovery rate. The simple and cost-effective sensing strategy to marine toxins could be applied in the fields of seafood safety and water quality control.

Dinophysis spp. abundance and toxicity events in South Cornwall, U.K.: Interannual variability and environmental drivers at three coastal sites

Dinophysis is a genus of dinoflagellates with the potential to cause diarrhoeic Shellfish Poisoning (DSP) in humans. The lipophilic toxins produced by some species of Dinophysis spp. can accumulate within shellfish flesh even at low cell abundances, and this may result in the closure of a shellfish farm if toxins exceed the recommended upper limit. Over the period 2014 to 2020 inclusive there were several toxic events along the South West coast of U.K. related to Dinophysis spp. The Food Standards Agency (FSA) monitoring programme measure Dinophysis cell abundances and toxin concentration within shellfish flesh around the coasts of England and Wales, but there are few schemes routinely measuring the environmental parameters that may be important drivers for these Harmful Algal Blooms (HABs). This study uses retrospective data from the FSA monitoring at three sites on the south Cornwall coast as well as environmental data from some novel platforms such as coastal WaveRider buoys to investigate potential drivers and explore whether either blooms or toxic events at these sites can be predicted from environmental data. Wind direction was found to be important in determining whether a bloom develops at these sites, and low air temperature in June was associated with low toxicity in the shellfish flesh. Using real time data from local platforms may help shellfish farmers predict future toxic events and minimise financial loss.

Harmful Algal Blooms and their impacts on shellfish mariculture follow regionally distinct patterns of water circulation in the western English Channel during the 2018 heatwave

Harmful algal blooms (HABs) can have severe ecological, societal and economic impacts upon marine ecosystems, human health and the seafood industry. We evaluated changes in marine plankton communities with prevailing physico-chemical conditions throughout an exceptionally warm summer (2018), to elucidate key factors governing HABs and their impacts on shellfish mariculture in the western English Channel. Despite warm, stable weather conditions and widespread seasonal stratification throughout the summer, divergent plankton community compositions were observed at two rope-grown mussel (Mytilus edulis) farms (St Austell Bay and Lyme Bay) and a long-term ecological research LTER site (Plymouth L4). There were significant differences between sites in the abundances of HAB species, including Dinophysis spp. and Karenia mikimotoi, whose cell counts bloomed in excess of UK Food Standards Agency (FSA) advisory 'trigger' levels at Plymouth L4 and St Austell Bay, but not at the Lyme Bay site. The K. mikimotoi bloom occurred over two weeks in August and comprised up to 88% of the standing phytoplankton biomass in St Austell Bay. Dinophysis spp. also bloomed here from May to September, constituting up to 28% of phytoplankton biomass. This protracted bloom resulted in concentrations of Dinophysis toxins 1 & 2 and pectenotoxins and okadaic acid in shellfish, which closed shellfish harvesting operations on farms located in St Austell Bay, and other shellfish sites in the west of the western English Channel (but not in the east of the region). Inter-site differences in the abundances of these and other HAB species were associated with variations in water circulation and co-occurring phytoplankton and zooplankton communities. Furthermore, plankton monitoring data obtained from the L4 site over the past 3 decades showed HAB species (including Dinophysis spp.) with abundances commonly occurring above advisory trigger levels during warmer periods, such as that coinciding with our study. Under projected climate warming these blooms are likely to continue to be governed by regionally distinct patterns of water circulation, which need to be taken into account in marine spatial planning, when assessing the suitability of new shellfish mariculture sites.

Progress on the investigation and monitoring of marine phycotoxins in China

Marine phycotoxins associated with paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), amnesic shellfish poisoning (ASP), neurotoxic shellfish poisoning (NSP), ciguatera fish poisoning (CFP), tetrodotoxin (TTX), palytoxin (PLTX) and neurotoxin β-N-methylamino-L-alanine (BMAA) have been investigated and routinely monitored along the coast of China. The mouse bioassay for monitoring of marine toxins has been progressively replaced by the enzyme-linked immunosorbent assay (ELISA) and liquid chromatography tandem mass spectrometry (LC-MS/MS), which led to the discovery of many new hydrophilic and lipophilic marine toxins. PSP toxins have been detected in the whole of coastal waters of China, where they are the most serious marine toxins. PSP events in the Northern Yellow Sea, the Bohai Sea and the East China Sea are a cause of severe public health concern. Okadaic acid (OA) and dinophysistoxin-1 (DTX1), which are major toxin components associated with DSP, were mainly found in coastal waters of Zhejiang and Fujian provinces, and other lipophilic toxins, such as pectenotoxins, yessotoxins, azaspiracids, cyclic imines, and dinophysistoxin-2(DTX2) were detected in bivalves, seawater, sediment, as well as phytoplankton. CFP events mainly occurred in the South China Sea, while TTX events mainly occurred in Jiangsu, Zhejiang and Fujian provinces. Microalgae that produce PLTX and BMAA were found in the phytoplankton community along the coastal waters of China.

Marine By-Products Tested as Feed for Almaco Jack Seriola rivoliana and Their Effect on Fatty Acids and Sterols in Different Tissues

Marine by-products can compose up to 70% of the total weight of products from fisheries, most of which are discarded. However, these by-products are rich in highly unsaturated fatty acids that are not synthetized by most marine animals produced by aquaculture. Here, we used three marine by-products (shrimp head, Catarina scallop viscera, and Pen shell viscera) to produce lipid-rich (72.9–144.6 g/kg) meals which were used to partially substitute commercial fishmeal (FM) on feeds that were used to grow Almaco Jack (Seriola rivoliana) juveniles for 10 weeks. The content of 20:5n-3 and 22:6n-3 in tissues of fish fed shrimp and Pen shell presented values similar to controls, but the former had a better effect on growth, lipid, and phytosterols levels. Catarina meal had lower concentration of 20:4n-6 and 22:6n-3 in feed but promoted higher proportion of 20:4n-6 in muscle and 22:6n-3 in liver, indicating a selective conservation in relation to other fatty acids. Catarina meal contained traces of 18:5n-3 (0.02 g/kg) indicating that scallops probably ingested dinoflagellates; after testing, phycotoxins like okadaic acid (OA) and dinophysistoxin 1 (DTX1) were detected by mouse bioassay, by lateral flow immunochromatography, and quantified by HPLC–MS/MS. The presence of these toxins at the detected concentrations (OA: 27.64 µg/g and DTX1: 10.31 µg/g) affected almaco jack juveniles, a setback that needs to be addressed before meal manufacturing from mollusks. Marine by-products rich in lipids can be used to reduce the use of FM in the diet, and their use improve the lipid content and growth compared to control diet with FM.Graphical Abstract

Specification of the Okadaic Acid Equivalent for Okadaic Acid, Dinophysistoxin-1, and Dinophysistoxin-2 Based on Protein Phosphatase 2A Inhibition and Cytotoxicity Assays Using Neuro 2A Cell Line

Diarrhetic shellfish poisoning (DSP) is a globally occurring disease threatening public health and trade. The causative toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1), and dinophysistoxin-2 (DTX2) are collectively called OAs, and are quantified using the LC-MS/MS method. The hazardous effect of total OAs is expressed as the sum of OA equivalents defined for respective OAs based on mouse lethality, produced by either intraperitoneal (OAip) or oral administration (OAor). OAs are potent inhibitors of protein phosphatase 2A (PP2A) and are cytotoxic, necessitating expansion of the concept of OA equivalents to all relevant bioactivities. In this study, we determined OA equivalents for respective OA members in PP2A inhibition and cytotoxicity assays. To secure result credibility, we used certified OAs, reference materials, and PP2A produced using genetic engineering. The relative ratio of the OA equivalents determined by PP2A inhibition assays for OA, DTX1, and DTX2 were 1.0:1.6:0.3, while the ratio determined using the cytotoxicity assays indicated 1.0:1.5:0.5. OA equivalents showed a similar tendency in the PP2A inhibition and cytotoxicity assays, and matched better with oral toxicity data than intraperitoneal toxicity in mice. The PP2A inhibition assay, which measures the core activity of the OAs, suggested a higher OA equivalent for DTX1 than that currently used.

Cyclodextrin polymers as passive sampling materials for lipophilic marine toxins in Prorocentrum lima cultures and a Dinophysis sacculus bloom in the NW Mediterranean Sea

Cyclodextrins, cyclic oligomers that form a conical structure with an internal cavity, are proposed as new and sustainable materials for passive sampling of lipophilic marine toxins. Two applicability scenarios have been tested. First, disks containing β-cyclodextrin-hexamethylene diisocyanate (β-CD-HDI) and β-cyclodextrin-epichlorohydrin (β-CD-EPI) polymers were immersed in Prorocentrum lima cultures for different days (2, 12 and 40). LC-MS/MS analysis showed capture of free okadaic acid (OA) and dinophysistoxin-1 (DTX1) by cyclodextrins at contents that increased with immersion time. Cyclodextrins resulted more efficient in capturing DTX1 than OA. In a second experiment, disks containing β-CD-HDI, β-CD-EPI, γ-CD-HDI and γ-CD-EPI were deployed in harbor waters of El Masnou (NW Mediterranean Sea) during a Dinophysis sacculus bloom in February 2020. Free OA and pectenotoxin-2 (PTX2) were captured by cyclodextrins. Toxin contents were higher at sampling points and sampling weeks with higher D. sacculus cell abundance. In this case, PTX2 capture with cyclodextrins was more efficient than OA capture. Therefore, cyclodextrins have provided information regarding the toxin profile of a P. lima strain and the spatial and temporal dynamics of a D. sacculus bloom, proven efficient as passive sampling materials for environmental monitoring.

Shellfish contamination with lipophilic toxins and dietary exposure assessments from consumption of shellfish products in Shenzhen, China

Lipophilic shellfish toxins (LSTs) can cause human illness and therefore represent a serious threat to public health. Shellfish are the main dietary source of LSTs, but very few studies have appraised dietary exposure to LSTs through shellfish consumption in China. We measured levels of multiple LSTs in shellfish samples sold in the principal wholesale seafood market in the southern coastal city of Shenzhen, and we estimated the potential for acute and chronic LST exposure of the Shenzhen population via ingestion of shellfish. LST contamination data were obtained from a total of 14 species of 188 commercial samples. Eleven individual LSTs, namely okadaic acid (OA), dinophysis toxin-1 and −2 (DTX1 and DTX2), pectenotoxin-2 (PTX2), yessotoxin and homo yessotoxin (YTX and hYTX), azaspiracid-1, −2 and −3 (AZA1, AZA2, AZA3), spirolides (SPXs), and gymnodimine (GYM), were determined using liquid chromatography electrospray-ionization tandem mass spectrometry (LC-ESI-MS/MS). More than two thirds of samples showed undetectable LSTs, while the detection rates (the proportion of samples with detectable LSTs) of individual LSTs ranged from 0% to 45.7%. Most shellfish samples had lower levels of LST contamination than the corresponding limits of detection (LODs), while some samples had levels of hYTX and GYM that exceeded the limits of quantification (LOQs). Overall, levels of LSTs in the 188 samples were below the regulatory limits set by most countries. Acute and chronic exposures of LST were estimated by a point-estimate modeling method that combined sample contamination data with consumption data from dietary survey of Shenzhen residents and consumption figures proposed by EFSA, the European Food Safety Authority. Seasonal variations in LST concentrations were noted in some instances. Overall, the estimated acute exposure to LSTs based on consumption of large-size shellfish portions and the maximum LSTs contamination level were below the provisional acute reference doses (ARfDs) proposed by the EFSA. Chronic exposure estimates based on mean and 99th percentile consumption of shellfish by Shenzhen residents and mean LSTs contamination levels in the collected samples were from 2452 to 74 times lower than those associated with estimated acute exposure levels.

Occurrence and Trophodynamics of Marine Lipophilic Phycotoxins in a Subtropical Marine Food Web.

Marine lipophilic phycotoxins (MLPs) are produced by toxigenic microalgae and cause foodborne illnesses. However, there is little information on the trophic transfer potential of MLPs in marine food webs. In this study, various food web components including 17 species of mollusks, crustaceans, and fishes were collected for an analysis of 17 representative MLPs, including azaspiracids (AZAs), brevetoxins (BTXs), gymnodimine (GYM), spirolides (SPXs), okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs), yessotoxins (YTXs), and ciguatoxins (CTXs). Among the 17 target MLPs, 12, namely, AZAs1-3, BTX3, GYM, SPX1, OA, DTXs1-2, PTX2, YTX, and the YTX derivative homoYTX, were detected, and the total MLP concentrations ranged from 0.316 to 20.3 ng g-1 wet weight (ww). The mean total MLP concentrations generally decreased as follows: mollusks (8.54 ng g-1, ww) > crustaceans (1.38 ng g-1, ww) > fishes (0.914 ng g-1, ww). OA, DTXs, and YTXs were the predominant MLPs accumulated in the studied biota. Trophic dilution of the total MLPs was observed with a trophic magnification factor of 0.109. The studied MLPs might not pose health risks to residents who consume contaminated seafood; however, their potential risks to the ecosystem can be a cause for concern.

Current Trends and Challenges for Rapid SMART Diagnostics at Point-of-Site Testing for Marine Toxins.

In the past twenty years marine biotoxin analysis in routine regulatory monitoring has advanced significantly in Europe (EU) and other regions from the use of the mouse bioassay (MBA) towards the high-end analytical techniques such as high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS). Previously, acceptance of these advanced methods, in progressing away from the MBA, was hindered by a lack of commercial certified analytical standards for method development and validation. This has now been addressed whereby the availability of a wide range of analytical standards from several companies in the EU, North America and Asia has enhanced the development and validation of methods to the required regulatory standards. However, the cost of the high-end analytical equipment, lengthy procedures and the need for qualified personnel to perform analysis can still be a challenge for routine monitoring laboratories. In developing regions, aquaculture production is increasing and alternative inexpensive Sensitive, Measurable, Accurate and Real-Time (SMART) rapid point-of-site testing (POST) methods suitable for novice end users that can be validated and internationally accepted remain an objective for both regulators and the industry. The range of commercial testing kits on the market for marine toxin analysis remains limited and even more so those meeting the requirements for use in regulatory control. Individual assays include enzyme-linked immunosorbent assays (ELISA) and lateral flow membrane-based immunoassays (LFIA) for EU-regulated toxins, such as okadaic acid (OA) and dinophysistoxins (DTXs), saxitoxin (STX) and its analogues and domoic acid (DA) in the form of three separate tests offering varying costs and benefits for the industry. It can be observed from the literature that not only are developments and improvements ongoing for these assays, but there are also novel assays being developed using upcoming state-of-the-art biosensor technology. This review focuses on both currently available methods and recent advances in innovative methods for marine biotoxin testing and the end-user practicalities that need to be observed. Furthermore, it highlights trends that are influencing assay developments such as multiplexing capabilities and rapid POST, indicating potential detection methods that will shape the future market.

Simultaneous determination of 12 lipophilic shellfish toxins in plasma and urine by ultra-high performance liquid chromatography-tandem mass spectrometry

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了优化,采用乙腈对尿液和血浆样品进行提取。采用Phenomenex Kinetex C18色谱柱(50 mm×3 mm, 2.6 μm)进行分离,以0.05%(v/v)氨水水溶液、90%(v/v)乙腈水溶液为流动相,以流速0.40 mL/min梯度洗脱时,12种目标化合物分离效果最好。串联质谱的离子源为电喷雾离子(ESI)源,采用多反应监测(MRM)模式检测。12种目标物的基质效应均在0.8~1.1之间,表明该前处理方法的基质干扰低,采用外标法可对化合物进行准确定量。12种贝类毒素的线性范围为0.03~36.25 μg/L,相关系数均大于0.995。尿液检测的方法定量限为0.23~0.63 μg/L,血浆检测的方法定量限为0.31~0.84 μg/L。3个加标水平的回收率为72.7%~124.1%,日内精密度为2.1%~20.0%,日间精密度为2.1%~15.3%。利用该方法检测健康人尿液和血浆样本,以及经腹腔注射12种贝类毒素的小鼠尿液和血液样本。20份健康人样本中未检出目标物,20份小鼠样本中12种贝类毒素均有检出。该方法操作简便,样品取样量少,方法灵敏高,适用于血浆和尿液中脂溶性贝类毒素的快速检测。

Selective analysis of the okadaic acid group in shellfish samples using fluorous derivatization coupled with liquid chromatography-tandem mass spectrometry

Okadaic acid (OA) group are diarrheal shellfish poison that accumulates in the midgut glands of shellfish. It is difficult to remove these poisons by normal cooking because they are thermally stable and hydrophobicity. Therefore, in order to prevent foodborne disease due to shellfish poison, analysis by liquid chromatography (LC)-tandem mass spectrometry (MS/MS) before shipment is necessary. Herein the selective analytical method for OA group in shellfish sample using fluorous derivatization coupled with LC-MS/MS was developed. OA group were derivatized with the fluorous alkylamine reagent by condensing agent, and the obtained derivatives were separated with fluorous LC column (Fluofix-II 120E, 250 × 2.0 mm i.d., 5 μm, Fujifilm Wako Pure Chemical). The derivatized OA group were selective retained by fluorous LC column and accurate analysis was enabled. The present method was applied to the analysis of OA and dinophysistoxin-1 (DTX-1) in scallop midgut gland which is the certified reference material provided by national metrology institute of Japan. As a result of analysis using the present method with DTX-2 as the internal standard, the quantitative value were in agreement with the certified value.