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Lipophilic shellfish toxins pose significant threats to the health of seafood consumers and public health. The symptoms of these kinds of toxins include severe diarrhea, abdominal cramps, nausea and gastrointestinal disorders. These symptoms could be hardly distinguished with many other symptoms of food poisoning and diseases. Therefore, a fast and accurate determination method in human biological samples is urgently needed for the accurate judgement of food poisoning incident, which is important for the investigation of public health emergencies and clinical treatment of poisoned patients. However, there were several flaws of the previous studies reported on the analysis of lipophilic shellfish toxins: (1) limited target compounds were covered; (2) the pre-treatment process was complex; (3) the sensitivity of the compound was low. In this study, a simple extraction method coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the simultaneous determination of 12 lipophilic shellfish toxins, including azaspir acid 1 (AZA1), azaspir acid 2 (AZA2), azaspir acid 3 (AZA3), dinophysistoxin 1 (DTX1), dinophysistoxin 2 (DTX2), gymnodimine (GYM), hyessotoxin (HYTX), okadaic acid (OA), pinnatoxin (Pntx), pectenotoxins 2 (PTX2), spirolides 1 (SPX1), yessotoxin (YTX), in plasma and urine. Firstly, the instrument conditions were optimized. Different additions in mobile phase were compared and 0.05% (v/v) ammonia solution was selected since it can improve the peak shape of YTX and HYTX, and increase the respondence by four times. Secondly, the volume of acetonitrile (0.2, 0.4, 0.6, 0.8, 1.0 mL) use for the extraction of the target compounds in plasma was optimized. Satisfactory recoveries were obtained when 0.6 mL of acetonitrile was used. At the same time, satisfactory recoveries were obtained when 0.9 mL of acetonitrile was used in urine samples. Finally, under the optimized conditions, the 12 compounds in plasma and urine samples were ultrasonically extracted with acetonitrile. Chromatographic separation was performed on a Phenomenex Kinetex C18 column (50 mm×3 mm, 2.6 μm) with 90% (v/v) acetonitrile aqueous solution and water containing 0.05% (v/v) ammonia as mobile phases. Gradient elution with a flow rate of 0.40 mL/min was employed. The 12 compounds were monitored in the multiple reactions monitoring (MRM) mode with electrospray ionization (ESI) under both positive and negative conditions. The matrix effects of the 12 compounds ranged from 0.8 to 1.1. Therefore, external standard calibration curves were used for the quantification. The 12 shellfish toxins showed good linear relationship in the range of 0.03-36.25 μg/L with the correlation coefficients greater than 0.995. The limits of detection (LODs, S/N =3) were 0.08-0.21 ng/mL for the urine samples and 0.10-0.28 μg/L for the plasma samples, respectively. The limit of quantitations (LOQs, S/N =10) were 0.23-0.63 μg/L for the urine samples and 0.31-0.84 μg/L for the plasma samples, respectively. The recoveries of the 12 compounds were in the range of 72.7%-124.1% at three spiked levels (i. e., LOQ, three times LOQ, and ten times LOQ). The intra-day and inter-day precisions were 2.1%-20.0% and 2.1%-15.3%, respectively. The method was applied in the detection of the 12 lipophilic shellfish toxins in the urine and plasma samples of healthy humans and mice previously injected with the 12 shellfish toxins intraperitoneally. None of the 12 toxins were found in the samples from healthy human, while all of the 12 lipophilic shellfish toxins were found in the urine and plasma samples collected from the poisoned mice in the range of 1.14-2.35 μg/L and 1.01-1.17 μg/L, respectively. The established method has the advantages of sensitive, quick, easy to operate, and of low sample volume. It can be used for the simultaneous determination of 12 lipophilic shellfish toxins in urine and plasma samples.

Brevetoxins were confirmed in urine specimens from patients diagnosed with neurotoxic shellfish poisoning (NSP) after consumption of gastropods that were recreationally harvested from an area previously affected by a Karenia brevis bloom. Several species of gastropods (Triplofusus giganteus, Sinistrofulgur sinistrum, Cinctura hunteria, Strombus alatus, Fulguropsis spirata) and one clam (Macrocallista nimbosa) from the NSP implicated gastropod collection area (Jewfish Key, Sarasota Bay, Florida) were examined for brevetoxins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA). All gastropods and the clam were contaminated with brevetoxins. Composite B-type toxin concentrations in gastropods ranged from 1.1 to 198 μg BTX-3 equiv./g by ELISA, levels likely capable of causing NSP in consumers. Several brevetoxin metabolites previously characterized in molluscan shellfish were identified in these gastropods. Brevetoxin analog profiles by ELISA were similar in the gastropod species examined. This work documents the occurrence of NSP through consumption of a type of seafood not typically monitored in Florida to protect human health, demonstrating the need to better assess and communicate the risk of NSP to gastropod harvesters in Karenia brevis endemic areas., (Published by Elsevier Ltd.)

Domoic acid (DA)-producing harmful algal blooms (HABs) have been present at unprecedented geographic extent and duration in recent years causing an increase in contamination of seafood by this common environmental neurotoxin. The toxin is responsible for the neurotoxic illness, amnesic shellfish poisoning (ASP), that is characterized by gastro-intestinal distress, seizures, memory loss, and death. Established seafood safety regulatory limits of 20 μg DA/g shellfish have been relatively successful at protecting human seafood consumers from short-term high-level exposures and episodes of acute ASP. Significant concerns, however, remain regarding the potential impact of repetitive low-level or chronic DA exposure for which there are no protections. Here, we report the novel discovery of a DA-specific antibody in the serum of chronically-exposed tribal shellfish harvesters from a region where DA is commonly detected at low levels in razor clams year-round. The toxin was also detected in tribal shellfish consumers' urine samples confirming systemic DA exposure via consumption of legally-harvested razor clams. The presence of a DA-specific antibody in the serum of human shellfish consumers confirms long-term chronic DA exposure and may be useful as a diagnostic biomarker in a clinical setting. Adverse effects of chronic low-level DA exposure have been previously documented in laboratory animal studies and tribal razor clam consumers, underscoring the potential clinical impact of such a diagnostic biomarker for protecting human health. The discovery of this type of antibody response to chronic DA exposure has broader implications for other environmental neurotoxins of concern.

In August 2014, a puffer fish poisoning incidence resulting in one fatality was reported in New Caledonia. Although tetrodotoxin (TTX) intoxication was established from the patients' signs and symptoms, the determination of TTX in the patient's urine, serum or plasma is essential to confirm the clinical diagnosis. To provide a simple cost-effective rapid screening tool for clinical analysis, a maleimide-based enzyme-linked immunosorbent assay (mELISA) adapted for the determination of TTX contents in human body fluids was assessed. The mELISA was applied to the analysis of urine samples from two patients and a response for the presence of TTX and/or structurally similar analogues was detected in all samples. The analysis by LC-MS/MS confirmed the presence of TTX but also TTX analogues (4-epiTTX, 4,9-anhydroTTX and 5,6,11-trideoxyTTX) in the urine. A change in the multi-toxin profile in the urine based on time following consumption was observed. LC-MS/MS analysis of serum and plasma samples also revealed the presence of TTX (32.9 ng/mL) and 5,6,11-trideoxyTTX (374.6 ng/mL) in the post-mortem plasma. The results provide for the first time the TTX multi-toxin profile of human samples from a puffer fish intoxication and clearly demonstrate the implication of TTX as the causative agent of the reported intoxication case., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Over the past 15 years or so, several Scottish harbour seal (Phoca vitulina) populations have declined in abundance and several factors have been considered as possible causes, including toxins from harmful algae. Here we explore whether a link could be established between two groups of toxins, domoic acid (DA) and saxitoxins (STXs), and the decline in the harbour seal populations in Scotland. We document the first evidence that harbour seals are exposed to both DA and STXs from consuming contaminated fish. Both groups of toxins were found in urine and faeces sampled from live captured (n = 162) and stranded animals (n = 23) and in faecal samples collected from seal haul-out sites (n = 214) between 2008 and 2013. The proportion of positive samples and the toxins levels measured in the excreta were significantly higher in areas where harbour seal abundance is in decline. There is also evidence that DA has immunomodulatory effects in harbour seals, including lymphocytopenia and monocytosis. Scottish harbour seals are exposed to DA and STXs through contaminated prey at potentially lethal levels and with this evidence we suggest that exposure to these toxins are likely to be important factors driving the harbour seal decline in some regions of Scotland., (Copyright © 2015 Elsevier Ltd. All rights reserved.)