Simultaneous determination of neurotoxic shellfish toxins (brevetoxins) in commercial shellfish by liquid chromatography tandem mass spectrometry

et al. 2021

Toxins

In this study, Karenia brevis 165 (K. brevis 165), a Chinese strain, was used to research brevetoxin (BTX) metabolites. The sample pretreatment method for the enrichment of BTX metabolites in an algal culture medium was improved here. The method for screening and identifying intracellular and extracellular BTX metabolites was established based on liquid chromatography-time-of-flight mass spectrometry (LC-ToF-MS) and liquid chromatography triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS). The results show that the recovery rates for BTX toxins enriched by a hydrophilic–lipophilic balance (HLB) extraction column were higher than those with a C18 extraction column. This method was used to analyze the profiles of extracellular and intracellular BTX metabolites at different growth stages of K. brevis 165. This is the first time a Chinese strain of K. brevis has been reported that can produce toxic BTX metabolites. Five and eight kinds of BTX toxin metabolites were detected in the cell and culture media of K. brevis 165, respectively. Brevenal, a toxic BTX metabolite antagonist, was found for the first time in the culture media. The toxic BTX metabolites and brevenal in the K. brevis 165 cell and culture media were found to be fully proven in terms of the necessity of establishing a method for screening and identifying toxic BTX metabolites. The results found by qualitatively and quantitatively analyzing BTX metabolites produced by K. brevis 165 at different growth stages show that the total toxic BTX metabolite contents in single cells ranged between 6.78 and 21.53 pg/cell, and the total toxin concentration in culture media ranged between 10.27 and 449.11 μg/L. There were significant differences in the types and contents of toxic BTX metabolites with varying growth stages. Therefore, when harmful algal blooms occur, the accurate determination of BTX metabolite types and concentrations will be helpful to assess the ecological disaster risk in order to avoid hazards and provide appropriate disaster warnings.

Section: Introductionmentioning

confidence: 99%

Profiling of Brevetoxin Metabolites Produced by Karenia brevis 165 Based on Liquid Chromatography-Mass Spectrometry

Shen

Song

et al. 2021

Toxins

“…(5) After further washing, 100 µL/well peroxidaseconjugated goat anti-mouse IgG (diluted 1:5000) was added and incubated at 37 • C for 30 min. (6) The plate was washed five times, 100 µL/well substrate solution was added to the reaction, and the plate was incubated at 37 • C for 15 min. (7) The reaction was stopped with 50 µL 2 M H 2 SO 4 , and the absorbance was detected at 450 nm on a Multiskan MK3 microplate reader.…”

Section: Elisa and Icelisa Protocolsmentioning

confidence: 99%

“…Several analytical methods have been established to detect brevetoxins, includi liquid chromatography-tandem mass spectrometry (LC-MS/MS) and receptor/antibod based immunoassays. For example, Shin et al (2018) developed an LC-MS/MS proto for the PbTx-1, PbTx-2, and PbTx-3 brevetoxins with a limit of quantification (LOQ) of μg/kg for each toxin [6]. Similarly, Wunschel et al (2018) developed an electrospray L MS/MS system for the same brevetoxins with an LOQ of 2.5 μg/kg for each toxin [7].…”

Section: Introductionmentioning

confidence: 99%

Development of a New Monoclonal Antibody against Brevetoxins in Oyster Samples Based on the Indirect Competitive Enzyme-Linked Immunosorbent Assay

Ding

et al. 2021

Foods

The consumption of shellfish contaminated with brevetoxins, a family of ladder-frame polyether toxins formed during blooms of the marine dinoflagellate Karenia brevis, can cause neurotoxic poisoning, leading to gastroenteritis and neurotoxic effects. To rapidly monitor brevetoxin levels in oysters, we generated a broad-spectrum antibody against brevetoxin 2 (PbTx-2), 1 (PbTx-1), and 3 (PbTx-3) and developed a rapid indirect competitive enzyme-linked immunosorbent assay (icELISA). PbTx-2 was reacted with carboxymethoxylamine hemihydrochloride (CMO) to generate a PbTx-2-CMO hapten and reacted with succinic anhydride (HS) to generate the PbTx-2-HS hapten. These haptens were conjugated to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) to prepare immunogen and coating antigen reagents, respectively, using the active ester method. After immunization and cell fusion, a broad-spectrum monoclonal antibody (mAb) termed mAb 1D3 was prepared. The 50% inhibitory concentration (IC50) values of the icELISA for PbTx-2, PbTx-1, and PbTx-3 were 60.71, 52.61, and 51.83 μg/kg, respectively. Based on the broad-spectrum mAb 1D3, an icELISA was developed to determine brevetoxin levels. Using this approach, the limit of detection (LOD) for brevetoxin was 124.22 μg/kg and recoveries ranged between 89.08% and 115.00%, with a coefficient of variation below 4.25% in oyster samples. These results suggest that our icELISA is a useful tool for the rapid monitoring of brevetoxins in oyster samples.

“…Three main types of methods can be used to analyze BTXs in microalgae cells, seawater samples, marine organisms (molluscs, fish), and sea spray/aerosol samples. (1) Physicochemical methods such as liquid chromatography-mass spectrometry (LC-MS/MS) enable either the targeted identification and quantification of BTXs for which chemical standards are available [18,[31][32][33][34], or the non-targeted detection of potential new BTX analogues [35].…”

Section: Introductionmentioning

confidence: 99%

“…Three main types of methods can be used to analyze BTXs in microalgae cells, seawater samples, marine organisms (molluscs, fish), and sea spray/aerosol samples. (1) Physico-chemical methods such as liquid chromatography-mass spectrometry (LC-MS/MS) enable either the targeted identification and quantification of BTXs for which chemical standards are available [ 18 , 31 , 32 , 33 , 34 ], or the non-targeted detection of potential new BTX analogues [ 35 ]. (2) Biochemical methods, such as specific binding tests (receptor binding assay (RBA) or immunological tests (radioimmunoassay (RIA), ELISA)), enable the overall quantification of BTXs [ 33 , 36 , 37 , 38 ].…”

Section: Introductionmentioning

confidence: 99%

Guidance Level for Brevetoxins in French Shellfish

et al. 2021

Brevetoxins (BTXs) are marine biotoxins responsible for neurotoxic shellfish poisoning (NSP) after ingestion of contaminated shellfish. NSP is characterized by neurological, gastrointestinal and/or cardiovascular symptoms. The main known producer of BTXs is the dinoflagellate Karenia brevis, but other microalgae are also suspected to synthesize BTX-like compounds. BTXs are currently not regulated in France and in Europe. In November 2018, they have been detected for the first time in France in mussels from a lagoon in the Corsica Island (Mediterranean Sea), as part of the network for monitoring the emergence of marine biotoxins in shellfish. To prevent health risks associated with the consumption of shellfish contaminated with BTXs in France, a working group was set up by the French Agency for Food, Environmental and Occupational Health & Safety (Anses). One of the aims of this working group was to propose a guidance level for the presence of BTXs in shellfish. Toxicological data were too limited to derive an acute oral reference dose (ARfD). Based on human case reports, we identified two lowest-observed-adverse-effect levels (LOAELs). A guidance level of 180 µg BTX-3 eq./kg shellfish meat is proposed, considering a protective default portion size of 400 g shellfish meat.