Loading...
Publication
Analysis of paralytic shellfish toxins in natural samples: liquid chromatography coupled to fluorescent detection versus liquid chromatography coupled to mass spectrometry
| Name: | Description: | Size: | Format: | |
|---|---|---|---|---|
| 2.58 MB | Adobe PDF |
Authors
Advisor(s)
Abstract(s)
As toxinas PSP (Paralytic Shellfish Poison), conhecidas pelos seus efeitos paralisantes, são produzidas por várias espécies de dinoflagelados marinhos e subsequentemente bioacumulam-se em bivalves filtradores. Estes bivalves tornam-se vetores para o consumo humano, podendo causar sintomas tóxicos como dormência, diarreia, náuseas e vómitos, e, em casos graves, levar à morte por paralisia respiratória. Estas toxinas são monitorizadas por diversos países para proteger a saúde pública. No entanto, algumas toxinas PSP não são monitorizadas nem regulamentadas, pois foram recentemente descobertas através de técnicas analíticas avançadas.
Neste estudo, comparamos o método AOAC de cromatografia líquida com deteção por fluorescência com o método desenvolvido no nosso laboratório, que utiliza cromatografia líquida acoplada à espectrometria de massa de alta resolução (LC-HRMS), seguida de extração de cromatogramas da massa exata (AMXIC) para quantificar as toxinas PSP regulamentadas e não regulamentadas. O procedimento AMXIC demonstrou ser seletivo, fornecendo perfis individuais claros tanto para toxinas regulamentadas quanto para não regulamentadas. Devido à indisponibilidade de CRMs (Certified Reference Materials) para esses análogos, foi realizada uma estimativa usando o procedimento AMXIC e a resposta ao análogo de toxina regulamentada mais semelhante. Analisaram-se extratos de amostras de mexilhão naturalmente contaminadas (Mytilus galloprovincialis) e do dinoflagelado marinho Gymnodinium catenatum utilizando os métodos mencionados. Além disso, os efeitos de matriz foram avaliados em extratos preparados com cartuchos de C18 e de carbono grafitizado, utilizando saxitoxina como composto de referência.
As toxinas PSP não regulamentadas com um grupo hidroxilo no C-11, conhecidas como toxinas M, foram detetadas na amostra de mexilhão contaminado, enquanto as toxinas GC foram observadas apenas na amostra de dinoflagelado. A identificação foi feita com base nas massas exatas e nos padrões de fragmentação. No entanto, nem todas as toxinas não regulamentadas presentes nas amostras foram identificadas devido à baixa intensidade dos sinais destes analitos após fragmentação. A falta de CRMs para essas toxinas não regulamentadas limita a sua identificação. Em conclusão, o método HPLC-FLD é eficaz para triagem, especialmente em análises de rotina. No entanto, o método LC-HRMS permite uma identificação mais precisa do perfil completo das toxinas PSP nas amostras, contribuindo para uma melhor avaliação da toxicidade total.
Paralytic shellfish toxins (PST) are produced by several marine dinoflagellate species that bioaccumulate in bivalves after feeding. These filter-feeder bivalves then serve as vector for human consumption and cause toxic symptoms such as numbness, diarrhea, nausea and vomiting leading to death due to respiratory paralysis. These toxins are monitored by different countries to protect national public health. However, only regulated PSTs are covered. On the other hand, non-regulated PSTs toxins, which are new analogues discovered in the recent years using advanced analytical techniques, may pose unknown threats. We compared the AOAC method, that uses liquid chromatography coupled to fluorescent detection, with the in-house-developed liquid chromatography-high resolution mass spectrometry (LC-HRMS) method, which involves full scan acquisition followed by accurate mass extracted ion chromatograms (AMXIC) to quantify the regulated and the non-regulated PSTs. The AMXIC procedure is selective as it gives clean individual profiles for regulated and non-regulated toxins. As CRMs for these analogues are not available, an estimate was obtained using AMXIC procedure and the response to the most similar regulated toxin analogue. Extracts from naturally contaminated mussel sample Mytilus galloprovincialis and marine dinoflagellate Gymnodinium catenatum were analyzed using the mentioned methods. Matrix effects evaluated in extracts prepared using C18 and graphitized carbon cartridges using saxitoxin as the reference compound. C-11 hydroxyl metabolites (so-called M-toxins) were detected in the contaminated mussel sample, while GC-toxins were only observed from the dinoflagellate sample. The identification was based on the exact expected m/z values and on the fragmentation patterns. However, not all identified non-regulated toxins were identified due to the low intensity of signals of searched analytes after fragmentation. The unavailability of CRM counterparts of non-regulated toxins limits to identify toxin analogue. Overall, the HPLC-FLD is very good as a screening method especially in routine analyze. The LC-HRMS method, on the other hand, is notable in establishing toxin profile of samples that contributes to better evaluation of overall toxicity.
Paralytic shellfish toxins (PST) are produced by several marine dinoflagellate species that bioaccumulate in bivalves after feeding. These filter-feeder bivalves then serve as vector for human consumption and cause toxic symptoms such as numbness, diarrhea, nausea and vomiting leading to death due to respiratory paralysis. These toxins are monitored by different countries to protect national public health. However, only regulated PSTs are covered. On the other hand, non-regulated PSTs toxins, which are new analogues discovered in the recent years using advanced analytical techniques, may pose unknown threats. We compared the AOAC method, that uses liquid chromatography coupled to fluorescent detection, with the in-house-developed liquid chromatography-high resolution mass spectrometry (LC-HRMS) method, which involves full scan acquisition followed by accurate mass extracted ion chromatograms (AMXIC) to quantify the regulated and the non-regulated PSTs. The AMXIC procedure is selective as it gives clean individual profiles for regulated and non-regulated toxins. As CRMs for these analogues are not available, an estimate was obtained using AMXIC procedure and the response to the most similar regulated toxin analogue. Extracts from naturally contaminated mussel sample Mytilus galloprovincialis and marine dinoflagellate Gymnodinium catenatum were analyzed using the mentioned methods. Matrix effects evaluated in extracts prepared using C18 and graphitized carbon cartridges using saxitoxin as the reference compound. C-11 hydroxyl metabolites (so-called M-toxins) were detected in the contaminated mussel sample, while GC-toxins were only observed from the dinoflagellate sample. The identification was based on the exact expected m/z values and on the fragmentation patterns. However, not all identified non-regulated toxins were identified due to the low intensity of signals of searched analytes after fragmentation. The unavailability of CRM counterparts of non-regulated toxins limits to identify toxin analogue. Overall, the HPLC-FLD is very good as a screening method especially in routine analyze. The LC-HRMS method, on the other hand, is notable in establishing toxin profile of samples that contributes to better evaluation of overall toxicity.
Description
Keywords
paralytic shellfish toxins saxitoxins liquid chromatography-high