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Anatoxin-a is a neurotoxin produced by various species of cyanobacteria such Anabaena, Planktothrix (formerly Oscillatoria), Aphanizomenon, Cylindrospermum, and Microcystis spp. This toxin acts as a mimic of the neurotransmitter acetylcholine and irreversibly binds the nicotinic acetylcholine receptor (NAChR) therefore affecting the functioning of the nervous system. Experimental studies have shown that in few minutes after drinking contaminated water, animals presented typical symptoms of anatoxin-a intoxication such as muscle fasciculation, gasping, violent convulsions, and death due to respiratory arrest. The estimated dosage needed to produce death in 50% of exposed mice (LD50) after interperitoneal administration was approximately 200 µg/kg. Anatoxin-a was first detected in Canada (1960s) and since then has been responsible for a number of animal and human fatalities, in different parts of the world. The search for efficient and reliable analytical methods for the control of such cyanotoxins is becoming more and more urgent due to their recent appearance and worldwide distribution. The present work, summarizes the development of an automated method combining Solid phase microextraction (SPME) with Gas Chromatography coupled with Mass Spectrometry (GC-MS) for the fast and sensitive determination of Anatoxin-a in water samples. This method is based on the direct derivatization of the analyte by adding isobutylchoroformate to the sample extract in alkaline conditions. The derivatized anatoxin-a was extracted by SPME procedure, submersing a PDMS fiber in an amber vial for 20 min under magnetic stirring. GC-MS is used to identify and quantify the analyte in the SIM mode (quantification ions underlined): 191,164 and 265. Parameters affected to the extraction such as: salt concentration, time of extraction, time of reaction and stirring speed, were initially evaluated. The results obtained in this study let us to conclude that this method is an efficient and fast alternative for the reliable control of anatoxin-a present in contaminated waters.

Dissertação de mestrado em Técnicas de Caracterização e Análise Química, Reunidas determinadas condições ambientais, algumas espécies fitoplanctónicas florescem massivamente libertando biotoxinas para o meio aquático. Por exposição a estas biotoxinas marinhas, alguns dos animais que se alimentam por filtração, nomeadamente moluscos bivalves, vão acumular e concentrar estas biotoxinas nos seus tecidos comestíveis. Entre estas biotoxinas marinhas encontram-se as toxinas amnésicas e um grande conjunto de toxinas designado por toxinas lipofílicas. O método oficial de análise de biotoxinas marinhas lipofílicas baseia-se na cromatografia líquida com detecção por espectrometria de massa em tandem (LCMS/ MS). Trata-se de um método multiresíduos a partir do qual é possível determinar quantitativamente um conjunto relativamente grande de biotoxinas marinhas lipofílicas que, por possuírem características comuns, designadamente o carácter lipofílico, por serem lábeis, polares e não voláteis, puderam ser agrupadas e analisadas num só passo. As toxinas amnésicas, compostas pelo ácido domóico (DA) e pelos seus isómeros, apresentam uma solubilidade relativa em solventes orgânicos, sendo que no procedimento LSE adoptado para a extracção do DA o solvente utilizado é uma mistura de metanol/água (1:1, v/v). Reconhecida a flexibilidade deste método multiresíduos em incorporar novas biotoxinas marinhas e com o conhecimento do carácter “semi-lipofílico” do DA, pretendeu-se encontrar um compromisso entre as toxinas lipofílicas e as toxinas amnésicas no que respeita ao rácio de metanol usado na extracção de ambas, e estudar a possibilidade de incluir estas toxinas no referido método. Este estudo experimental envolveu quatro procedimentos de extracção LSE de materiais de referência contaminados por toxinas ASP, que variam ligeiramente do método multitoxinas lipofílicas, designadamente na proporção de metanol usado para este fim, com o objectivo de estudar o referido proposto. As análises UPLC-MS/MS foram levadas a cabo recorrendo um espectrómetro de massa triplo quadrupolo com ionização por ESI em modo positivo com uma monitorização por SRM., When certain environmental conditions are combined, some of phytoplankton species bloom massively releasing biotoxins to the aquatic environment. From exposure to these marine biotoxins, some of the animals that feed by filtering, including bivalve molluscs, will accumulate and concentrate these biotoxins in their edible tissues. Among these biotoxins are amnesic toxins and a host of toxins known as lipophilic toxins. The official method of analysis of lipophilic biotoxins is based on liquid chromatography with tandem mass spectrometry detection (LCMS/ MS). Based on this multiresidue method, it is possible to quantify a relatively large amount of lipophilic biotoxins that, by having common characteristics, namely lipophilic character, to be labile, polar and non-volatile could be pooled and analysed in a one step. Amnesic toxins, comprising the domoic acid (DA) and its isomers, show a relative solubility in organic solvents, and the LSE procedure adopted for the DA extraction make use of a mixture of methanol/water (1:1, v/v). Recognized the flexibility of this multiresidue method in incorporating new biotoxins and with the knowledge of the DA "semi-lipophilic” character, it was intended to find a compromise between the lipophilic toxins and amnesic toxins as regards of the methanol ratio used for both extraction, and study the possibility of including these toxins in the multiresidue method. This experimental study involving four LSE procedures applied to reference materials contaminated by toxins ASP, ranging from slightly lipophilic multitoxinas method, namely the ratio of methanol used for this purpose, with the aim of studying the above proposed. UPLC-MS/MS analyzes were carried out using a triple quadrupole mass spectrometer with ESI ionization in positive mode and monitored by SRM.