Chemical methods for detecting phycotoxins: LC and LC/MS/MS

Abstract

Phycotoxins are natural products which are generally synthesized by marine microalgae, especially those belonging to the dinoflagellates group. Approximately 20 species of dinoflagellates and a smaller number of diatoms are currently known to produce phycotoxins and these account for less than 2% of all microalgae species. They are known to produce intoxication syndromes thr oughout the food chain from tropical to polar latitudes (Hallegraeff, 1993). Marine biotoxins are non-proteinaceous compounds with vastly differing structures, whose mol ecular weights lie between 250-3500 daltons. Their physico-chemical proper ties vary according to thei r polarity, lipophylic nature, thermal stability, sensitivity to pH, oxygen and light, etc. Danger of biotoxins poisoning for humans lies in their acute and chro nic effects. Consum ption of seafood contaminated with marine biotoxins may cause serious diseases that affect: the nervous system in paralytic shellfish poisoning (PSP), the intestinal system in diarrheic shellfish poisoning (DSP), and loss of memory in amne sic shellfish poisoning (ASP). Other well known toxins that have been identifie d in seafood from several countries are Brevetoxins (BTXs), Ciguatoxins (CTXs), Palytoxins (PLTXs) and tetrodotoxins (TTXs). Their mode of action is not clearly known, (Hu, et al. , 2001; Miles, et al. , 2000) and involves a variety of receptors and metabolic processes, wherein some activate or block sodium channels, while ot hers activate calcium channels. Some of them are glutamate agonists or inhi bitors of protein phosphatases. On the other hand, this group of pharmacol ogically active marine products includes some very powerful of the so-called "fast acting toxins" such as spirolides (SPXs) and gymnodimines (GYMs). Risk assessment for the presence of phycotoxins has not been an easy matter until now. Pragmatic decisi ons are normally taken during a bloom. The fact that marine biotoxins pose a risk to human health has led many countries to establish regulations for controlling the presence of these toxins in products of marine origin. Several countries have set specific lim its for certain marine toxins in bivalves and fish. Methodology seems to be a comm on problem when implementing safe and effective control. The first empirical scientific observations on harmful algal blooms and toxic mussels were reported in 1937. Mouse bioassay was the method developed at the time for detecting toxins produced by su ch microalgae and has been the standard method applied for years. All analytical methods described below have been theref ore evaluated against bioassays. The mouse bioassay method has an unquestionabl e value in sanitary control and food safety because it examines the response to toxins in a living organism and permits detection of new toxins. However, there is a growing concern nowadays due to poor specificity of the method and for reasons of animal welfare. Alternative assays and chemical analysis have been developed, whic h can be valuable tools for determination of biotoxins. The liquid chromatography (LC) method with ultraviolet (UV) and/or fluorescence detection (FLD) was the first anal ytical technique to be used. During the last decade, the introduction of LC coupled with mass spectrometry (MS) methods has led to significant progress in the field of marine toxins analyses. Optimisation and validation of these methods and development of standards and reference materials is currently the main target in the field of marine toxins. This chapter focuses on the different ch emical methods used for detecting and quantifying biotoxins and furthermore looks into single-laboratory validation and inter- laboratory validation studies that ha ve been carried out or are ongoing.