Lighting up shellfish toxins

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  • Published: Feb 23, 2004
  • Author: David Secko
  • Channels: Sample Preparation / HPLC
thumbnail image: Lighting up shellfish toxins

Many of us enjoy a good seafood meal, but the consequences of a dodgy batch can be extremely unpleasant. When this happens, there's a chance that a marine algal toxin is to blame.

Marine algal toxins, sometimes referred to as phycotoxins ('phyco' meaning seaweeds and algae), are natural toxic chemicals produced by small microscopic phytoplankton. When shellfish ingest phytoplankton during their natural filter feeding, these algal toxins can accumulate to harmful levels, creating a health hazard if consumed. In fact, of the reported foodborne diseases in the United States it is estimated that 10% result from ingesting naturally occurring algal toxins. Thus, the detection of algal toxins in shellfish has become a major health concern. Now, a new study has compared the use of different fluorimetric HPLC methods for detecting a type of algal toxin responsible for diarrhetic shellfish poisoning, with the hopes of improving the sensitivity of their detection.

Phycotoxins as natural contaminants in the marine environment is of very special interest for many places worldwide whose main economic resource is the seafood industry. This is the case of Galicia in the NW of Spain, one of the main mussel producers in the world says Ana Gago-Martinez, from the Universidad de Vigo, the author of the study.

Shellfish poisoning that results from ingesting these phycotoxins can be broken down into four types: paralytic shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP), amnesic shellfish poisoning (ASP), and diarrhetic shellfish poisoning (DSP). Each type of poisoning is linked to a different set of toxins, such as derivatives of saxitoxin causing PSP, domoic acid causing ASP and a group of high molecular weight polyethers (e.g. okadaic acid) causing DSP.

DSP toxins are well recognized in Galicia and analytical tools with enough sensitivity to detect these toxins at trace levels have to be available, Gago-Martinez explains.

Several techniques have been described for the detection of marine biotoxins including cytotoxicity assays, channel binding assays, enzyme-immunoassays and chromatographic methods. One of the most common methods involves a mouse bioassay, where the survival time of mice injected with shellfish extract is monitored. But, this technique turns out to be not very sensitive or selective and Gago-Martinez remarks, the search for a sensitive technique is a main interest for scientists working in this field of research.

This search has brought many researchers to chromatographic methods, particularly liquid chromatography with fluorescence detection (HPLC-FLD). However DSP toxins are not naturally fluorescent, making their derivatization necessary before HPLC-FLD analysis.

In the study, Gago-Martinez and colleagues tested several derivatization reagents to determine their main advantages and disadvantages. 9-anthracenyldiazomethane (ADAM), 9-chloromethylanthracene (CA) and 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone (BrDMEQ) were tested for the derivatization of phytoplankton extracts from the southwest coast of Ireland. ADAM derivatization gave good sensitivity with a quantitation limit of 0.714 ng and the ability to separate the rare DSP toxins 7-epi-PTX2SA and DTX2, a finding that was closely modeled by sensitivity and selectivity of BrDMEQ. CA derivatization gave no reaction for phytoplankton extracts, but worked for the derivatization of naturally contaminated shellfish. The results point to the use of ADAM and BrDMEQ for the analysis of acidic polyether DSP toxins by HPLC-FLD.

Gago-Martinez cautions that inefficient clean-up of samples used for HPLC-FLD is still a problem, suggesting that more selective removal of interferences, perhaps by immunoaffinity based approaches, is still a consideration for future work.

HPLC-FLD is a relatively simple and inexpensive tool in comparison with HPLC-MS. It is fast and sensitive, and should be considered as a clear replacement for the mouse bioassay, concludes Gago-Martinez. Which must come as some relief to the mice.

Does your crab supper harbour a nasty surprise?

Harmful algae - a few among many

Thousands of species of algae form the base of the marine food chain and only a few produce toxins. If shellfish consume these toxic algae, toxins can accumulate to levels that are harmful.

Under certain conditions, the species that do produce toxins 'bloom'. This is a burst of very fast growth that forms a mass called a Harmful Algae Bloom (HAB). Some such blooms are called 'red tide' due to pigments produced by the algae.

Diarrhetic shellfish poisoning (DSP) is a form of toxic syndrome resulting from the ingestion of toxic algae, such as Dinoflagellates, which normally causes non-fatal - but unpleasant - gastrointestinal upset.

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