Tissue distribution, effects of cooking and parameters affecting the extraction of azaspiracids from mussels, Mytilus edulis, prior to analysis by liquid chromatography coupled to mass spectrometry Philipp Hess a, * , Lai Nguyen b , John Aasen b , Myra Keogh a , Jane Kilcoyne a , Pearse McCarron a , Tore Aune b a Marine Environment and Food Safety Services, Marine Institute, Biotoxins, Galway Technology Park, Parkmore, Galway, Ireland b Department of Food Safety and Infection Biology, Norwegian School of Veterinary Science, P.O. Box 8146, N-0033 Oslo, Norway Received 17 September 2004; revised 18 March 2005; accepted 21 March 2005 Abstract This study used liquid chromatography coupled to mass spectrometry to identify some parameters important in the analysis of azaspiracids. The first aspect was the distribution of azaspiracids within mussels, in particular the content in the digestive gland as compared to the remaining tissues. In our study, azaspiracids accumulated in the digestive gland, similar to other lipophilic toxins. The ratio of toxin in the digestive gland compared to the whole mussel was on average circa 5, both for a bulk sample collected in Norway in 2004 and for 28 samples from Ireland collected over 3 years (2001–2003). These results may justify the practise to only analyse the digestive gland, a step considered necessary to achieve adequate detection limits for azaspiracids both in the mouse bioassay and other analytical techniques. Steaming of mussels as a sample pre-treatment was found to be another parameter affecting the result. Azaspiracids concentrated indirectly, i.e. through the loss of water or juice from the matrix. The cooked shellfish tissues had a concentration of azaspiracids 2-fold higher than the uncooked shellfish, both for whole flesh and for digestive gland tissue. This finding is of particular importance since it may affect the maximum guidance level at which shellfish may be allowed for human consumption. Finally, parameters affecting the extraction efficiency were studied, including the nature of the extraction solvent, the sample- to-solvent ratio and replicate extraction. The largest differences were observed between different solvents and between different sample-to-solvent ratios, while the effect of replicate extraction was minimal if large sample-to-solvent ratios were used. Duplicate extraction using 100% methanol was found to be the best combination of parameters. q 2005 Elsevier Ltd. All rights reserved. Keywords: Shellfish toxins; Azaspiracid poisoning toxins; Steaming; Methanol; Acetone; Food safety 1. Introduction Azaspiracids are a group of recently discovered toxins that cause azaspiracid poisoning, an illness similar to diarrhetic shellfish poisoning, in humans following Toxicon 46 (2005) 62–71 www.elsevier.com/locate/toxicon 0041-0101/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2005.03.010 * Corresponding author. Tel.: C353 91 730400; fax: C353 91 730470. E-mail address: philipp.hess@marine.ie (P. Hess).