Approaches to the evaluation of matrix effects in the liquid chromatography-mass spectrometry (LC-MS) analysis of three regulated lipophilic toxin groups in mussel matrix (Mytilus edulis).

Liquid chromatography coupled to mass spectrometry (LC-MS) is seen as an integral part of methods of choice for the replacement of animal tests in the determination of lipophilic shellfish toxins. However, these techniques are prone to matrix effects that need to be considered when developing and validating methods. The analysis of shellfish is a challenging task due to the complexity of the shellfish matrix and the number of shellfish species encountered in monitoring laboratories. Therefore, it is crucial that the cause and the extent of matrix effects is fully understood in order to apply corrective measures to the analytical method and to develop efficient sample clean-up steps. This paper presents different approaches to evaluate matrix effects associated with the analysis of okadaic acid (OA), azaspiracid-1 (AZA1) and pectenotoxin-2 (PTX2) in cooked and raw mussel flesh. Post-extraction addition and standard addition experiments were carried out and analysed using various LC-MS methods. Gradient and isocratic elution were compared and ultra-performance liquid chromatography (UPLC), using C8 and C18 Acquity BEH columns, was evaluated for the extent of matrix effects. When matrix effects were observed, OA and PTX2 were always prone to signal enhancement and AZA1 to signal suppression. For all the toxins studied, matrix effects were dependent on chromatographic conditions. UPLC separation using a C8 column significantly reduced matrix effects compared to the other conditions assessed. Furthermore, sample dilution has proven to be an efficient way of reducing matrix effects associated with OA analysis.

Toxicological comparison of diverse Cylindrospermopsis raciborskii strains: evidence of liver damage caused by a French C raciborskii strain.

The freshwater cyanobacterium Cylindrospermopsis raciborskii is known to produce toxic effects in several countries. Acute and chronic exposures to C. raciborskii in Australia have been linked to liver damage (hepatotoxicity) with concomitant effects on the kidneys, adrenal glands, small intestine, lungs, thymus, and heart. The alkaloid cylindrospermopsin, which produces these toxic effects, is thought to be a potent inhibitor of protein synthesis. C. raciborskii strains producing cylindrospermopsin or analogue alkaloids have also been reported in Florida, USA, and Thailand. Brazilian isolates of C. raciborskii are also toxic but act by a different mechanism, causing acute death in mice with neurotoxic symptoms similar to those induced by the saxitoxins. In this article we compare the toxicity in the mouse of a C. raciborskii French strain with C. raciborskii strains from various other sources (Australia, Brazil, Mexico, and Hungary). We tested the toxicity of cell extracts by a mouse bioassay. Acute, fatal neurotoxicity was produced by the Brazilian strain, which was confirmed by liquid chromatography with fluorescence detection of the cell extracts, which revealed the presence of saxitoxin, neosaxitoxin, and decarbamoylsaxitoxin, along with two unidentified compounds. Acute hepatotoxicity with severe liver, kidney, and thymus damage was observed with the Australian cylindrospermopsin-producing strain. The Mexican and Hungarian strains were not found to be toxic to mice in our experimental conditions. No animals died after exposure to the extracts of the French C. raciborskii strain. Histological examination of the liver revealed moderate, multifocal necrosis characterized by small areas of hepatocellular necrosis, combined with disorganization of the parenchyma and congestion of the inner sinusoid. These symptoms and lesions resembled those induced by cylindrospermopsin, but the chemical analysis performed by liquid chromatography coupled with either a diode array detector or a mass spectrometer demonstrated that this toxin was not present in our culture extract.

A French monitoring programme for determining ochratoxin A occurrence in pig kidneys.

Ochratoxin A is a carcinogen and nephrotoxin which can enter the food chain resulting in human exposure. As pig herds are exposed to ochratoxin A through their feed, their kidneys, livers and pork meat are considered as a possible route of exposure for humans. France, an important producer of pork and pork products, set up a national monitoring programme which included the training of six routine public laboratories in the analysis of ochratoxin A using an immunoaffinity step followed by a HPLC-fluorimetric detection. The programme randomly sampled 300 healthy and 100 nephropathic pig kidneys in 1997 and 710 healthy pig kidneys in 1998. Less than 10% of samples were significantly contaminated by ochratoxin A : in the 1997 survey, 1% of samples contained 0.40-1.40 microg kg(-1) of ochratoxin A and in the 1998 survey 7.6 % exhibited ochratoxin A levels in the range 0.5-5 microg kg(-1). In the case of nephropathic kidneys, only traces of ochratoxin A (0.16 to 0.48 microg kg(-1)) were detected in six samples out of 100. Even if not a major route of exposure for humans, pigs are clearly exposed to this mycotoxin and monitoring of pork products and of feed for swine is necessary.