Gonzalo Álvarez

First identification of azaspiracid and spirolides in Mesodesma donacium and Mulinia edulis from Northern Chile

In an attempt to evaluate the risk for human consumption associated to the accumulation of lipophilic toxins by two commercially important bivalves: macha (Mesodesma donacium) and clam (Mulinia edulis) in Coquimbo Bay (Chile), monitoring of these species was carried out from March to September 2008. The samples were analyzed by liquid chromatography-mass spectrometry (LC-MS) to detect okadaic acid, dinophysistoxins, pectenotoxins, azaspiracids, yessotoxins and spirolides. Low levels of Azaspiracid-1 and 13-desmethyl C spirolide were found in both species. The toxins were detected at different dates throughout the monitoring period and in some cases both toxins were detected in the same sample. In all cases, the concentration of the toxins was below the limit of quantification of the technique used and therefore these detections are only indicative of a potential risk. This is the first report of the occurrence of these groups of toxins in Chile and suggests that it is necessary to monitor routinely these substances to warrant public health and shellfish exportations.

Automated on-line solid-phase extraction coupled to liquid chromatography–tandem mass spectrometry for determination of lipophilic marine toxins in shellfish

Automated on-line solid-phase extraction (SPE) coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for fast determination of lipophilic marine toxins in shellfish samples. The direct coupling of an on-line SPE column to LC-MS/MS was accomplished using column switching techniques. Suitable chromatographic separation was performed on a reversed-phase C18 column under alkaline conditions (pH 11). The selected reversed-phase C18 SPE column allowed rapid and efficient on-line desalting of hydrolysed shellfish samples, avoiding signal suppression during mass spectrometry detection. Furthermore, the on-line SPE procedure allowed reducing matrix effects observed in raw and hydrolysed shellfish extracts. The proposed method was evaluated in terms of linearity, precision, accuracy and limits of detection (LODs). Quantitative recovery (97-102%) and satisfactory inter-day precision (RSD<8%) were achieved for all target compounds. LODs in the sub-μgkg-1 level (0.37-0.68μgkg-1) were obtained for all toxins except for okadaic acid, which showed a value of 2.75μgkg-1. Several mussel samples from North-western Spain were finally analysed in order to demonstrate the applicability of the method. Okadaic acid was the predominant toxin in all samples, although other lipophilic toxins were also detected.

Sensitive determination of domoic acid in shellfish by on-line coupling of weak anion exchange solid-phase extraction and liquid chromatography–diode array detection–tandem mass spectrometry

An automated method based on the use of on-line solid-phase extraction (SPE) coupled to liquid chromatography with diode array detection and tandem mass spectrometry (LC-DAD-MS/MS) has been developed for the determination of domoic acid in shellfish. The on-line coupling of SPE and liquid chromatography was accomplished by a column switching approach. A weak anion exchange (WAX) sorbent was selected for the SPE procedure, allowing a selective cleanup of shellfish extracts. High sensitivity was achieved by on-line pre-concentration of large volume injections (50-1000μL). A simple protein precipitation cleanup with acetone was used to efficiently remove proteins from shellfish extracts, preventing possible column clogging during chromatographic separation. The proposed method showed good performance in terms of linearity, limits of detection (LODs), precision and accuracy. Quantitative recovery and satisfactory inter-day precision (RSD ⩽7%) was achieved. LODs in the in the sub-nanogram per gram level (0.23-0.32ngg-1) were obtained with MS/MS detection. Matrix effects were assessed by post-extraction addition method and no signal enhancement or suppression was observed during MS detection. This method was successfully applied to the determination of domoic acid in several shellfish samples, including scallops, oysters, mussels, Manila clams, common cockles and razor clams.

Morphological and toxicological studies of Pseudo-nitzschia species from the central coast of Peru

Currently, there is little information on the genus Pseudo-nitzschia in Peruvian coastal waters available and some species have been misidentified. This is the first study of Pseudo-nitzschia species found in several blooms off the central coast of Peru. The cultures obtained and identified by scanning electron microscopy revealed the presence of Pseudo-nitzschia subpacifica and P. pungens, the first record of P. subpacifica in Peruvian coastal waters. Neither P. subpacifica nor P. pungens cultures contained domoic acid (DA) in detectable amounts using HPLC–MS/MS. Our results suggest that Pseudo-nitzschia species are common off the central coast of Peru. The detection of non-toxic strains of Pseudo-nitzschia does not necessarily mean that other populations or strains of this genus in Peru cannot produce DA. Research is needed to evaluate other strains from different locations along the Peruvian coast and to explore whether environmental factors or genetic variability affect the production of DA.

Accumulation and Biotransformation of Dinophysis Toxins by the Surf Clam Mesodesma donacium

Surf clams, Mesodesma donacium, were shown to accumulate toxins from Dinophysis acuminata blooms. Only pectenotoxin 2 (PTX2) and some of its derivatives were found, and no toxins from the okadaic acid group were detected. PTX2 seems to be transformed to PTX2 seco-acid (PTX2sa), which was found in concentrations more than ten-fold those of PTX2. The seco-acid was transformed to acyl-derivatives by esterification with different fatty acids. The estimated amount of these derivatives in the mollusks was much higher than that of PTX2. Most esters were originated by even carbon chain fatty acids, but some originated by odd carbon number were also found in noticeable concentrations. Some peaks of toxin in the bivalves did not coincide with those of Dinophysis abundance, suggesting that there were large differences in toxin content per cell among the populations that developed throughout the year. The observed depuration (from the digestive gland) was fast (more than 0.2 day−1), and was faster for PTX2 than for PTX2sa, which in turn was faster than that of esters of PTX2sa. PTX2 and PTX2sa were distributed nearly equally between the digestive gland and the remaining tissues, but less than 5% of the palmytoyl-esters were found outside the digestive gland.