A comparative study for PSP toxins quantification by using MBA and HPLC official methods in shellfish.

Commission Regulation (EC) N° 2074/2005 recognises the biological method as the reference method for Paralytic Shellfish Poisoning (PSP) toxins detection in molluscs. It was amended by Commission Regulation (EC) N° 1664/2006 that accepted the so-called Lawrence method as an alternative to the reference method. The goal of this study was to compare AOAC Official Methods of Analysis 959.08 (Biological method) and 2005.06 (Prechromatographic Oxidation and Liquid Chromatography with fluorescence detection) in samples with different toxin profiles. The influence of extraction solvent in the total samples toxicity was also evaluated. A total of 40 samples including mussels, clams, scallops, razor-clams, cockles, oysters and barnacles were analysed by both official methods. Samples were selected with Alexandrium and Gymnodinium toxic profiles, from different origin and including several presentations: fresh, frozen, canned and boiled. Acetic and hydrochloric acid extractions were performed in all samples and the extracts were simultaneously analysed by both methods. Most samples were naturally contaminated and two samples were spiked. Comparison of both official methods, mouse bioassay (MBA) with HCl extraction and Liquid Chromatography with fluorescence detection (HPLC-FLD) with acetic acid extraction, led to an 85% of consistent results regarding compliance with legal limit, including samples below and above it. The linear correlation coefficient was r² = 0.69 and the paired t test (two tails, α = 0.05) indicated that there were not significant differences among both sets of data. Nevertheless, toxicity differences were found in several samples. In 15 out of 18 shellfish with a Gymnodinium toxic profile, higher toxicity levels were obtained by MBA. This fact was more evident in 7 samples, partially related to the lack of standards and the impossibility of analysing dc-NEO, C1, 2 and GTX6 at the beginning of the study. However, other factors concerning the extraction and SPE clean-ups steps may also contribute. By contrast, 9 samples presented a much higher total toxicity by HPLC-FLD than by MBA. These higher results obtained by HPLC-FLD could not only be due to the use of the highest toxicity equivalency factor (TEF) for isomers oxidated into products that coelute when total toxicity of these samples were calculated. Further analyses of results obtained by HPLC-FLD and by MBA with both extracts were done separately.

Assessment of the hydrolysis process for the determination of okadaic acid-group toxin ester: presence of okadaic acid 7-O-acyl-ester derivates in Spanish shellfish.

The contamination of different types of shellfish by okadaic acid (OA)-group toxin esters is an important problem that presents serious risk for human health. During previous investigations carried out in our laboratory by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS), the occurrence of a high percentage of esters in relation to the total OA equivalents has been observed in several shellfish species. The determination of these kinds of toxins using LC/MS or other chemical methods requires a hydrolysis step in order to convert the sterified compounds into the parent toxins, OA, dinophysistoxins-1 (DTX-1) and dinophysistoxins-2 (DTX-2). Most of the hydrolysis procedures are based on an alkaline hydrolysis reaction. However, despite hydrolysis being a critical step within the analysis, it has not been studied in depth up to now. The present paper reports the results obtained after evaluating the hydrolysis process of an esterified form of OA by using a standard of 7-O-acyl ester with palmitoyl as the fatty acid (palOA). Investigations were focused on checking the effectiveness of the hydrolysis for palOA using methanol as solvent standard and matrices matched standards. From the results obtained, no matrix influence on the hydrolysis process was observed and the quantity of palOA converted into OA was always above 80%. The analyses of different Spanish shellfish samples showed percentages of palOA in relation to the total OA esters ranging from 27% to 90%, depending on the shellfish specie.

Lipophilic toxin profile in Galicia (Spain): 2005 toxic episode.

By the end of 2005, a toxic episode of phytoplankton origin in bivalve shellfish led to the closing down of several shellfish production areas in Galicia (northwestern region of Spain). During this time, different kinds of shellfish were collected and analysed by LC-MS/MS to search for the following lipophilic toxins: okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs), azaspiracids (AZAs) and spirolides. Samples were analysed before alkaline hydrolysis in order to investigate the presence of free OA and DTXs, AZAs, PTXs and spirolides, and after alkaline hydrolysis to detect OA and DTXs esters. All of the samples were found to be contaminated with OA and/or DTX-2, as well as esterified forms of these diarrhetic shellfish poison (DSP) toxins, at levels around and above European regulatory limit (160 microg of okadaic acid equivalents/kg). The analyses of mussels and razor clam also revealed the presence of 13-desmethyl spirolide C (SPX-1) at levels below 31 microg/kg. Likewise, in many of the samples different levels of pectenotoxin-2 secoacid (PTX-2sa) were detected. DSP toxin esters represent practically the 100% of the total OA equivalents for scallops, clams, razor clams and cockles.

Influence of the sample toxic profile on the suitability of a high performance liquid chromatography method for official paralytic shellfish toxins control.

An HPLC-FLD method, involving pre-chromatographic oxidation of the PSP toxins with hydrogen peroxide and periodate, has been AOAC validated through a collaborative trial and adopted as AOAC Official Method. This method could be a candidate for replacing the mouse bioassay (MBA) for the Official Control of PSP toxins at European level, once accepted by the legislation. An interlaboratory exercise has been organized by the CRLMB to evaluate its "fitness for purpose" for the Official Control of PSP toxins in the EU laboratories. Eighteen EU laboratories took part in the study and had to analyze six bivalve mollusc samples with several PSP toxic profiles. The performance of the participant laboratories in the application of this method was compared with that obtained at the collaborative trial. Information on problems/drawbacks encountered by participants in the application of this method was also sought. The HPLC validated method is only applicable for Official PSP Control for certain samples. This depends on sample PSP toxic profile. Results obtained for samples where only GTX2,3 and STX were present were satisfactory and in agreement with MBA results. Results obtained for a sample with a toxic profile dominated by GTX6 and suspected to contain also C1,2 and C3,4 were not satisfactory. GTX5 and dc-STX could be quantified, although the results achieved (total toxicity) were lower than those obtained by MBA. It can be also useful as a screening method, complementary to MBA, helping in the reduction of the animals used. However, the lack of several PSP standards, the fact that the method is not validated for all the PSP toxins, and several drawbacks found in its application are a handicap to fully implement it for Official PSP Control as a viable replacement for bioassay.

First evidence of spirolides in Spanish shellfish.

During the months of November and December 2005, a harmful algal bloom in the northwestern region of Spain caused the accumulation of different algal toxins in several types of shellfish. Many of the shellfish were contaminated at levels above European regulatory limit, presenting serious risks for human health. The analysis of mussels sampled in the affected areas to search for lipophilic toxins, using liquid chromatography tandem mass spectrometry (LC-MS/MS), showed the presence of free okadaic acid (OA) and dinophysistoxin-2 (DTX-2) as well as esters of these toxins. The results also revealed the presence of minor amounts of 13-desmethyl spirolide C (SPX-1) in the analysed samples, although this toxin has never been reported before in Spain. The combination of different MS modes of operation, just as enhanced MS (EMS) and MS(3) experiments, allowed to confirm the first occurrence of spirolides in Spanish shellfish.

Histamine and biogenic amine production by Morganella morganii isolated from temperature-abused albacore.

Histamine-producing bacteria were isolated from albacore stored at 0, 25, 30, and 37 degrees C. They were screened using Niven's differential medium, and their histamine production was confirmed by high-pressure liquid chromatography analysis. The optimum temperature for growth of histamine-producing bacteria was 25 degrees C. The bacterium producing the highest level of histamine was isolated from fish abused at 25 degrees C. It was identified as Morganella morganii by morphological, cultural, biochemical, and antimicrobial characteristics and by the Vitek microbial identification system. The M. morganii isolate was inoculated into tuna fish infusion broth medium, and the effect of temperature was determined for microbial growth and formation of histamine and other biogenic amines. The isolate produced the highest level of histamine, 5,253 ppm, at 25 degrees C in the stationary phase. At 15 degrees C, histamine production was reduced to 2,769 ppm. Neither microbial growth nor histamine formation was detected at 4 degrees C. To determine whether the isolate can also produce other biogenic amines that can potentiate histamine toxicity, production of cadaverine, putrescine, serotonin, tryptamine, tyramine, phenylethylamine, spermidine, and spermine by the isolate was also monitored. Cadaverine, putrescine, and phenylethylamine were detected with microbial growth in the tuna fish infusion broth medium. The optimum temperature for cadaverine, putrescine, and phenylethylamine formation was found to be 25 degrees C, as it was for histamine.

Histamine and cadaverine production by bacteria isolated from fresh and frozen albacore (Thunnus alalunga).

Two hundred twenty-seven bacterial strains were isolated from fresh and frozen albacore stored either at -18 or -25 degrees C and investigated for their abilities to produce biogenic amines. As a preliminary screening, all 227 strains were tested in either Niven or Niven modified medium, which allowed the selection of 25 presumptive histamine-producing strains. High-pressure liquid chromatography revealed that only 10 of the 25 strains selected were able to produce low histamine concentrations (<25 ppm) in tryptic soy broth medium supplemented with 2% histidine. None of the 25 strains tested produced putrescine or spermine, whereas 6 strains produced spermidine. Histamine production by Stenotrophomonas maltophilia strain 25MC6 was not prevented at 4 degrees C, and the levels of this amine reached concentrations of 25.8 ppm after 6 days. Three S. maltophilia strains showed strong lysine-decarboxylating activity. Their cadaverine formation capacity was determined by high-pressure liquid chromatography in tryptic soy broth supplemented with 1% lysine; this revealed that the three S. maltophilia strains tested produced more than 700 ppm of cadaverine during the first 24 h of incubation at 37 degrees C. S. maltophilia strain 15MF, initially obtained from fresh albacore tuna, produced up to 2,399 ppm and 4,820 ppm of cadaverine after 24 and 48 h of incubation at 37 degrees C, respectively. To our knowledge, this is the first report on histamine and cadaverine production by strains of the species S. maltophilia, previously known as Pseudomonas and Xanthomonas maltophilia, isolated from fresh and frozen albacore tuna.

Changes in biogenic amines and microbiological analysis in albacore (Thunnus alalunga) muscle during frozen storage.

Albacore specimens of extra quality were analyzed for their biogenic amine contents after 1, 3, 6, and 9 months of frozen storage at -18 degrees C or -25 degrees C. A high-performance liquid chromatography method involving a linear elution gradient was optimized for the identification and determination of putrescine, cadaverine, histamine, spermidine, and spermine in albacore tuna. Putrescine was the biogenic amine that showed the highest increase, reaching concentrations of 59.04 ppm (815% of the initial level) and 68.26 ppm (942% of the initial level) in the white muscle of albacore after 9 months of frozen storage at -18 and -25 degrees C, respectively. Cadaverine, histamine, and spermidine concentrations were below 3, 5, and 11 ppm, respectively, after 9 months of frozen storage, while spermidine underwent a significant decrease at both storage temperatures. Microbiological analysis confirmed the absence of species of Enterobacteriaceae in 75% of the albacore specimens after 9 months of frozen storage; coliforms were always below 3 CFU/g. The survival rate of the psychrotrophic microorganisms after 9 months of frozen storage at -25 degrees C was 4.6%, while 38.9 and 92.1% of the aerobic mesophiles present in the white muscle of albacore before freezing survived 9 months of storage at -18 and -25 degrees C, respectively.