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An interlaboratory collaborative study to validate a colorimetric phosphatase inhibition assay for quantitative determination of the okadaic acid (OA) toxins group in molluscs, OkaTest, was conducted. Eight test materials, including mussels, scallops, clams, and cockles, were analyzed as blind duplicates. Blank samples and materials containing different OA toxin levels ranging from 98 to 275 microg/kg OA equivalents were included. The study was carried out by a total of 16 laboratories from 11 different countries. Values obtained for repeatability relative standard deviations (RSDr) ranged from 5.4 to 11.2% (mean 7.5%). Reproducibility RSD (RSD(R)) values were between 7.6 and 13.2% (mean 9.9%). The Horwitz ratio (HorRat) values ranged between 0.4 and 0.6. A recovery assay was also carried out using a sample spiked with OA. A mean recovery of 98.0% and an RSD of 14.5% were obtained. The results obtained in this validation study indicate that the colorimetric phosphatase inhibition assay, OkaTest, is suitable for quantitative determination of the OA toxins group. OkaTest could be used as a test that is complementary to the reference method for monitoring the OA toxins group.

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

AOAC Official Method(SM) 2005.06 for the determination of saxitoxin (STX)-group toxins in shellfish by LC with fluorescence detection with precolumn oxidation was previously validated and adopted First Action following a collaborative study. However, the method was not validated for all key STX-group toxins, and procedures to quantify some of them were not provided. With more STX-group toxin standards commercially available and modifications to procedures, it was possible to overcome some of these difficulties. The European Union Reference Laboratory for Marine Biotoxins conducted an interlaboratory exercise to extend AOAC Official Method 2005.06 validation for dc-GTX2,3 and to compile precision data for several STX-group toxins. This paper reports the study design and the results obtained. The performance characteristics for dc-GTX2,3 (intralaboratory and interlaboratory precision, recovery, and theoretical quantification limit) were evaluated. The mean recoveries obtained for dc-GTX2,3 were, in general, low (53.1-58.6%). The RSD for reproducibility (RSD(r)%) for dc-GTX2,3 in all samples ranged from 28.2 to 45.7%, and HorRat values ranged from 1.5 to 2.8. The article also describes a hydrolysis protocol to convert GTX6 to NEO, which has been proven to be useful for the quantification of GTX6 while the GTX6 standard is not available. The performance of the participant laboratories in the application of this method was compared with that obtained from the original collaborative study of the method. Intralaboratory and interlaboratory precision data for several STX-group toxins, including dc-NEO and GTX6, are reported here. This study can be useful for those laboratories determining STX-group toxins to fully implement AOAC Official Method 2005.06 for official paralytic shellfish poisoning control. However the overall quantitative performance obtained with the method was poor for certain toxins.

An LC/MS/MS method has been developed, assessed, and intralaboratory-validated for the analysis of the lipophilic toxins currently regulated by European Union legislation: okadaic acid (OA) and dinophysistoxins 1 and 2, including their ester forms; azaspiracids 1, 2, and 3; pectenotoxins 1 and 2; yessotoxin (YTX), and the analogs 45 OH-YTX, Homo YTX, and 45 OH-Homo YTX; as well as for the analysis of 13-desmetil-spirolide C. The method consists of duplicate sample extraction with methanol and direct analysis of the crude extract without further cleanup or concentration. Ester forms of OA and dinophysistoxins are detected as the parent ions after alkaline hydrolysis of the extract. The validation process of this method was performed using both fortified and naturally contaminated samples, and experiments were designed according to International Organization for Standardization, International Union of Pure and Applied Chemistry, and AOAC guidelines. With the exception of YTX in fortified samples, RSDr below 15% and RSDR were below 25%. Recovery values were between 77 and 95%, and LOQs were below 60 microg/kg. These data together with validation experiments for recovery, selectivity, robustness, traceability, and linearity, as well as uncertainty calculations, are presented in this paper.

The application of ultra-performance rapid resolution LC on a 1.8 microm particle-size column coupled with tandem MS (RRLC-MS/MS) is described for the analysis of amnesic shellfish poisoning (ASP) toxins in shellfish. Complete resolution among domoic acid (DA) and the isomers was achieved in less than 3 min. The method was intralaboratory validated for direct analysis of crude extracts without further cleanup. It showed LODs ranging from 0.05 to 0.09 mg/kg and a working range that complied with the current regulatory level for DA of 20 mg/kg, and with the level of 4.5 mg/kg recently proposed by the European Food Safety Authority. Confirmatory capabilities were demonstrated according to the Commission Decision 2002/657/EC criteria. The results obtained by RRLC-MS/MS agreed with those provided by the reference LC-UV method, both intralaboratory for the analysis of blind samples (R2 = 0.9751) and interlaboratory through participation in the proficiency test for ASP toxins during 2009 (z-score = -0.962 and 0.177 for low- and high-contaminated samples, respectively). RRLC-MS/MS provided fast analysis and additional confirmatory capabilities for direct analysis of crude extracts while the performance and reliability of the results were maintained, even in very complex matrixes.

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.

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.

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.

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.

Raw cow's milk collected from dairy farms in the province of Leon, Spain, was examined for aflatoxin M(1) (AFM(1)). The samples were analysed with a commercial competitive enzyme-linked immunosorbent assay (ELISA) kit and high-performance liquid chromatography (HPLC). The concentrations of AFM(1) in the milk extracts were initially estimated by ELISA, with recovery rates of 74.6-109% for artificially contaminated milk at levels of 10-80 ng x l(1). Samples found to contain more than 10 ng x l(1) were further quantified with HPLC. The mean recovery for this method was 89.3%. The quantification limit was 10 ng x l(1) for both ELISA and HPLC. Although AFM(1) was confirmed in only 3.3% of the samples, the concentrations in all these cases were lower than the maximum limit applicable to these products pursuant to European Union legislation. Both methods were validated with reference material certified by the Community Bureau of Reference.