European Union Reference Laboratory for Marine Biotoxins-EURLMB, Spanish Food Safety and Nutrition Agency, Ministry of Health, Social Policy and Equality, CITEXVI, Campus Universitario de Vigo, Fonte das Abelleiras, Lagoas-Marcosende, 36310 Vigo, Spain.
Toxicon. 2012 Oct;60(5):864-73. doi: 10.1016/j.toxicon.2012.05.022. Epub 2012 Jun 7.
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.
委员会法规（EC）第 2074/2005 号承认生物方法是贝类麻痹性贝类毒素（PSP）检测的参考方法。该法规经委员会法规（EC）第 1664/2006 号修订，该法规接受了所谓的劳伦斯方法作为参考方法的替代方法。本研究的目的是比较 AOAC 官方分析方法 959.08（生物方法）和 2005.06（预色谱氧化和荧光检测的液相色谱法）在具有不同毒素特征的样品中的应用。还评估了提取溶剂对总样品毒性的影响。总共分析了 40 个样品，包括贻贝、蛤、扇贝、文蛤、贻贝、牡蛎和藤壶。这些样品分别来自不同的产地，包括新鲜、冷冻、罐装和煮过的贻贝、蛤、扇贝、文蛤、贻贝、牡蛎和藤壶。使用这两种官方方法分析了所有样品。对所有样品进行了乙酸和盐酸提取，并同时使用这两种方法进行了提取分析。大多数样品是自然污染的，有两个样品是添加的。通过小鼠生物测定（MBA）与 HCl 提取和液相色谱与荧光检测（HPLC-FLD）与乙酸提取的比较，对于符合法定限量的结果，包括低于和高于法定限量的结果，两种方法的一致性达到 85%。线性相关系数为 r²=0.69，双侧配对 t 检验（α=0.05）表明两组数据之间没有显著差异。然而，在几个样品中发现了毒性差异。在具有 Gymnodinium 毒素特征的 18 种贝类中，有 15 种贝类的 MBA 获得了更高的毒性水平。这一事实在 7 个样本中更为明显，这部分与缺乏标准以及在研究开始时无法分析 dc-NEO、C1、2 和 GTX6 有关。然而，与提取和 SPE 净化步骤有关的其他因素也可能起作用。相比之下，9 个样品的 HPLC-FLD 总毒性比 MBA 高得多。HPLC-FLD 获得的这些更高的结果不仅可能是由于使用了氧化为共洗脱产物的异构体的最高毒性等效因子（TEF），当计算这些样品的总毒性时。分别对 HPLC-FLD 和 MBA 与两种提取物获得的结果进行了进一步分析。
