Studies on Standardization and Automation of Chemical Analysis of Paralytic Shellfish Toxins.

麻痹性贝类中毒是影响许多国家贝类养殖业的严重毒理问题。目前，毒素的测定采用小鼠生物测定法，但由于生物测定法的缺点，迫切需要替代的方法。我们建立了一种柱后衍生化高效液相色谱方法，可以高灵敏度地分析几乎所有的甲壳毒素类似物。然而，由于缺乏标准毒素，公共卫生机构对这些方法的使用受到了阻碍。本研究对高效液相色谱分析的标准化进行了研究，包括提取和净化过程。重点介绍了参比标准的新制备方法。并对自动化系统的发展进行了探讨。研究结果如下：1.通过将氧化液中的磷酸二氢钠改为磷酸二氢钾，解决了氧化剂中沉淀导致高效液相色谱分析不稳定的问题。2.建立了一套双壳贝类试液的制备方法，包括酸提取、ODS柱净化和超滤。建立了从浮游植物和海水样品中提取毒素的方法。3.建立了包括新柱层析在内的多步柱层析分离纯化双壳类毒素的高效方法。用高效液相和核磁共振对毒素进行纯度测定后，通过燃烧分析测定氮含量来确定毒素的浓度。4.根据化学数据确定各组分的特定毒性，这是根据化学数据评价海产品毒性的关键。该系统具有良好的可靠性，每天可分析20个以上的样品。

Paralytic shellfish poisoning is a serious toxicological problem affecting the shellfish industries in many countries. Currently, determination of the toxins has been performed using the mouse bioassay, but alternative methods have been strongly required due to the drawbacks of the bioassay. We have developed an post column derivatization HPLC method which enable to analyze almost all saxitoxin analogs at high sensitivity. However, utilition of the methods by public health institutions has been hampered due to the lack of standard toxins. In this study, the standardization of the HPLC analysis, including extraction and cleanup procedures, was inverstigated. Special emphasis was laid on the new preparative methods of the reference standards. Development of automated system was also explored. Results of the studies are summarized as follows.1.Problem of precipitation in the oxidizing reagent which causes erratic operation of HPLC was solved by replacing sodium phosphate to potassium phosphate in the oxidizing solution.2.A protocol for the preparation of test solution from bivalves was achieved, including acid extraction and cleanup with an ODS cartridge column and ultrafiltration. Extraction methods from phytoplankton and seawater samples were also developed.3.Effective toxins purification method from toxic bivalves was developed using several steps of column chromatography including new ones. After purity was determined by HPLC and NMR,concentration of the toxin was determined by measurement of nitrogen content with a combustion analysis.4.Specific toxicity of each components, which is essential to evaluate seafood toxicity from the chemical data, were determined.Complete automated system with column and mobile phase switching mechanism was developed. The system showed good reliability and could perform the analysies of more than 20 samples perday.