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

麻痹性贝类毒素化学分析标准化和自动化研究。

• 批准号: 07556049
• 负责人: OSHIMA Yasukatsu
• 金额: $ 2.75万
• 依托单位: Tohoku Unicersity
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07556049/
• 关键词: HPLC analysis; Paralytic shellfish toxins; Reference standards; Automation

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.

麻痹性贝类中毒是影响许多国家贝类产业的严重毒理学问题。目前，毒素的测定已经使用小鼠生物测定法进行，但由于生物测定法的缺点，强烈需要替代方法。我们已经开发了一种柱后衍生HPLC方法，使分析几乎所有的石房蛤毒素类似物在高灵敏度。然而，由于缺乏标准毒素，公共卫生机构对这些方法的利用受到阻碍。本研究对高效液相色谱分析的标准化进行了研究，包括提取和净化程序。特别强调了参比标准品的新制备方法。还探讨了自动化系统的发展。主要研究结果如下：1.将氧化剂中的磷酸钠替换为磷酸钾，解决了氧化剂沉淀导致HPLC操作不稳定的问题; 2.建立了双壳贝类样品的酸提取、ODS柱净化和超滤的方法。建立了浮游植物和海水样品中毒素的提取方法。3.建立了一种有效的双壳贝类毒素的柱层析纯化方法。采用高效液相色谱法和核磁共振法测定毒素纯度，燃烧分析法测定氮含量，从而确定毒素的含量。4.确定了各组分的毒性，并建立了一套完整的自动化系统，包括柱和移动的相切换机制。该系统具有良好的可靠性，每天可对20多个样品进行分析。

项目成果

H.Ravn,Y.Oshima他3名: "Standardized extraction method for paralytic shellfish toxins in phytoplankton" J.Appl.Phycol.7. 589-594 (1995)

H.Ravn、Y.Oshima 和其他 3 人：“浮游植物中麻痹性贝类毒素的标准化提取方法”J.Appl.Phycol.7 (1995)。

Y. Oshima: "Postcolumn derivatization liquid chromatographic method for paralytic shellfish toxins." Journal of A. O. A. C. International. 78. 528-532 (1995)

Y. Oshima：“麻痹性贝类毒素的柱后衍生液相色谱法。”

Y.Oshima: "Post-column derivatization HPLC method for the analysis of PSP." J.Assoc.Offic.Anal.Chem.International. 78. 795-799 (1995)

Y.Oshima：“用于分析 PSP 的柱后衍生 HPLC 方法。”

Ravn, H., Anthoni, U., Christophersen, C., Nielsen, P.H., and Oshima, Y.: Standardized extraction method for paralytic shellfish toxins in phytoplankton.J.Appl.Phycol.7. 589-594 (1995)

Ravn, H.、Anthoni, U.、Christophersen, C.、Nielsen, P.H. 和 Oshima, Y.：浮游植物中麻痹性贝类毒素的标准化提取方法。J.Appl.Phycol.7。

Oshima, Y: "Post-column derivatization HPLC method for the analysis of PSP." J.Assoc.Offic.Anal.Chem.International. 78. 795-799 (1995)

Oshima, Y：“用于分析 PSP 的柱后衍生 HPLC 方法。”