麻痹性贝类毒素GTX1&4和GTX2&3标准样品的制备技术
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摘要
贝类毒素标准样品的制备是保障贝类质量安全的基础,本研究以有毒贻贝(Mytilus edulis)为原料,探究麻痹性贝类毒素标准样品的制备技术。以1%乙酸溶液提取贻贝中的麻痹性贝类毒素,采用羟丙基葡聚糖凝胶柱(Sephardex LH20)净化后收集目标馏分,再经高效液相色谱以TSK-GEL Hillic Amide-80色谱柱(4.6 mm×250.0 mm,5.0μm)分离、纯化,根据麻痹性贝类毒素的保留时间收集色谱峰流出液,连续制备合并馏分,40℃减压蒸馏浓缩近干,用75%乙腈水(含0.25%甲酸)溶解,即制得毒素标准样品溶液。分别采用四极杆/离子阱复合质谱与标准物质的多级质谱信息进行匹配,以高分辨质谱获得的精确质量数进行定性确证,样品溶液所含的麻痹性贝类毒素为4种膝沟藻毒素,分别为膝沟藻毒素-1&-4 (Gonyautxins-1&-4, GTX1&4)和膝沟藻毒素-2&-3(Gonyautxins-2&-3, GTX2&3)。均匀性实验中,经F检验表明在95%的置信区间范围内样品均匀性良好。稳定性考察实验中,标准样品在4℃和-18℃两种环境条件下储存6个月稳定性较好。标准样品经8家实验室联合定值,并评定了定值结果的不确定度。本研究制得的麻痹性贝类毒素标准样品为GTX 1(3 663μg/L)>X4(1 147μg/L)和GTX 2(1 884μg/L)>X3(797μg/L)的混合溶液,溶剂为75%乙腈水(含0.25%甲酸),为国内贝类毒素标准样品研发提供了技术支撑。[中国渔业质量与标准,2019,9(2):62-70]
The preparation of shellfish poison reference materials is the basis for ensuring the quality and safety of shellfish. The preparation technology of paralytic shellfish poisons(PSPs) from the mussel contaminated naturally with PSPs was studied. The PSPs from mussel samples were extracted with 1% acetic acid aqueous solution, cleaned-up by Sephardex LH20 gel column. Then the crude PSPs solution was separated and purified by HPLC coupled with TSK-GEL Hillic Amide-80 column(4.6 mm×250.0 mm,5.0 μm). The aqueous solution was collected based on specific retention time and concentrated by vacuum distillation at 40 ℃. Then the obtained purified PSP solutions were dissolved in 75% acetonitrile(containing 0.25% formic acid) for subsequent analysis. The reference material was confirmed to contain GTX1&4 and GTX2&3 by LC-MS/MS and HRMS(high resolution mass spetrum). According to homogeneity analysis, the results were validated by F-test statistical method. The 6-months stability results indicated that the PSPs reference material was stable at 4 ℃and-18 ℃, respectively. The definition value was certificated by eight laboratories and the uncertainty evaluation was analyzed. The prepared reference material is an instrument calibration solution which contains a mixed solution of GTX 1(3 663 μg/L)>X4(1 147 μg/L)and GTX 2(1 884 μg/L)>X3(797 μg/L)dissolved in filtered 75% acetonitrile aqueous solution containing 0.25% formic acid. [Chinese Fishery Quality and Standards, 2019, 9(2):62-70]
The preparation of shellfish poison reference materials is the basis for ensuring the quality and safety of shellfish. The preparation technology of paralytic shellfish poisons(PSPs) from the mussel contaminated naturally with PSPs was studied. The PSPs from mussel samples were extracted with 1% acetic acid aqueous solution, cleaned-up by Sephardex LH20 gel column. Then the crude PSPs solution was separated and purified by HPLC coupled with TSK-GEL Hillic Amide-80 column(4.6 mm×250.0 mm,5.0 μm). The aqueous solution was collected based on specific retention time and concentrated by vacuum distillation at 40 ℃. Then the obtained purified PSP solutions were dissolved in 75% acetonitrile(containing 0.25% formic acid) for subsequent analysis. The reference material was confirmed to contain GTX1&4 and GTX2&3 by LC-MS/MS and HRMS(high resolution mass spetrum). According to homogeneity analysis, the results were validated by F-test statistical method. The 6-months stability results indicated that the PSPs reference material was stable at 4 ℃and-18 ℃, respectively. The definition value was certificated by eight laboratories and the uncertainty evaluation was analyzed. The prepared reference material is an instrument calibration solution which contains a mixed solution of GTX 1(3 663 μg/L)>X4(1 147 μg/L)and GTX 2(1 884 μg/L)>X3(797 μg/L)dissolved in filtered 75% acetonitrile aqueous solution containing 0.25% formic acid. [Chinese Fishery Quality and Standards, 2019, 9(2):62-70]
引文
[1] 邱江兵.双壳贝类对麻痹性贝毒的代谢转化及其生理生化相应[D].青岛:中国海洋大学,2014.
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[3] 吴海燕,郭萌萌,邴晓菲,等.液相色谱-四极杆/线性离子阱复合质谱测定双壳贝类中麻痹性贝类毒素[J].海洋与湖沼,2017,48(3):508-515.
[4] 傅博强,陈敏璠,唐治玉,等.生物毒素检测方法及标准物质研究进展[J].生命科学,2016,28(1):51-63.
[5] 国家食品药品监督管理总局,国家卫生和计划生育委员会.GB 5009.213—2016 食品安全国家标准贝类中麻痹性贝类毒素的测定[S].北京:中国标准出版社,2016.
[6] 朱敬萍,金雷,张小军,等.小鼠生物法检测麻痹性贝类毒素技术探讨及应用[J].中国渔业质量与标准,2017,7(2):30-35.
[7] 黄宗锈,林健,陈冠敏,等.麻痹性贝类毒素小鼠生物法检测中位数法与均数法差异性比较[J].中国卫生检验杂志,2018,18(2):369-370.
[8] 许道艳,刘磊,刘仁沿,等.麻痹性贝毒单克隆抗体的制备和酶联免疫检测方法的建立[J].中国免疫学杂志,2013,29(1):69-73.
[9] 彭志兰,罗海军,王维洁,等.舟山海域麻痹性贝类毒素污染情况及其2种检测方法比较[J].食品安全质量检测学报,2017,8(4):1436-1440.
[10] 于兵,曹际娟,尤永莉,等.ELISA与小白鼠生物法检测贝类中麻痹性贝毒的比较[J].检验检验科学,2005,15(1):32-35.
[11] 李芳,李雪梅,李献刚,等.贝类毒素检测方法研究概况[J].食品研究与开发,2015,36(23):184-186.
[12] 黄海燕,付英斌,赵昆山,等.麻痹性贝类毒素细胞检测法的建立[J].中国卫生检验杂志,2010,4:779-781.
[13] 于慧娟,蔡友琼,黄宣运,等.10种麻痹性贝类毒素的固相萃取及液相色谱-串联质谱测定法[J].海洋渔业,2015,37(4):364-371.
[14] 李兆永,陈军辉,王帅,等.麻痹性贝毒素电喷雾质谱负离子模式分析特征[J].质谱学报,2014,35(1):8-15.
[15] 梅晓颀,何秀平,王江涛.分子印迹固相萃取联用高效液相色谱分离测定海水中的膝沟藻毒素GTX1,4[J].分析化学,2016,44(2):212-217.
[16] 中华人民共和国国家质量监督检验检疫总局,中国国家标准化管理委员会.GB/T 15000.3—2008 标准样品工作导则(3) 标准样品定值的一般原则和统计方法[S].北京:中国标准出版社,2008.
[17] 邴晓菲,吴海燕,王群,等.麻痹性贝类毒素在栉孔扇贝体内的代谢轮廓[J].中国水产科学,2017,24(3):623-632.
[18] 中国合格评定国家认可委员会.CNAS-CL04:2017 标准物质/标准样品生产者能力认可准则[S].北京:中国质检出版社,2017.
[19] 傅博强,唐治玉,王晶,等.腹泻性贝类毒素基体标准物质的研制[J].化学分析计量,2014,23(3):1-4.
[20] 陈伟珠,谢全灵,张怡评,等.4,9-脱水河豚毒素国家标准样品的研制[J].化学分析计量,2014,23(3):1-4.
[2] 周名江,李钧,于仁诚,等.赤潮藻毒素研究新进展[J].中国海洋药物,1999,18(3):48-54.
[3] 吴海燕,郭萌萌,邴晓菲,等.液相色谱-四极杆/线性离子阱复合质谱测定双壳贝类中麻痹性贝类毒素[J].海洋与湖沼,2017,48(3):508-515.
[4] 傅博强,陈敏璠,唐治玉,等.生物毒素检测方法及标准物质研究进展[J].生命科学,2016,28(1):51-63.
[5] 国家食品药品监督管理总局,国家卫生和计划生育委员会.GB 5009.213—2016 食品安全国家标准贝类中麻痹性贝类毒素的测定[S].北京:中国标准出版社,2016.
[6] 朱敬萍,金雷,张小军,等.小鼠生物法检测麻痹性贝类毒素技术探讨及应用[J].中国渔业质量与标准,2017,7(2):30-35.
[7] 黄宗锈,林健,陈冠敏,等.麻痹性贝类毒素小鼠生物法检测中位数法与均数法差异性比较[J].中国卫生检验杂志,2018,18(2):369-370.
[8] 许道艳,刘磊,刘仁沿,等.麻痹性贝毒单克隆抗体的制备和酶联免疫检测方法的建立[J].中国免疫学杂志,2013,29(1):69-73.
[9] 彭志兰,罗海军,王维洁,等.舟山海域麻痹性贝类毒素污染情况及其2种检测方法比较[J].食品安全质量检测学报,2017,8(4):1436-1440.
[10] 于兵,曹际娟,尤永莉,等.ELISA与小白鼠生物法检测贝类中麻痹性贝毒的比较[J].检验检验科学,2005,15(1):32-35.
[11] 李芳,李雪梅,李献刚,等.贝类毒素检测方法研究概况[J].食品研究与开发,2015,36(23):184-186.
[12] 黄海燕,付英斌,赵昆山,等.麻痹性贝类毒素细胞检测法的建立[J].中国卫生检验杂志,2010,4:779-781.
[13] 于慧娟,蔡友琼,黄宣运,等.10种麻痹性贝类毒素的固相萃取及液相色谱-串联质谱测定法[J].海洋渔业,2015,37(4):364-371.
[14] 李兆永,陈军辉,王帅,等.麻痹性贝毒素电喷雾质谱负离子模式分析特征[J].质谱学报,2014,35(1):8-15.
[15] 梅晓颀,何秀平,王江涛.分子印迹固相萃取联用高效液相色谱分离测定海水中的膝沟藻毒素GTX1,4[J].分析化学,2016,44(2):212-217.
[16] 中华人民共和国国家质量监督检验检疫总局,中国国家标准化管理委员会.GB/T 15000.3—2008 标准样品工作导则(3) 标准样品定值的一般原则和统计方法[S].北京:中国标准出版社,2008.
[17] 邴晓菲,吴海燕,王群,等.麻痹性贝类毒素在栉孔扇贝体内的代谢轮廓[J].中国水产科学,2017,24(3):623-632.
[18] 中国合格评定国家认可委员会.CNAS-CL04:2017 标准物质/标准样品生产者能力认可准则[S].北京:中国质检出版社,2017.
[19] 傅博强,唐治玉,王晶,等.腹泻性贝类毒素基体标准物质的研制[J].化学分析计量,2014,23(3):1-4.
[20] 陈伟珠,谢全灵,张怡评,等.4,9-脱水河豚毒素国家标准样品的研制[J].化学分析计量,2014,23(3):1-4.