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.
作者
郭萌萌
彭吉星
吴海燕
郑关超
姚琳
翟毓秀
谭志军
GUO Mengmeng;PENG Jixing;WU Haiyan;ZHENG Guanchao;YAO Lin;ZHAI Yuxiu;TAN Zhijun(Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao 266071,China)
出处
《中国渔业质量与标准》
2019年第2期62-70,共9页
Chinese Fishery Quality and Standards
基金
中国水产科学研究院基本科研业务费(2016HY-ZD1101)
关键词
麻痹性贝类毒素
膝沟藻毒素
标准样品
高效液相色谱
制备
均匀性
稳定性
不确定度
paralytic shellfish poisons
gonyautxins
reference material
high performance liquid chromatography
preparation technology
homogeneity analysis
stability
uncertainty
