分散固相萃取结合UPLC-MS/MS测定闽东地区贝类中河豚毒素

Determination of Tetrodotoxin in Shellfish from Eastern Fujian by Using the Dispersive Solid Phase Extraction Combined with UPLC-MS/MS

建立了分散固相萃取结合UPLC-MS/MS测定闽东地区贝类中河豚毒素含量的分析确证方法。对样品前处理及色谱条件进行优化,贝类均质样品用90%甲醇0.5%乙酸溶液提取,离心后上清液用石墨化碳黑(GCB)和C18混合粉末快速净化。上清液经尼龙膜过滤以及Waters AQUITY UPLC BEH HILIC色谱柱分离,以乙腈0.1%甲酸溶液作为流动相梯度洗脱,采用UPLC-MS/MS分析,外标法基质标准曲线定量。结果表明:在优化的试验条件下,贝类河豚毒素在5.4 min内完成色谱分离分析,在0.5~100 ng·mL^(-1)范围内线性关系良好,相关系数(r^(2))=0.995;方法的检出限(LOD)为5μg·kg^(-1),定量下限(LOQ)为15μg·kg^(-1),在15、30、150μg·kg^(-1)3个不同加标水平下,贝类河豚毒素的加标回收率为89.4%~99.0%,相对标准偏差(RSD,n=6)为7.2%~15.2%。该方法准确、简单快捷、前处理成本低、实用性强,能满足目前快速定量检测贝类中河豚毒素含量的技术要求。

In this paper,a confirmation method for the determination of tetrodotoxin content in shellfish in eastern Fujian by using the dispersive solid phase extraction combined with UPLC-MS/MS was established.The pretreatment for the samples was carried out and the chromatographic conditions were optimized.The homogeneous samples of shellfish were extracted with 90%methanol and 0.5%acetic acid solution.After the centrifugation,the supernuant was quickly purified with the mixed powder of graphitized carbon black(GCB)and C18.Then,the supernatant was filtered through the nylon membrane and separated by using the Waters AQUITY UPLC BEH HILIC chromatographic column.The acetonitrile 0.1%formic acid solution was used as the mobile phase for the gradient elution.And the UPLC-MS/MS analysis was performed and quantified by using the matrix-matched standard curve of external standard method.The results showed that under the optimized experimental conditions,the separation and analysis of chromatography of tetrodotoxin from shellfish were completed within 5.4 min.The linear relationship was good in the range of 0.5-100 ng·mL^(-1),with the correlation coefficient(r^(2))=0.995.The limit of detection(LOD)of the method was 5μg·kg^(-1),and the lower limit of quantitation(LOQ)was 15μg·kg^(-1).At three different spiked levels of 15,30,150μg·kg^(-1),the recoveries of tetrodotoxin in shellfish were 89.4%-99.0%,and the relative standard deviations(RSD,n=6)were 7.2%-15.2%.The method was accurate,simple and fast,with low pretreatment cost and strong practicability,which could meet the technical requirements of the rapid quantitative detection of tetrodotoxin content in shellfish.