免疫亲和柱结合超高效液相色谱-串联质谱法测定牛奶中的16种真菌毒素比较研究

Comparative study of immunoaffinity columns combined with ultra performance liquid chromatography-tandem mass spectrometry for determination of 16 kinds of mycotoxins in milk

目的通过比较5种不同商品化多合一免疫亲和柱的可检测目标毒素种类、回收率和稳定性,筛选性能最优的免疫亲和柱,建立免疫亲和前处理-超高效液相色谱-串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)测定牛奶中16种真菌毒的方法。方法牛奶样品分别经5种不同多毒素免疫亲和柱进行净化富集,用优化后的UPLC-MS/MS,在多反应监测模式下测定,同位素内标法定量,筛选覆盖牛奶中真菌毒素污染种类全面、回收率和稳定性最佳的免疫亲和柱,并进行免疫亲和柱性能评价和方法学验证,最终将方法应用于实际样品检测。结果免疫亲和柱A对牛奶中16种真菌毒素在低、中、高3个添加水平均具有良好的准确度和精密度,加标回收率在83.6%~126.8%之间,相对标准偏差为0.2%~18.4%。柱A内各毒素残留水平低于仪器检出限,柱容量在21.8~1317.5 ng之间。使用免疫亲和柱A富集净化样品,各目标毒素在线性范围内线性良好,相关系数均大于0.99,定量限为0.0010~0.2000ng/g。应用该方法对牛奶质控样品和实际样品进行检测,质控样品测定值均在标示范围内;实际样品中能够检出较低水平的黄曲霉毒素M_(1)、去环氧脱氧雪腐镰刀菌烯醇和伏马毒素B1。结论本研究验证筛选免疫亲和柱A应用于牛奶中16种真菌毒素的测定,其柱残留和柱容量能够满足牛奶中真菌毒素污染的测定;经方法学验证该方法准确可靠、灵敏度高,适用于牛奶中16种真菌毒素的日常检测。

Objective To screen the immunoaffinity column with the best performance by comparing the detectable target toxin types,recovery rates and stabilities of 5 kinds of different commercial multi-in-one immunoaffinity columns,establish a method for the determination of 16 kinds of mycotoxins in milk by immunoaffinity pretreatment-ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).Methods The milk samples were purified and enriched by 5 kinds of different multi-toxin immunoaffinity columns respectively.The optimized UPLC-MS/MS was used for determination in the multi-reaction monitoring mode and quantitation by isotope internal standard method.The immunoaffinity column with comprehensive coverage of the mycotoxin pollution types in milk and the optimal recovery rate and stability was screened.The performance evaluation and methodological verification of the immunoaffinity column were conducted.Finally,the method was applied to actual sample detection.Results The immunoaffinity column A showed good accuracy and precision at low,medium and high addition levels for the 16 kinds of mycotoxins in milk,with the recoveries of spiked samples within the range of 83.6%-126.8%,and the relative standard deviations of 0.2%-18.4%.The residual levels of each toxin in column A were below the limit of detection with a column capacity of 21.8-1317.5 ng.The purified samples were enriched with immunoaffinity column A,and the linearity of each target toxin was good in the linear range,with the correlation coefficient greater than 0.99 and the limits of quantification of 0.0010-0.2000 ng/g.The method was applied to the detection of milk quality control sample and actual samples.The measured values of quality control samples were within the marked range.Lower levels of aflatoxin M_(1),de-epoxy-deoxynivalenol and fumonisin B1could be detected in actual samples.Conclusion This study verified that screening immunoaffinity column A is applied to the determination of 16 kinds of mycotoxins in milk,and its column residue and column capacity can meet the determination of mycotoxin pollution in milk.The method is proved to be accurate,reliable and sensitive by the methodology,and is suitable for the daily detection of 16 kinds of mycotoxins in milk.