高通量高效快速净化方法结合UPLC-MS/MS测定粮食中黄曲霉毒素

Determination of aflatoxins in grains using the high-throughput high efficient clean-up method and ultra-high performance liquid chromatography-tandem mass spectrometry

建立粮食中黄曲霉毒素B1、B2、G1、G2快捷高效的确证检测方法。通过对色谱及质谱分析条件、震荡提取方式、稀释倍数分别优化确定满足黄曲霉毒素B1、B2、G1、G2检测方法的最优条件,同时,开发一款自动过柱仪与快速净化柱配合使用对粮食中杂质进行高通量的高效净化,并采用超高效液相色谱-串联质谱(UPLC-MS/MS)进行检测分析。粮食样品经84%乙腈水溶液提取、Speedy Prep®-Myco1流穿式净化柱净化,使用ACQUITY UPLC BEH C18色谱柱分离,以0.1%甲酸水和乙腈作为流动相进行梯度洗脱,质谱采用电喷雾正离子模式和多反应监测模式(multiple reaction monitoring,MRM)采集信号,外标法定量。对于大米、小黄米、小麦、黄豆、面粉、大麦、黑米、花生、玉米基质,本方法黄曲霉毒素B1、B2、G1、G2的检出限和定量限分别为0.06~0.12μg/kg和0.20~0.40μg/kg,在其线性范围内相关系数R≥0.9993,在0.2、0.4、1、10、20、40μg/kg添加回收率为72.37%~118.4%,相对标准偏差(RSD,n=6)为0.64%~14%,回收率和精密度良好。该方法前处理操作简单、稳定性好,适用于粮食中黄曲霉毒素B1、B2、G1、G2的检测。

A simple and efficient method for determination of aflatoxins B1,B2,G1 and G2 in grains was established.By optimizing the chromatographic and mass spectrometry analysis conditions,oscillation extraction mode and dilution ratio,the optimal conditions satisfying the detection methods of aflatoxin B1,B2,G1 and G2 were determined respectively.At the same time,an high-throughput automatic presser was developed and used in combination with the flow-through clean-up column for high-throughput and efficient purification of impurities in grain.Ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)was used for detection and analysis.The grinded grain sample was extracted by 84%acetonitrile solution and purified by Speedy Prep®-Myco 1 clean-up column.During UPLC-MS/MS quantification through external standard method,the purified extract was gradient eluted by ACQUITY UPLC BEH C18 column with 0.1%formic acid solution in water and acetonitrile as the mobile phase.Mass spectrometric signal collection was performed by electrospray positive ion and multiple reaction monitoring(MRM)mode.For rice,small yellow rice,wheat,yellow bean,flour,barley,black rice,peanut and corn,the limits of detection(LODs)and the limits of quantification(LOQs)of aflatoxins B1,B2,G1 and G2 were 0.06-0.12μg/kg and 0.20-0.40μg/kg with correlation coefficients higher than 0.9993 in their linear ranges.At spiked levels of 0.2,0.4,1,10,20 and 40μg/kg,aflatoxins B1,B2,G1 and G2 were obtained with recoveries between 72.37%to 118.4%and relative standard deviations(RSD,n=6)between 0.64%to 14%.The recoveries and precision were good.This method is simple and stable,and is suitable for the detection of aflatoxin B1,B2,G1 and G2 in grain.