作者：何洁1,2, 余婷婷1, 梁松1, 王冰3, 李鹏4, 殷果3, 王炳志1, 徐梅1, 马红圳1, 闫研3, 严义勇1
作者单位：1. 深圳市易瑞生物技术股份有限公司，广东 深圳 518101;
2. 深圳职业技术大学集成电路关键材料研究院，广东 深圳 518055;
3. 深圳药品检验研究院，广东 深圳 518055;
4. 澳门大学中华医药研究院 中药质量研究国家重点实验室，澳门 999078
关键词：黄曲霉毒素;超高效液相色谱-串联质谱;粮食;高通量自动过柱仪;高效前处理
摘要：
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0.06~0.12 μg/kg和0.20~0.40 μg/kg，在其线性范围内相关系数R≥0.9993，在0.2、0.4、1、10、20、40 μg/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。
Determination of aflatoxins in grains using the high-throughput high efficient clean-up method and ultra-high performance liquid chromatography-tandem mass spectrometry
HE Jie1,2, YU Tingting1, LIANG Song1, WANG Bing3, LI Peng4, YIN Guo3, WANG Bingzhi1, XU Mei1, MA Hongzhen1, YAN Yan3, YAN Yiyong1
1. Shenzhen Bioeasy Biotechnology Co., Ltd., Shenzhen 518101, China;
2. Institute of Critical Materials for Integrated Circuits, Shenzhen Polytechnic University, Shenzhen 518055, China;
3. Shenzhen Institute for Drug Control, Shenzhen 518055, China;
4. State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau 999078, China
Abstract: 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 0.06-0.12 μg/kg and 0.20-0.40 μg/kg with correlation coefficients higher than 0.9993 in their linear ranges. At spiked levels of 0.2, 0.4, 1, 10, 20 and 40 μg/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.
Keywords: aflatoxin;ultra-high performance liquid chromatography-tandem mass spectrometry;grain;high-throughput automatic presser;high efficient sample preperation
2024, 50(5):62-70 收稿日期: 2022-10-27;收到修改稿日期: 2022-12-09
基金项目: 广东省粤港湾联合创新领域项目(2021A0505080003)；深圳市技术攻关重点项目(JSGG20191115141601721)
作者简介: 何洁(1987-),女,安徽合肥市人,高级工程师,博士,研究方向为多孔微球材料开发及应用研究、食品安全等。
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