王丹 李琳 聂丹丹 王守彬
摘 要:目的:建立CI-LLME-超高效液相色谱-串联质谱法检测果蔬中8种杀菌剂残留量的方法。方法:使用冷冻诱导液液微萃取前处理技术结合三重四极杆分析技术检测果蔬产品中8种杀菌剂的残留量。结果:在给定的浓度范围内8种杀菌剂存在良好的线性关系(R2>0.990);方法检出限为0.04~1.83 μg·kg-1,定量限为0.12~5.49 μg·kg-1。10.0 μg·L-1、20.0 μg·L-1、100.0 μg·L-1添加水平的回收率为81.5%~117.1%,精密度(RSD)为0.05%~6.12%,重复性实验标准偏差为0.045~0.887,RSD为0.47%~9.47%,均符合国家标准要求。结论:该前处理技术具备一步式样品前处理能力,简单快速、成本较低,灵敏度高且重现性好,因此该技术可用于检测果蔬中多种微量杀菌剂的含量。
关键词:杀菌剂;果蔬;冷冻诱导液液微萃取;超高效液相色谱-串联质谱法
Abstract: Objective: To create a method for detecting the residues of eight fungicides in fruits and vegetables using CI-LLME-ultra high performance liquid chromatography-tandem mass spectrometry. Method: The residue of eight fungicides in fruit and vegetable products was detected using liquid-liquid freeze-induced liquid-liquid microextraction pre-treatment technology combined with triple quadrupole analysis technology. Result: Good linear relationships (R2>0.990) were observed for the eight fungicides in the given concentration ranges; the limits of detection of the method were in the range of 0.04~1.83 μg·kg-1, and the limits of quantification of the method were in the range of 0.12~5.49 μg·kg-1. The recoveries were in the range of 81.5%~117.1% at the spiked levels of 10.0 μg·L-1, 20.0 μg·L-1, 100.0 μg·L-1 and precision of the method was in the range of 81.5%~117.1%, the precision (RSD) was 0.05%~6.12%, and the standard deviation of repeatability experiment was 0.045~0.887 with the RSD of 0.47%~9.47%. Conclusion: This pre-treatment technology has the capability of one-step sample pretreatment, which is simple, fast, cost-effective, sensitive, and reproducible. Therefore, this technology can be used to detect the content of various trace fungicides in fruits and vegetables.
Keywords: fungicides; fruits and vegetables; liquid liquid freezing induced microextraction; ultra high performance liquid chromatography-tandem mass spectrometry
隨着生活水平的提高,人们对农产品的质量安全以及农产品中农药的使用越来越关注。因此,加强农药残留分析方法的研究至关重要,目前在检测杀菌剂残留量前,通常会采用分散固相萃取方法对样品进行处理。分散固相萃取方法是在水和乙腈溶液中加入无水硫酸镁等物质,实现乙腈层的分离[1-3]。
研究显示,利用水和有机溶液进行冷冻诱导液液微萃取(Cryoinduced Liquid-Liquid Microextraction,CI-LLME),能够减少盐的使用量,从而降低可能引入的杂质,在多种食品基质中得到了应用[4-6]。本研究优化了CI-LLME检测方法,使该方法可以快速准确地检测果蔬中的杀菌剂。
1 材料与方法
1.1 仪器与设备
Aglient 1290-6460超高效液相-三重四极杆分析仪、Aglient RRHD Eclipse plus C18分析柱,安捷伦公司。
1.2 材料与试剂
水果和蔬菜样品为市场销售产品。
液质专用乙腈、甲酸(赛默飞);无水硫酸镁、C18、PSA粉末(Aglient);屈臣氏蒸馏水;8种杀菌剂标准品(100 μg·mL-1,农业农村部环境保护科研监测所)。
1.3 方法
1.3.1 标准系列溶液配制
分别量取一定量的杀菌剂标准溶液(100 μg·mL-1),用乙腈溶液稀释10倍后,摇匀制成混合标准品储备液(10 μg·mL-1)。再取不同量的混合标准储备液,用初始流动相配制成浓度为0.5 ng·mL-1、1.0 ng·mL-1、5.0 ng·mL-1、10.0 ng·mL-1、20.0 ng·mL-1、40.0 ng·mL-1、80.0 ng·mL-1和100.0 ng·mL-1的标准系列溶液。
1.3.2 樣品制备
准确称量5 g水果、蔬菜样品,放入50 mL BD管中,加入乙腈溶液10 mL旋涡,混合6 min,加入4 g无水硫酸镁和1 g氯化钠混合5 min,以8 000 r·min-1冷冻离心4 min,取1.5 mL上清液,加入装有250 mg无水硫酸钠、50 mg PSA、25 mg C18粉的混合净化剂中,混匀40 s,以8 000 r·min-1冷冻离心4 min,取1.0 mL上清液加入5 mL BD管中,加入1.5 mL纯水混匀,进行冷冻诱导液液微萃取、纯化,-80 ˚C冷冻6 min,提取上层有机相,以8 000 r·min-1冷冻离心4 min后,过0.22 μm滤膜上机。
1.4 仪器分析条件
(1)液相色谱条件。流动相:A为0.05%甲酸水溶液,B为乙腈;洗脱梯度:80%A,0~0.01 min;70%A,0.01~3.00 min;15%A,3~5 min;70%A,5.0~7.5 min;10%A,7.5~10.0 min;80%A,10~15 min;柱温:40 ℃;流速:0.2 mL·min-1;进样方式:自动进样;进样体积:5 μL。
(2)质谱条件。扫描方式:正离子扫描;离子源:ESI;检测方式:多反应监测;雾化器压力:30 psi;鞘气温度:300 ℃;流速:11 L·min-1;毛细管电压:4 000 V;喷嘴电压:250 V。8种杀菌剂的相关参数见表1。
2 结果与分析
2.1 方法线性、检出限和定量限
按照1.4仪器条件测定1.3.1配制的标准系列溶液,以浓度为横坐标、峰面积为纵坐标绘制标准曲线,8种杀菌剂标准曲线线性相关系数见表2。线性回归方程的相关系数为0.993 1~0.999 0,表明冷冻诱导液液微萃取技术联合液相三重四极杆分析仪检测8种杀菌剂能够获得良好的线性表现。以3倍信噪比计算得到检出限为0.04~1.83 μg·kg-1,以10倍信噪比得出定量限为0.12~5.49 μg·kg-1,符合国标要求。
2.2 加标回收率
在空白样品中分别加入1.0 μg·L-1、20.0 μg·L-1、100.0 μg·L-1 3种浓度标准物质,3次进样测定。图1展示了8种杀菌剂TIC图,加标回收率结果见表3。样品加标回收率在81.5%~117.1%,相对标准偏差(RSD)在0.05%~6.12%。
2.3 样品重复性实验
对已知浓度的样品进行6次连续进样检测,结果见表4。标准偏差在0.045~0.887,相对标准偏差(RSD)在0.47%~9.47%,均符合国标要求。
3 结论
冷冻诱导液液微萃取前处理技术结合UPLC-MS/MS分析技术检测果蔬中8种杀菌剂线性关系良好,检出限和定量限均符合国标要求,且方法重现性令人满意。冷冻诱导液液微萃取前处理技术快速、高效,易操作,节约了试剂和时间成本。冷冻诱导液液微萃取前处理技术结合UPLC-MS/MS分析技术能够实现样品提取、富集和净化一步式处理,适用于果蔬中多种杀菌剂的检测。
参考文献
[1]吕冰,尹帅星,陈达炜,等.QuEChERS-四极杆/静电场轨道阱高分辨质谱测定动物性食品中氟虫腈及其代谢物残留[J].分析测试学报,2017,36(12):1424-1430.
[2]ZHANG C Y,DENG Y C,ZHENG J F,et al.The application of the QuEChERS methodology in the determination of antibiotics in food : a review[J].Trends in Analytical Chemistry,2019,118:517-537.
[3]PERESTRELO R,SILVA P,PORTO-FIGUEIRA P,et al.QuEChERS-Fundamentals, relevant improvements, applications and future trends[J].Analytica Chimica Acta,2019,1070:1-28.
[4]LI S H,CHEN D W,LV B.et al.Enhanced sensitivity and effective cleanup strategy for analysis of neonicotinoids in complex dietary samples and the application in the total diet study[J].Journal of Agricultural and Food Chemistry,2019,67(9):2732-2740.
[5]CHUNG S W C,LAM C H.Development of a 15-classes multiresidue method for analyzing 78 hydrophilic and hydrophobic veterinary drugs in milk, egg and meat by liquid chromatography-tandem mass spec-trometry[J].Analytical Methods,2015,7(16):6764-6776.
[6]WANG F Q,LI S H,FENG H,et al.An enhanced sensitivity and clean-up strategy for the nontargeted screening and targeted determina- tion of pesticides in tea using modified dispersive solid-phase ex-traction and cold-induced acetonitrile aqueous two-phase sys-tems coupled with liquid chromatography-high resolution mass spectrometry[J].Food Chemistry,2019,275:530-538.