超高效液相色谱-四极杆串联飞行时间质谱分析牛乳及婴幼儿配方乳粉中500种农药残留

Analysis of 500 Pesticide Residues in Milk and Infant and Young Children Formula Milk Powder by Ultra-high Performance Liquid Chromatography-Quadrupole-Time of Flight Mass Spectrometry

目的:应用超高效液相色谱-四极杆串联飞行时间质谱建立多种类农药的定性筛查和定量分析方法,并用于市售牛乳及婴幼儿配方乳粉样品的测试分析。方法:开发并验证了一种采用QuEChERS提取和Captiva EMR-Lipid萃取柱净化的稳定可靠工作流程,通过Luna Omega Polar C_(18)超高效液相色谱柱(2.1 mm×100 mm,1.6μm)分离,经2 mmol/L甲酸铵和0.01%甲酸溶液(A)及2 mmol/L甲酸铵和0.01%甲酸-甲醇溶液(B)为流动相进行梯度洗脱。采用商品化的农药认证标准物质,基于碰撞诱导解离和电子激活解离碎裂技术,并结合Zeno SWATH DIA采集模式,建立了一种单针进样即可对500种农药化合物同时进行定性筛查与定量分析的检测方法。采用OS软件通过质量偏差、保留时间偏差、差异同位素比、谱库相似程度4个因子获得的综合得分进行定性筛查。采用基质外标法,通过提取离子色谱峰面积进行定量分析。结果:500种目标农药及其代谢物的综合得分为90.4~99.4分,所得结果均大于定性筛查的最低限(90分),检出限在0.025~0.5μg/kg之间。选择GB 2763-2021《食品中农药最大残留限量》标准中牛乳项下共103种限用农药及其代谢物绘制基质添加标准曲线,相关系数在0.99以上,3个水平加标回收率在72.7%~114.5%之间,测定值的相对标准偏差(n=6)为2.1%~13.6%,定量限在0.15~3μg/kg之间。结论:该方法灵敏、准确,适用于牛乳及婴幼儿配方乳粉中多类别农药的筛查确证及高概率检出农药的定量分析,可显著降低检测成本,具有实际应用价值。

Objective:An ultra-high liquid chromatography-quadrupole-time of flight mass spectrometry(UPLC-QTOF-MS)was established and applied for the screening and quantitation of pesticide residues in raw milk and infant and young children formula milk powder.Methods:A stable and reliable pretreatment procedure involving quick,easy,cheap,effective,rugged and safe(QuEChERS)extraction and Captiva EMR-lipid cleanup was developed and validated.The samples were separated on a Luna Omega Polar C_(18)(2.1 mm×100 mm,1.6μm)by gradient elution using mobile phase A consisting of 2 mmol/L ammonium formate and 0.01%formic acid and mobile phase B consisting of 2 mmol/L ammonium formate and 0.01%formic acid.By analyzing commercial pesticide certified reference materials,a single injection method using two fragmentation techniques,collision-induced dissociation(CID)and electron activated dissociation(EAD)was developed for the simultaneous qualitative screening and quantitative analysis of 500 pesticide compounds,in which mass spectrometry data were collected using the Zeno sequential window acquisition of all theoretical fragment ion spectra(SWATH)data-independent acquisition(DIA)mode.Comprehensive scores obtained by the OS software from four factors,including mass deviation,retention time deviation,differential isotope ratio and spectrum library similarity,were used for qualitative screening.The matrix-matched external standard method was used for quantitative analysis based on the peak area of precursor ions in the extracted ion chromatogram(XIC).Results:The comprehensive scores of the 500 pesticides and their metabolites ranged from 90.4 to 99.4 points,higher than the minimum score of qualitative screening(90 points),and the limits of detection(LOD)were in the range of 0.025-0.5μg/kg.The correlation coefficients(r^(2))of the matrix-matched calibration curves for 103 pesticides and their metabolites with maximum residue limits(MRL)in milk set by the national standard of China GB 2763-2021 were above 0.99.The average recoveries ranged from 72.7%to 114.5%at three spiked concentration levels with relative standard deviations(RSDs)of 2.1%-13.6%(n=6),and the limits of quantitation(LOQ)were in the range of 0.15-3μg/kg.Conclusion:This method was sensitive and accurate,and could be used for the screening identification of multiple pesticides and their metabolites and the quantitative analysis of restricted pesticides with high detection rate in raw milk and infant formula milk powder at low cost.