同位素内标-超高效液相色谱串联质谱法检测麦芽中11种真菌毒素

Simultaneous determination of 11 mycotoxins in malt by isotope internal standard-UPLC-MS/MS

采用同位素内标法定量,建立了同时测定麦芽药材中11种真菌毒素污染水平的超高效液相色谱串联质谱法(UPLC-MS/MS)。采用乙腈-水-乙酸(80∶19∶1)溶剂系统超声提取药材中真菌毒素,离心、过滤后,加入[^(13)C_(17)]-AFB_1及[^(13)C_(18)]-ZEN标记的真菌毒素混合同位素标准溶液,以含0.1%甲酸的甲醇溶液-2 mmol·L^(-1)乙酸铵水溶液为流动相于Phenomenex Kinetex C_(18)色谱柱(100 mm×2.1 mm,2.6μm)上梯度洗脱分离,以电喷雾离子源正负离子切换模式多反应监测方式检测(MRM)。结果表明,11种真菌毒素在采用基质添加内标稳定同位素法定量时于各自的浓度范围内线性关系良好,r>0.999 1,加标回收率为75.0%~117.0%,RSDs<5.1%,检测限(LODs)和定量限(LOQs)分别为0.05~30μg·kg^(-1)和0.15~87.5μg·kg^(-1),远低于欧盟规定的真菌毒素最大允许残留量(MRLs)。所测的20批麦芽样品中,9批检测到真菌毒素残留,通过相同保留时间处的特征离子二级碎片排除了假阳性结果。该方法的样品前处理过程简便快捷、检测方法重现性好、灵敏度高,适用于复杂麦芽基质中多种真菌毒素的同时定性定量测定。

A suitable ultra-high performance liquid chromatography coupled with tandem mass spectrometry（UPLC-MS/MS） method was developed for the determination of 11 mycotoxins with isotope internal standard in malt. The mycotoxins in malt were extracted and purified by one-step ultrasonic extraction procedure using acetonitrile/water/acetic acid（80∶19∶1）, and then detected and confirmed by UPLC-MS/MS, and quantified by isotope labeled AFB1（[^13C17]-AFB1） and ZEN（[^13C18]-ZEN） internal standards. Rapid separation of the 11 mycotoxins was successfully achieved on a Phenomenex Kinetex C18 column（100 mm × 2.1 mm, 2.6 μm） with gradient elution using the mobile phase of methanol containing 0.1% formic acid and 2 mmol·L^-1 ammonium acetate in water. Simultaneous acquisition was performed in multiple reaction monitoring（MRM） mode with electrospray ionization（ESI） source operated in both positive and negative ionization modes. The established method provided a good linearity for the 11 mycotoxins within their respective linear ranges with correlation coefficients all higher than 0.999 1. The average recoveries ranged from 75.0% to 117.0% with relative standard deviations（RSDs） below 5.1%. The limits of detection（LODs） and quantitation（LOQs） ranged from 0.05 to 30 μg·kg^（-1） and 0.15 to 87.5 μg·kg^（-1）, respectively, which were below the maximum residue levels（MRLs） set by the European Union. Twenty malt samples were analyzed and nine samples were detected with mycotoxins, which were confirmed according to the same fragment ions found in positive samples and the standards at the same retention time. This study has demonstrated that the one-step extraction procedure of mycotoxins from complex matrices coupled to UPLC-MS/MS method is simple, quick, accurate and sensitive for quantitative and qualitative analysis of multiple mycotoxins in malt.