液相色谱-串联质谱法测定蜂王浆中双甲脒及其代谢物的残留量

Simultaneous Determination of Amitraz and Its Metabolites in Royal Jelly by HPLC-MS/MS

采用固相萃取-液相色谱-串联质谱法(SPE-LC-MS/MS)测定蜂王浆中双甲脒、单甲脒、2,4-二甲基苯基甲酰胺和2,4-二甲基苯胺的残留量。样品经氨水稀释、氨化乙腈沉淀蛋白并提取,通过中性Al_2O_3固相萃取柱净化,以液相色谱-串联质谱多反应监测正离子模式检测,同位素稀释内标法和外标法定量。结果表明,双甲脒、单甲脒、2,4-二甲基苯基甲酰胺和2,4-二甲基苯胺的定量限分别为0.05、0.5、5.0和0.5μg/kg,在空白蜂王浆基质溶液0～300μg/kg范围内绘制线性工作曲线,线性相关系数大于0.997,对空白蜂王浆进行添加浓度5.0、10、100、200μg/kg的实验,总体回收率为50.5%～110%,相对标准偏差为0.8%～15.0%。该方法简便、快捷,定量限能够满足目前国内外残留限量要求,可为蜂王浆中双甲脒及其代谢物残留量的测定提供参考。

Royal jelly is a popular nutritional supplement, its quality directly affects consumer’s health. In this work, solid phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry(SPE-LC-MS/MS) was developed for determination of amitraz, N-2,4-dimethyl phenyl-N-methylformamidine(DMPF), N-(2,4-dimethylphenyl) formamide(DMF) and 2,4-dimethylaniline(DMA) in royal jelly. The protein of royal jelly was precipitated, and the extracts were dissolved and distilled with ammonia solution and ammonia of acetonitrile solution. The supernatant solution was cleaned up with solid phase extraction column of N-Al2O3 cartridges and eluted with ammonia of acetonitrile solution into a glass culture tube, the eluate evaporated to dryness and reconstituted in acetonitrile solution, and then filtered through nylon membrane into a glass LC vial for LC-MS/MS analysis. The solution was separated by Agilent Eclipse XDB-C18 column(150 mm×4.6 mm×5 μm) and the quantitative detection was performed on LC-MS/MS by multiple reaction monitoring(MRM) mode under positive electrospray ionization(ESI+). Isotopes dilution internal standard method or external standard method were used to determine the residue contents in sample. The limits of quantification(LOQs, S/N=10) of amitraz, DMPF, DMF and DMA are 0.05, 0.5, 5.0 and 0.5 μg/kg, respectively. Single-laboratory method validation data were determined, the calibration standard curves concentration are 0, 5.0, 10, 100, 200, 300 μg/kg and correlation coefficients are more than 0.997. Appropriate amounts of the standard target compounds were spiked into the 'blank' royal jelly and fixed at four final concentration levels of 5.0, 10, 100 and 200 μg/kg, the recoveries and relative standard deviations(RSDs) using spiked matrix calibration standard curves are 50.5%-110% and 0.8%-15.0%, respectively. This method is convenient, rapid and effective, it is suitable for determination and confirmation of amitraz and its metabolites in royal jelly sample, and in line with the regulations of European Union(EU).