Department of Pharmacology and Toxicology, National Veterinary Research Institute, Aleja Partyzantów 57, 24-100 Puławy, Poland.
Molecules. 2022 Sep 5;27(17):5741. doi: 10.3390/molecules27175741.
The aim of this study was to develop a method for the determination of glyphosate, its metabolite aminomethylphosphonic acid (AMPA), and glufosinate ammonium residues in beebread samples, which could then be used to assess bees' exposure to their residues. The complexity of beebread's matrix, combined with the specific properties of glyphosate itself, required careful selection and optimization of each analysis step. The use of molecularly imprinted solid-phase extraction (MIP-SPE) by AFFINIMIP glyphosate as an initial clean-up step significantly eliminated matrix components and ensured an efficient derivatization step. Colorless beebread extracts were derivatized by the addition of 9-fluorenylmethyl chloroformate (FMOC-Cl). After derivatization, in order to remove FMOC-OH and residual borate buffer, a solid-phase extraction (SPE) clean-up step using Oasis HLB was carried out. Instrumental analysis was performed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The method was validated according to the SANTE/11312/2021 guideline at concentrations of 5, 10, and 100 µg/kg, and satisfactory recovery (trueness) values (76-111%) and precision (RSDr) ≤ 18% were obtained. The limit of quantification (LOQ) was 5 µg/kg for AMPA and glufosinate ammonium and 10 µg/kg for glyphosate. The method was positively verified by the international proficiency test. Analysis of beebread samples showed the method's usefulness in practice. The developed method could be a reliable tool for the assessment of beebread's contamination with residues of glyphosate, its metabolite AMPA, and glufosinate ammonium.
本研究旨在开发一种测定蜂粮样品中草甘膦、其代谢物氨甲基膦酸（AMPA）和草铵膦残留量的方法，以便评估蜜蜂接触这些残留的情况。蜂粮基质的复杂性，再加上草甘膦本身的特殊性质，需要仔细选择和优化每个分析步骤。采用 AFFINIMIP 草甘膦作为分子印迹固相萃取（MIP-SPE）的初始净化步骤，可显著消除基质成分，并确保有效的衍生化步骤。无色蜂粮提取物通过添加 9-芴甲基氯甲酸酯（FMOC-Cl）进行衍生化。衍生化后，为了去除 FMOC-OH 和残留的硼酸盐缓冲液，使用 Oasis HLB 进行固相萃取（SPE）净化步骤。仪器分析采用液相色谱-串联质谱法（LC-MS/MS）进行。该方法按照 SANTE/11312/2021 准则在 5、10 和 100 µg/kg 浓度下进行验证，得到了令人满意的回收率（准确度）值（76-111%）和精密度（RSDr）≤18%。AMPA 和草铵膦的定量限（LOQ）为 5 µg/kg，草甘膦的定量限为 10 µg/kg。该方法通过国际能力验证得到了正向验证。对蜂粮样品的分析表明，该方法在实际应用中具有实用性。所开发的方法可作为评估蜂粮中草甘膦、其代谢物 AMPA 和草铵膦残留污染的可靠工具。
