Department of Molecular Systems Biology, Helmholtz Centre for Environmental Research-UFZ, Leipzig, Germany.
Department of Environmental Immunology, Helmholtz Centre for Environmental Research-UFZ, Leipzig, Germany.
Rapid Commun Mass Spectrom. 2020 Apr 15;34(7):e8668. doi: 10.1002/rcm.8668.
Glyphosate is one of the most widely used herbicides and it is suspected to affect the intestinal microbiota through inhibition of aromatic amino acid synthesis via the shikimate pathway. In vitro microbiome bioreactors are increasingly used as model systems to investigate effects on intestinal microbiota and consequently methods for the quantitation of glyphosate and its degradation product aminomethylphosphonic acid (AMPA) in microbiome model systems are required.
An optimized protocol enables the analysis of both glyphosate and AMPA by simple extraction with methanol:acetonitrile:water (2:3:1) without further enrichment steps. Glyphosate and AMPA are separated by liquid chromatography on an amide column and identified and quantified with a targeted tandem mass spectrometry method using a QTRAP 5500 system (AB Sciex).
Our method has a limit of detection (LOD) in extracted water samples of <2 ng/mL for both glyphosate and AMPA. In complex intestinal medium, the LOD is 2 and 5 ng/mL for glyphosate and AMPA, respectively. These LODs allow for measurement at exposure-relevant concentrations. Glyphosate levels in a bioreactor model of porcine colon were determined and consequently it was verified whether AMPA was produced by porcine gut microbiota.
The method presented here allows quantitation of glyphosate and AMPA in complex bioreactor fluids and thus enables studies of the impact of glyphosate and its metabolism on intestinal microbiota. In addition, the extraction protocol is compatible with an untargeted metabolomics analysis, thus allowing one to look for other perturbations caused by glyphosate in the same sample.
草甘膦是应用最广泛的除草剂之一，它通过抑制芳香族氨基酸合成途径中的莽草酸途径，被怀疑会影响肠道微生物群。体外微生物组生物反应器越来越多地被用作研究肠道微生物群影响的模型系统，因此需要在微生物组模型系统中定量分析草甘膦及其降解产物氨甲基膦酸（AMPA）的方法。
优化后的方案通过简单的甲醇：乙腈：水（2:3:1）提取，无需进一步富集步骤，即可同时分析草甘膦和 AMPA。草甘膦和 AMPA 通过酰胺柱进行液相色谱分离，并使用 QTRAP 5500 系统（AB Sciex）进行靶向串联质谱法鉴定和定量。
我们的方法在提取水样中的草甘膦和 AMPA 的检测限（LOD）均<2ng/mL。在复杂的肠道培养基中，草甘膦和 AMPA 的 LOD 分别为 2ng/mL 和 5ng/mL。这些 LOD 允许在与暴露相关的浓度下进行测量。测定了猪结肠生物反应器模型中的草甘膦水平，从而验证了 AMPA 是否由猪肠道微生物群产生。
本文介绍的方法允许在复杂的生物反应器液中定量分析草甘膦和 AMPA，从而能够研究草甘膦及其代谢物对肠道微生物群的影响。此外，提取方案与非靶向代谢组学分析兼容，因此可以在同一样品中寻找草甘膦引起的其他干扰。
