University of Hawaii Cancer Center, Analytical Biochemistry Shared Resource, 701 Ilalo Street, Honolulu, HI, 96813, USA.
Anal Bioanal Chem. 2020 Dec;412(30):8313-8324. doi: 10.1007/s00216-020-02966-1. Epub 2020 Oct 3.
Aminomethylphosphonic acid (AMPA) is the main metabolite of glyphosate (GLYP) and phosphonic acids in detergents. GLYP is a synthetic herbicide frequently used worldwide alone or together with its analog glufosinate (GLUF). The general public can be exposed to these potentially harmful chemicals; thus, sensitive methods to monitor them in humans are urgently required to evaluate health risks. We attempted to simultaneously detect GLYP, AMPA, and GLUF in human urine by high-resolution accurate-mass liquid chromatography mass spectrometry (HRAM LC-MS) before and after derivatization with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) or 1-methylimidazole-sulfonyl chloride (ImS-Cl) with several urine pre-treatment and solid phase extraction (SPE) steps. Fmoc-Cl derivatization achieved the best combination of method sensitivity (limit of detection; LOD) and accuracy for all compounds compared to underivatized urine or ImS-Cl-derivatized urine. Before derivatization, the best steps for GLYP involved 0.4 mM ethylenediaminetetraacetic acid (EDTA) pre-treatment followed by SPE pre-cleanup (LOD 37 pg/mL), for AMPA involved no EDTA pre-treatment and no SPE pre-cleanup (LOD 20 pg/mL) or 0.2-0.4 mM EDTA pre-treatment with no SPE pre-cleanup (LOD 19-21 pg/mL), and for GLUF involved 0.4 mM EDTA pre-treatment and no SPE pre-cleanup (LOD 7 pg/mL). However, for these methods, accuracy was sufficient only for AMPA (101-105%), while being modest for GLYP (61%) and GLUF (63%). Different EDTA and SPE treatments prior to Fmoc-Cl derivatization resulted in high sensitivity for all analytes but satisfactory accuracy only for AMPA. Thus, we conclude that our HRAM LC-MS method is suited for urinary AMPA analysis in cross-sectional studies.
氨甲基膦酸（AMPA）是草甘膦（GLYP）和洗涤剂中膦酸的主要代谢物。草甘膦是一种在世界范围内广泛使用的合成除草剂，单独使用或与草铵膦（GLUF）一起使用。公众可能会接触到这些潜在的有害化学物质；因此，迫切需要灵敏的方法来监测人体中的这些化学物质，以评估健康风险。我们试图通过高分辨率精确质量液相色谱-质谱（HRAM LC-MS）在衍生化前和衍生化后同时检测人尿中的草甘膦、氨甲基膦酸和草铵膦，衍生化试剂分别为 9-芴甲氧羰基氯（Fmoc-Cl）或 1-甲基咪唑-磺酰氯（ImS-Cl），并经过多次尿液预处理和固相萃取（SPE）步骤。与未衍生化尿液或 ImS-Cl 衍生化尿液相比，Fmoc-Cl 衍生化在所有化合物的方法灵敏度（检测限；LOD）和准确性方面达到了最佳组合。在衍生化之前，草甘膦的最佳步骤涉及 0.4 mM 乙二胺四乙酸（EDTA）预处理，然后进行 SPE 预清洗（LOD 为 37 pg/mL），对于 AMPA 则不涉及 EDTA 预处理和 SPE 预清洗（LOD 为 20 pg/mL），或 0.2-0.4 mM EDTA 预处理而无需 SPE 预清洗（LOD 为 19-21 pg/mL），对于草铵膦，则涉及 0.4 mM EDTA 预处理和无需 SPE 预清洗（LOD 为 7 pg/mL）。然而，对于这些方法，准确性仅对于 AMPA 是足够的（101-105%），而对于草甘膦（61%）和草铵膦（63%）则适中。在 Fmoc-Cl 衍生化之前进行不同的 EDTA 和 SPE 处理，可使所有分析物具有高灵敏度，但 AMPA 的准确性仅令人满意。因此，我们得出结论，我们的 HRAM LC-MS 方法适合于横断面研究中尿氨甲基膦酸的分析。
