Improved Chromatography and MS-Based Detection of Glyphosate and Aminomethylphosphonic Acid Using iTrEnDi

Glyphosate (GLY), a synthetic, nonselective systemic herbicide that is particularly effective against perennial weeds, is the most used weedkiller in the world. There are growing concerns over GLY accumulation in the environment and the attendant human health-associated risks, and despite increased attention in the media, GLY and its breakdown product aminomethylphosphonic acid (AMPA) remain elusive to many analytical strategies. Chemical derivatization coupled with high-performance liquid chromatography-mass spectrometry (HPLC-MS) addresses the challenge of quantifying low levels of GLY and AMPA in complex samples. Here we demonstrate the use of in situ trimethylation enhancement using diazomethane (iTrEnDi) to derivatize GLY and AMPA into permethylated products ([GLY^Tr]⁺ and [AMPA^Tr]⁺, respectively) prior to analysis via HPLC-MS. iTrEnDi produced quantitative yields and resulted in a 12-340-fold increases in HPLC-MS-based sensitivity for [GLY^Tr]⁺ and [AMPA^Tr]⁺, respectively, compared with underivatized counterparts. The limits of detection of derivatized compounds were found to be 0.99 ng/L for [GLY^Tr]⁺ and 1.30 ng/L for [AMPA^Tr]⁺, demonstrating significant sensitivity improvements compared to previously established derivatization techniques. iTrEnDi is compatible with the direct derivatization of Roundup formulations. Finally, as proof of principle, a simple aqueous extraction followed by iTrEnDi enabled the detection of [GLY^Tr]⁺ and [AMPA^Tr]⁺ on the exterior of field-grown soybeans that were sprayed with Roundup. Overall, iTrEnDi ameliorates issues relating to low proton affinity and chromatographic retention, boosting HPLC-MS-based sensitivity and enabling the elucidation of elusive analytes such as GLY and AMPA within agricultural systems.