A simple liquid chromatography-high resolution mass spectrometry method for the determination of glyphosate and aminomethylphosphonic acid in human urine using cold-induced phase separation and hydrophilic pipette tip solid-phase extraction

Abstract

Recently, the phenomenon of acute poisoning events caused by glyphosate (GLY) had frequently occurred all over the world. The present work reported a simple liquid chromatography-high resolution mass spectrometry (LC-HRMS) method for direct determination of GLY and its metabolite aminomethylphosphonic acid (AMPA) in human urine by combining cold-induced phase separation (CIPS) with hydrophilic pipette tip solid-phase extraction (PTSPE). First, a urine sample was mixed with acetonitrile at a 80% concentration to precipitate proteins. After centrifugation, the mixture was performed a CIPS at −20 °C to enrich GLY and AMPA (six-fold) in the lower water phase which was further performed PTSPE procedure. PTSPE as a miniaturized procedure of SPE, combined with a manual accu-jet® Pro Pipette Controller, was used to extract GLY and AMPA, in which a new type of hydrophilic adsorbent (HILIC powder) based on amide-modified silica was selected as the adsorption of GLY and AMPA. The key factors including the type and the amount of adsorbent, the loading extraction solution, the type and volume of eluent, and the number of aspirating/dispensing cycles were investigated in detail. Meanwhile, the selectivity and sensitivity of GLY and AMPA analysis were improved by the use of LC-HRMS based on targeted single ion monitoring (tSIM) mode without tedious derivatization. This method made a full use of the advantages of these techniques by combining efficient enrichment, effective extraction and selective separation in a simple way. Finally, a comprehensive validation of the method was rigorously executed and the results indicated that the validated method afforded desired linearity, precision, accuracy, and sensitivity.