LAUSR

Abstract The increasing usage of nanopesticides in agriculture poses a concern to plant crops due to unknown implications of engineered nanomaterials (ENMs). Targeted metabolomics can provide both qualitative and quantitative information at a molecular level to investigate the response of plants to emerging environmental stressors, such as nanopesticides. Here we describe a detailed protocol for the extraction and analysis of plant metabolites, specifically 23 amino acids in plants, using hydrophilic interaction liquid chromatography coupled to triple quadrupole mass spectrometry (HILIC-LC-MS/MS). Sufficient separation of 23 amino acids was achieved, without the need for derivatization, on an HILIC column with an MS/MS detector in a single run of 12 min with high sensitivity, selectivity and robustness and low LOD (0.005–15 ng/mL) and LOQ (0.02–50 ng/mL). A simple and efficient method to effectively extract amino acids from plant tissues was developed with a high recovery rate (80–120%). The protocol was then applied to determine the levels of amino acids in cucumber plants exposed to various environmentally-relevant levels of nano copper (nCu at 0, 200, 400, and 800 mg/kg soil; harvested in 60 days). Dose-dependent changes in amino acid levels were found; 13 amino acids were upregulated due to nCu stress, particularly, tyrosine increased 6.1, 8.2, and 11.0 fold after exposure to 200, 400 and 800 mg/kg nCu, respectively. The change in amino acid levels suggests an active defense response of the cucumber plant to nCu stress. We demonstrate that the HILIC-LC-MS/MS method is an effective and efficient technique to analyze underivatized amino acids in plant samples.