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Untargeted metabolites profiling from maize was extracted and analyzed using UPLC−qTOFMS. A 100 mg of freeze−dried sample was extracted in 1 mL of 80% methanol in water (v/v) and 0.05 mL of 2−chlorophenylalanine (100 μg/mL) and 0.05 mL of 3,4,5−trimethoxycinnamic acid (100 μg/mL) as internal standards. The sample was shaken at 4 °C overnight, and then centrifuged at 4 °C and 16,000 × g for 5 min. A total of 0.9 mL of the supernatant was evaporated in a vacuum centrifuge concentrator for 30 min. During evaporation, the left pellet was added 0.5 mL of 80% methanol in water, and then shaken using a thermomixer at 30 °C for 30 min for re-extraction. The re-extracted mixture was centrifuged at 4 °C and 16,000 × g for 5 min. The evaporation of the first mixture was paused, and the supernatant (0.5 mL) of the re-extracted mixture was transferred into a tube containing the first mixture. The combined supernatants were evaporated in a vacuum centrifugal concentrator for 3 h. A total of 0.3 mL of 80% methanol in water was added to the pellet. The mixture was filtered through a 0.2 μm syringe filter into a 2 mL autosampler vial.

Finally, the sample was injected for UPLC−qTOFMS analysis in an ACQUITY UPLC I−Class PLUS system (Waters, Milford, MA, USA). A Luna Omega Polar C18 column (150 mm × 2.1 mm, 1.6 μm; 00F−4748−AN, Phenomenex, Torrance, CA, USA) was used, and the injection volume was 10 μL. The column temperature was 40 °C, and the column flow was 0.3 mL/min. Solvent A was composed of 0.1% formic acid in water, and Solvent B was composed of 0.1% formic acid in acetonitrile. The binary gradient elution system of solvents A and B was as follows: 0 min, A 90% / B 10%; 6 min, A 80% / B 20%; 24 min, A 0% / B 100 %; 27 min, A 0% / B 100%; 28 min, A 90% / B 10%; 30 min, A 90%, B 10%.

Mass spectrometry data were acquired separately in positive and negative ion modes. The ion−source and the desolvation gas temperatures were adjusted to 100 °C and 250 °C. Nitrogen and argon were used as desolvation and collision gases, respectively. The cone and desolvation gas flows were adjusted to 50 and 600 L/h, respectively. The capillary and cone voltages were 3.0 kV and 40 V, respectively. The low and high collision energies were 6 eV and 10–30 eV, respectively. Mass spectrometry data were acquired in continuum MSE mode. The scanned mass range was 50–1200 m/z. Mass calibration was performed by direct infusion of 0.5 mM sodium formate solution in a 90:10 isopropanol:water mixture before measurements. Additionally, a solution of 200 pg/μL leucine enkephalin in 1:1 acetonitrile:water containing 0.1% formic acid was applied as a lock mass for accurate mass correction (m/z 556.2771 in positive and m/z 554.2615 in negative ion mode).