Dissolve 4 mg of TMAE in 1 mL of 80% methanol (Merck) solution. After mixing, centrifuge the solution at 8000 r/min for 10 min and collect the supernatant. The supernatant samples were filtered using nylon membranes (13 mm × 0.22 μm, ANPEL Laboratory Technologies Inc.) and separated on an Agilent 1290 Infinity UHPLC (Agilent Technologies, Santa Clara, CA, USA) in series with an Agilent 6530 Accurate-Mass Q-TOF LC/MS (Agilent Technologies, Santa Clara, CA, USA) system equipped with Agilent Eclipse XDB-C18 (4.6 × 150 mm, 5 μm). The UHPLC-Q/TOF-MS test conditions are based on the method of Pieczykolan, A. et al. (38 (link)) with some modifications. The mobile phases used in this experiment were water containing 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B). The gradient elution program followed the schedule below: 0-1.5 min, 13% B; 2-15 min, 20% B; 16-23 min, 25% B; 25-28 min, 33% B; 30-33 min, 60% B; 34-37 min, 13% B. The flow rate was 0.35 mL/min, and the column temperature was maintained at 25°C. The negative ion source voltage was set at -4.5 kV, the capillary temperature at 500°C, the spray gas at 55 psi, and the Curtain Gas at 30 psi. The mass-to-charge ratios of primary and secondary fragments of isolated compounds were screened in the MS-DIAL database and literature reports to identify compounds in extracts (39 (link), 40 (link)).
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