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50 μL of each sample was added with 200 μL cold (−20 °C) methanol:ethanol mix (v:v; 1:1) in a new polypropylene tube. After vortexing for 5 min, samples were centrifuged to remove the protein pellet at 14,000 rpm for 10 min at 4 °C. The supernatants were transferred into autosampler vials and 4 µL of each sample was injected into the Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific, Bremen, Germany).

An ACQUITY UPLC HSS T3 Column (2.1 × 100 mm, 1.8 μm, Waters) was connected to separate and detect of the analytes. The mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in ACN:MeOH, 7:3). The gradient started at 5% B for 1 min, increased linearly to 80% B over 12 min, was maintained at 80% B for 0.5 min, and then returned to 5% B at 13.5 min with a total runtime of 15 min. The flow rate was set to 0.4 mL/min. The column oven temperature was maintained at 40°C, and the autosampler was kept at 4°C, with an injection volume of 4 μL.

LC-MS/MS analysis was performed in a Vanquish Flex UHPLC system equipped with an Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) for metabolomics analysis. The ESI source conditions were set as follows: sheath gas flow rate was 40 Arb, the auxiliary gas flow rate was 10 Arb, the sweep gas flow rate was 1 Arb, the vaporizer temperature was 350 °C, and the spraying voltage was − 3.0 kV (negative).