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For urinary metabolite extraction, 10 μl of urine samples stored in an ultra-low-temperature freezer were thawed at 4 °C. To each thawed sample, 21.9 μl of water, 164.1 μl of acetonitrile, and 4 μl of an internal standard solution were added and mixed for approximately 1 minute. The mixture was the incubated at 4 °C for 1 hour to extract metabolites, followed by centrifugation at 12,700 rpm for 20 minutes at 4 °C. After centrifugation, 180 ul of the supernatant was transferred to a new microcentrifuge tube, and 45 ul of this supernatant was subsequently transferred to an autosampler vial for analysis.

LC separations were carried out on an SeQuantZIC-HILIC column (2.1 mm × 100 mm, particle size 3.5 μm, 200 Å, Merck, Germany). Column temperature and flow rate were set to 40 °C and 0.3 ml/min, respectively. The mobile phases used were 0.1% formic acid and 10 mM ammonium acetate in an acetonitrile : water mixture (90:10, v/v) (A) and acetonitrile : water mixture (50:50, v/v) (B). The liner gradients were as follows: 1% B, 0.0–1.0 min; 1–99% B, 1.0–14.0 min; 99% B, 14.0–16.0 min; 99–1% B, 16.0–16.1 min; 1% B, 16.1–20.1 min. The injection volume of sample was 1 μl using partial loop mode for both positive and negative ionization polarity modes. Quality control (QC) samples, pooling identical aliquots of the samples, were measured regularly throughout the run for data reproducibility.

The MS was operating in both positive and negative ionization modes. The mass scan range was set to m/z 50–1,000. The MS parameters were configured as follows: nebulizer gas pressure, 2.2 bar; dry gas flow rate, 10.0 L/min; dry gas temperature, 220 °C, capillary voltage, ±4,500 V; and end plate offset, 500 V. The MS/MS method was performed to obtain the fragmentation patterns of metabolites. Nitrogen served as the collision gas, and precursor ion fragmentation was induced using a collision energy of 20 eV in both positive and negative ionization modes. Precise mass data was acquired at a rate of 8 spectra/sec in both modes. In positive ionization mode, an external standard with [M+H]+ 622.0289 m/z was used, and the mass range of 621.00–623.00 m/z was excluded to prevent fragmentation of the standard. Similarly, in negative ionization mode, an external standard with [M+CH3COOH-H]- 680.0344 m/z was used, and the mass range of 679.00–681.00 m/z was excluded.