4.3. Measurement of Tryptophan, Tryptamine, and Serotonin

The content of tryptophan, tryptamine, and serotonin was determined using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system as previously described [48]. The freeze-dried samples were crushed using a mixer mill (MM 400; Retsch, Haan, Germany) with zirconia beads for 1 min at 30 Hz, and 0.1 g of the dry powder was extracted overnight at 4 °C with 1 mL of 70% aqueous methanol containing 0.1 mg/L lidocaine (internal standard). Following centrifugation at 10,000× g for 10 min, the lipid-soluble extracts were absorbed, and 0.4 mL of each extract was mixed and filtered (SCAA-104, 0.22 μm pore size; Angel, Shanghai, China) before the LC-MS analysis.

The targeted metabolic profiling analysis was conducted using scheduled multiple reaction monitoring (MRM) via an LC-ESI-QQQ-MS/MS system (LCMS-8060, SHIMADZU, Kyoto, Japan). The UPLC (Shim-pack UFLC SHIMADZU CBM30A system) conditions were as follows: column, shim-pack GISS C18 (pore size 1.9 μm, dimensions 2.1 × 100 mm); solvent system, water (0.04% acetic acid), acetonitrile (0.04% acetic acid); gradient program, 95:5 v/v at 0 min, 5:95 v/v at 12.0 min, 5:95 v/v at 13.2 min, 95:5 v/v at 13.3 min, 95:5 v/v at 15.0 min; flow rate, 0.40 mL min⁻¹; temperature, 40 °C; injection volume: 2 μL. The ESI source operation parameters were as follows: nebulizing gas flow, 3 L min⁻¹; heating gas flow, 10 L min⁻¹; interface temperature, 500 °C; DL temperature, 250 °C; heat block temperature, 400 °C; drying gas flow, 10 L min⁻¹. The recorded data were processed with LabSolutions 5.91 software.