*2.1.3.1 Reagents and instrumentation*

The simultaneous analytical method developed can be adapted to major metabolites of TRP including melatonin in clinical sample.

The analytical targets of developed method are major metabolites, such as tryptophan (TRP), L-5-hydroxytryptophan (5-HTP), serotonin (5-HT), kynurenine (KYN), 5-hydroxy-tryptophol, tryptophol, 5-hydroxyindoleacetic acid (5-HIAA), indole-3-acetic acid, anthranilic acid (AA), kynurenic acid (KYNA), quinaldic acid, indole-3-butyric acid, 3-hydroxykynurenine (3-HKYN), 3-hydroxyanthranilic acid (3-HAA), xanthurenic acid (XA), melatonin, and quinolinic acid (QA). Each compound was purchased from major chemical regent manufacturers, such as FUJIFILM Wako chemical (Osaka, Japan) and Sigma-Aldrich (St. Louis, MO, USA).

Metabolite analysis was performed by a liquid chromatograph tandem mass spectrometer, the LCMS-8060 quadrupole mass spectrometer combined with Nexera X2 liquid chromatograph system (Shimadzu Corporation, Kyoto, Japan).

The targets are separated by reversed-phase chromatography using C18 analytical column, L-Columns ODS2 (2.1 mm × 150 mm, CERI, Tokyo, Japan) with a gradient elution. Mobile phases were 0.1% formic acid solution and acetonitrile with the gradient elution by 5% concentration of acetonitrile in 3 min and then 5–95% in 6 min, followed by 5% in 3 min at a total flow rate of 0.4 mL/ min. The temperature of the column was 40°C. Electrospray ionization (ESI) was used as mostly positive ionization with multi-reaction monitoring (MRM) detection.

Flow rate of the neutralizer and the drying gas were 2 L/min and 10 mL/min, respectively. Temperature of desolvation line (heated capitally tube) was 250°C. ESI interface was used at 400°C with 10 L/min of heating gas flow. Each MRM transition was optimized using each standard solution. Optimized results were shown in **Table 1**.

All mother solutions of 1 mg/mL had been stocked under −80°C, and standard samples for calibration curve were prepared prior to use as mixture solution by consideration of each range of measurement concentration.
