Masslynx software version 4

Brain and serum serotonin concentrations of both the pCPA-treated mice and Tph2-/- mice were determined by UPLC-QTof-MS. Mice brain were weighed and homogenized in cold methanol (−20 °C) (4 mL methanol per gram of brain tissue) while 100 μL serum samples were mixed with 400 μL cold methanol. The homogenate or the mixture was then centrifuged at 10,000 xg for 15 min. The supernatant was dried under nitrogen and resuspended with 150 μL pure water, 120 μL chloroform and 30 μL isopropanol. After centrifugation, the upper aqueous layer was injected into the UPLC-QTof-MS system for analysis. UPLC-QTof-MS analysis was performed under the same conditions used in the non-targeted metabolic profiling. The serotonin concentrations were calculated using the Waters Masslynx software (version 4.1) based on the standard sample.

LC-MS/MS analysis was performed on ACQUITY UPLC I-Class coupled with triple quadrupole mass spectrometer ACQUITY XEVO TQD (Waters, Prague, Czech Republic) with electrospray ionization (ESI) source in both positive and negative mode. MS/MS analysis was performed in the multiple reaction monitoring (MRM) mode. The electrospray capillary voltage was set to 2.0 kV, desolvation gas flow was 650 l/hr with a temperature 350 °C, and the source temperature was set to 150 °C. MassLynx software version 4.1 (Waters) was used for the data acquisition, and TargetLynx XS (Waters) was used for processing.

Biomarker analyses were conducted on 360 participants with available plasma samples (174 in 3-C and 186 in AMI). Plasma 3-MH and 1-MH concentrations (µmol/L) were determined simultaneously by ultra-performance liquid-chromatography tandem mass spectrometry (UPLC-MS/MS; Acquity Ultra Performance LC system, Quattro Premier XE mass spectrometer, MassLynx Software Version 4.1 (all Waters Corporation, Milford, MA, USA)) according to Kochlik et al. [28 (link)].
The eGFR, as an estimation of kidney function, was calculated according to Levey et al. [33 (link)], taking into account sex, age and plasma creatinine concentrations of the participants, as follows: $\mathrm{Female}:\le 62\mathsf{\mu }\mathrm{mol}/\mathrm{L}\mathrm{creatinine}:eGFR=144\times {(Crea/0.7)}^{-0.329}\times {0.993}^{Age}$
$\mathrm{Female}:>62\mathsf{\mu }\mathrm{mol}/\mathrm{L}\mathrm{creatinine}:eGFR=144\times {(Crea/0.7)}^{-1.209}\times {0.993}^{Age}$
$\mathrm{Male}:\le 80\mathsf{\mu }\mathrm{mol}/\mathrm{L}\mathrm{creatinine}:eGFR=141\times {(Crea/0.9)}^{-0.411}\times {0.993}^{Age}$
$\mathrm{Male}:>80\mathsf{\mu }\mathrm{mol}/\mathrm{L}\mathrm{creatinine}:eGFR=141\times {(Crea/0.9)}^{-1.209}\times {0.993}^{Age}$
Four ratios were calculated: 3-MH/Crea, 1-MH/Crea, 3-MH/eGFR and 3-MH/1-MH.

The UPLC-MS/MS system was composed of an Acquity^TM UPLC system (Waters, Milford, MA, USA) equipped with a column compartment, refrigerated autosampler, binary solvent manager, and tandem quadrupole mass spectrometer with an API source (XEVO TQ-MS) connected to MassLynx software version 4.1 (Waters, Milford, MA, USA). The separations were conducted on an Atlantis T3 column (4.6 mm × 50 mm, 3 µm) from Waters maintained at 40 °C. The mobile phase was composed of 0.3% acetic acid in water (A) and 0.3% acetic acid in acetonitrile (B), and the following gradient elution was applied: 2% B at 0 min, 2–30% B at 0–4 min, held at 30% B for 3 min, 30–98% B at 7–7.1 min, 98% B until 8 min, 98–2% B at 8–8.1 min, and 2% B until 10 min. The process was conducted with an injection volume of 5 µL and a flow rate of 0.5 mL/min. The following parameters were adjusted to determine the maximized ionization: mass spectrometry desolvation temperature and gas flow were set at 625 °C and 1100 L/h, respectively; the selective ion monitoring mode was [M + H]⁺ ions for all catechins and ethyl gallate, except for EGCG, which used [M − H]^– ions. The detected ion cone voltages and collision energy in each electrospray mode was 25 V, 25 V for EGCG; 25 V, 15 V for ECG; 20 V, 15 V for EGC; 20 V, 15 V for EC; and 18 V, 12 V for ethyl gallate, respectively.

A Waters triple-quadrupole mass spectrometer (Waters Corporation, Prague, Czech Republic)with an electrospray ionization source in negative mode (ESI-) was used. The mass spectrometer was operated with the following parameters: capillary voltage 3 kV, source temperature 150 °C, desolvation temperature 350 °C, cone gas 50 L/h, and desolvation gas 800 L/h. The source cone voltages and collision energies were manually optimized for each SRM transition. The chromatographic apparatus consisted of a Waters ACQUITY UPLC system with a binary gradient pump, autosampler and column thermostat (Waters, Prague, Czech Republic). Chromatographic separation was performed on an ACQUITY UPLC BEH C18 (2.1 × 50 mm, 1.7 μm particle size) column. The column temperature was maintained at 30 °C. Mobile phase A consisted of 0.1% formic acid in water. Mobile phase B consisted of 100% acetonitrile. The elution started at 20% B (0–0.5 min), increasing to 90% B (0.5–2 min) to 90% B (2–3 min), returning to 20% B and re-equilibrating at 3–4 min. The flow rate was 0.5 mL/min, and the injection volume was 5 μL. MassLynx software version 4.1 (Waters Corporation, Prague, Czech Republic) was used for instrument control and data acquisition and analysis.