Tryptophan metabolites were
analyzed according to Koper et al.25 (link) Samples
were centrifuged (21700g, 10 min, 4 °C) and
diluted in 0.1% formic acid, followed by filtration using a 0.22 μm
cellulose filter (Phenomenex) and high-resolution mass spectrometry
(HRMS) analysis. A silica modified Luna Polar C18 column (50 ×
2.1 mm, 1.6 μm, Phenomenex) was used for the chromatographic
separation of Trp and tryptophan metabolites. The mobile phases consisted
of water (A) and acetonitrile (B) both with 0.1% v/v of formic acid,
and the following gradient (min/%B) was used: (0/2), (0.50/2), (9.5/70),
and (12/70). The flow rate was 200 μL/min, the column temperature
was 40 °C, and 5 μL was injected. The U-HPLC system (Accela
1250, Thermo Fisher, Bremen, Germany) was interfaced to an Exactive
Orbitrap HRMS (Thermo), and the analytes were detected through a heated
electrospray interface (HESI-II) in positive mode by scanning the
ions listed in Table S1 in the m/z range of 50–400. Analytical
performances, mass spectrometry optimization, and linearity range
were monitored according to Koper et al.25 (link) Each sample was analyzed in duplicate, and the concentrations are
given in micromolars.