Kynurenine Metabolite Analysis

Kynurenine metabolites were analyzed with the modified method of Virág and Wang [22, 23]. The standard compounds (tryptophan (PHR1176), L-kynurenine (K8625), kynurenic acid (K3375), and quinolinic acid ({"type":"entrez-protein","attrs":{"text":"P63204","term_id":"52783587"}}P63204)) were purchased from Sigma Aldrich (USA). The colon and brain tissues were homogenized using a bullet blender (Next Advance Inc.) with a D.W./MeOH/ACN (1:2:2). The homogenized tissue and blood plasma was mixed with trifluoroacetic acid and centrifuged at 20,000 ×g for 15 min. The supernatant lyophilized using a vacuum-tray freeze dryer (Operon). Powdered extract was dissolved with MeOH/ACN (1:1) and centrifuged at 20,000 ×g for 15 min. The supernatants were injected into an ultraperformance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry system (UPLC-Q-TOF-MS, Vion, Waters Corp., USA). The extracted samples were separated on the Acquity UPLC BEH C18 Column (2.1 mm × 100 mm, 1.7 μm; Waters Corp.) equilibrated with water: acetonitrile (ACN) containing 0.1% formic acid. Elution was carried out at a flow rate of 0.35 ml/min for 12 min. Mass spectrometric detection was conducted by multiple reaction monitoring (MRM) with an electrospray ionization (ESI) source in the positive mode. The LC/MS conditions were as follows: ramp collision energy, 20–45V; oven temperature, 40°C; capillary voltage, 3kV; and mass range, 50–1500. The MRM conditions for the analytes are shown in Table 1.

Multiple reaction monitoring (MRM) conditions in the positive ESI mode.