2.9 Estimation of kynurenine metabolites
The concentrations of Trp, Kyn, 3-HK and KA were measured using an HPLC–MS/MS method as described by Han et al. (Han, Qin et al., 2019) with slight modifications. Briefly, tissue was transferred into a new EP tube, then mixed with 90 μl of prechilled (4 °C) methanol (0.1% formic acid)-aqueous (8:2, v/v) mixture and 10 μl of IS (2-Cl-Phe; 1 μg mL-1). The mixture was homogenized using an ultrasonic homogenizer followed by centrifugation at 20,000 ×g for 20 min at 4°C. After centrifugation, the separated supernatant was transferred into a 2 ml autosampler, and 10 μl was injected into the system at a flow rate of 0.4 ml min-1. The standard curve was prepared using the same procedure as the brain sample.
The HPLC–MS/MS system consisted of a Dionex UltiMate 3000 Ultra-HPLC system (Thermo, San Jose, CA, USA) and an API 4000Q Trap mass spectrometer (AB SCIEX, Foster City, USA) equipped with an electrospray ionization (ESI) source interface. The optimized mass spectrometric parameters were set as follows: curtain gas, 15 psi; collision gas, 2; ion spray voltage, 5500 V for positive mode or -4500 V for negative mode; ion source temperature, 600°C; ion source gas 1, 55 psi; ion source gas 2, 55 psi. Accurate quantification was operated in multiple reaction monitoring (MRM) mode; the transitions were m/z 205.1→146.1 for Trp (positive), m/z 209.1→94.1 for Kyn (positive), m/z 225.1→208.1 for 3-HK (positive), m/z 188.0→144.0 for KA (negative). The declustering potential (DP) was set at 40, 60, 45 and -40 V, and the collision energy (CE) was 24, 22, 14 and -22 V for Trp, Kyn, 3-HK and KA, respectively.
Chromatographic separation was performed on an Ultimate XB-C18 column (100 mm × 2.1 mm, 5 μm, Welch Materials, Inc.). Mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile. The temperature of the autosampler was set at 4 °C. Gradient separation was set as follows: 0-1 min, 5% B; 1-3 min, 5-60% B; 3.1-5 min, 5% B for column equilibration. The analysis was performed in a total run time of 5 min. Under these conditions, the retention times were 3.53, 1.97, 0.89 and 3.68 min for Trp, Kyn, 3-HK and KA, respectively. The above data were recorded and analysed using AB SCIEX Analyst 1.6 software.
Tryptophan (Trp, purity≥99.5%, Cat# 93659), kynurenine (Kyn, purity≥98%, Cat# K8625), 3-hydroxy-DL-kynurenine (3-HK, purity≥98%, Cat# 148776) and kynurenic acid (KA, purity≥98%, Cat# K3375) were purchased from Sigma‒Aldrich (St. Louis, MO, USA). 2-Chloro-L-phenylalanine (2-Cl-Phe, purity=98%, Cat# C105993) used as an internal standard (IS) was purchased from Aladdin Inc. (Shanghai, China). HPLC-grade acetonitrile and methanol were obtained from Fisher Chemical (Fisher Scientific, Shanghai, China).