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).