Brain kynurenine metabolite quantification

Dissected brain tissue was snap frozen on dry ice and stored at −80°C until analysis. Brain regions were prepared according to the protocol for IDO activity in brain tissue. Samples were prepared by mixing 450 μg brain protein 1:2 (v/v) with methanol containing 2% acetic acid and then centrifuging the mixture at 12,000×g for 10 minutes at 4°C. Supernatants were filtered through a Phenomenex Phree Phospholipid extraction column and a 0.2 μm filter. The samples were then run on a Waters Acquity UPLC-MS/MS with a methanol/2% acetic acid mobile phase gradient on a 2 × 100−mm Waters BEH C¹⁸ column. The sample (5 μl) was injected with a total flow rate of 0.3 ml/min and a total run time of 4.5 minutes. Analytes were verified using two M+H parent-daughter transitions. Tryptophan parent m/z = 205.13 and daughter m/z = 118.03 and 146.07; kynurenic acid parent m/z = 190.07 and daughter m/z = 88.96 and 116.03; kynurenine parent m/z = 209.04 and daughter m/z = 94.02 and 146.03; 3-HK parent m/z = 224.98 and daughter m/z = 110.04 and 162.05. Concentrations were quantified using QuanLynx (Waters) software from serial dilutions of standards.