6400 series

Whole blood was collected in Ethylenediaminetetraacetic acid (EDTA) vacutainers and processed within 4 hours of collection. Soluble CD14 was measured in plasma using the Quantikine sCD14 kit (R&D Systems, USA) according to the manufacturer’s instructions as previously described^{60 (link)}. Kynurenine and tryptophan concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS, Agilent, 6400 series) as previously described^{61 (link)}. The HPLC conditions used were as follows: the column was a Synergi Polar RP column (75 mm × 4.6 mm) from Phenomenex (CA, USA) and the mobile phase was composed of 0.1% formic acid in ultrapure water and a mixture of methanol and acetonitrile (7% v/v) with gradient program. The flow rate was set at 1.0 ml/min and the run time for each sample was 6 min.

Tryptophan and kynurenine levels were measured by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described in Huang et al. [37 ]. Briefly, trifluoroacetic acid was added into the plasma samples for protein plasma precipitation before analysis. A total of 5μl sample was injected into the LC-MS/MS system (Agilent, 6400 series). The HPLC conditions were as follows: the column was a Synergi Polar RP column (75 x 4.6mm) from Phenomenex (CA, USA) and the mobile phase was composed of formic acid in ultrapure water and a mixture of methanol and acetonitrile (7% v/v) with gradient program. The flow rate was set at 1.0 ml/min and the run time for each sample was 6 min. The LC-MS/MS method was validated according to the International Council for Harmonization (ICH) guidelines. K/T ratio was calculated by dividing the concentration kynurenine to that of tryptophan, both measured in ng/ml.