A validated liquid chromatography‐high resolution‐tandem mass spectrometry method for the simultaneous quantitation of tryptophan, kynurenine, kynurenic acid, and quinolinic acid in human plasma

Abstract

Tryptophan (TRP) catabolism via the kynurenine pathway is considered to represent a major link between inflammation and various diseases, including neurodegenerative disorders, depression, schizophrenia, multiple sclerosis, cardiovascular disease, and cancer. The kynurenine pathway and levels of TRP and its metabolites kynurenine (KYN), kynurenic acid (KYNA) and quinolinic acid (QUIN) are well regulated under physiological conditions but may be altered as part of the activated immune response. A simple, sensitive, and specific liquid chromatography-time of flight mass spectrometry method was developed for determining levels of the four compounds in human plasma samples. The workflow involves protein precipitation with acetonitrile, chromatographic separation on a Phenomenex Luna NH2 column by applying a linear 6 min gradient of 50-5% acetonitrile in aqueous ammonium acetate solution (5mM, pH 9.5), and mass spectrometric detection with high-resolution tandem mass spectrometry. Charcoal-treated plasma served as surrogate matrix for external standard calibration. Stable-isotope-labeled analogues were used as internal standards. The calibration ranges were 0.5-50μg/ml for TRP, 20-1000ng/mL for KYN und QUIN, and 1-50ng/mL for KYNA. Validation proved fitness of the developed workflow for the intended purpose. The established method was applied to the quantification of the four targets in 100 authentic plasma samples.