Development and validation of a single analytical method for the determination of tryptophan, and its kynurenine metabolites in rat plasma

It is highly beneficial to monitor the activity of the kynurenine pathway in a large series of samples with high accuracy and reliability in a single experimental protocol. We have developed a rapid specific solid-phase extraction (SPE)–liquid chromatography–electrospray ionization tandem mass spectrometry method for assaying tryptophan, kynurenine, kynurenic acid (KYNA), 3-hydroxyanthranilic acid (3OHAA), anthranilic acid and quinolinic acid (QA) in rat plasma. We also evaluated picolinic acid (PA) in this method, but it presented with unacceptable validation parameters. The assay involves pre-purification by SPE followed by chromatographic separation by C18 reversed phase chromatography. Mass spectrometric detection was performed using a mass spectrometer in positive and negative electrospray ionization; with a flow rate of 0.2 mL/min and an injection volume of 10 μL. Total run time including sample clean-up was 12 min. The assay method was found to be linear (R2 > 0.95) and all the validation parameters were within acceptance range. The developed technique also demonstrated a significant elevation in plasma tryptophan, kynurenine, anthranilic acid and QA, and a significant decrease in KYNA, in rats subjected to post-weaning social isolation rearing, a putative animal model of relevance for depression and schizophrenia. This method can therefore be applied to measure metabolites of the kynurenine pathway in plasma accurately and precisely by LC–MS/MS, thereby helping to realize new opportunities in pharmacological and diagnostic research.