Simultaneous detection of stable isotope-labeled and unlabeled l-tryptophan and of its main metabolites, l-kynurenine, serotonin and quinolinic acid, by gas chromatography/negative ion chemical ionization mass spectrometry

A method for the detection of unlabeled and 15N2-labeled l-tryptophan (l-Trp), l-kynurenine (l-Kyn), serotonin (5-HT) and quinolinic acid (QA) in human and rat plasma by GC/MS is described. Labeled and unlabeled versions of these four products were analyzed as their acyl substitution derivatives using pentafluoropropionic anhydride and 2,2,3,3,3-pentafluoro-1-propanol. Products were then separated by GC and analyzed by selected ion monitoring using negative ion chemical ionization mass spectrometry. l-[13C11, 15N2]-Trp, methyl-serotonin and 3,5-pyridinedicarboxylic acid were used as internal standards for this method. The coefficients of variation for inter-assay repeatability were found to be approximately 5.2% for l-Trp and 15N2-Trp, 17.1% for l-Kyn, 16.9% for 5-HT and 5.8% for QA (n = 2). We used this method to determine isotope enrichments in plasma l-Trp over the course of a continuous, intravenous infusion of l-[15N2]Trp in pregnant rat in the fasting state. Plasma 15N2-Trp enrichment reached a plateau at 120 min. The free Trp appearance rate (Ra) into plasma was 49.5 ± 3.35 µmol/kg/h. The GC/MS method was applied to determine the enrichment of 15N-labeled l-Trp, l-Kyn, 5-HT and QA concurrently with the concentration of non-labeled l-Trp, l-Kyn, 5-HT and QA in plasma. This method may help improve our understanding on l-Trp metabolism in vivo in animals and humans and potentially reveal the relative contribution of the four pathways of l-Trp metabolism. Copyright © 2014 John Wiley & Sons, Ltd.