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.