Pentafluorobenzylation Method for Quantification of Acidic Tryptophan Metabolites Using Electron Capture Negative Ion Mass Spectrometry

An improved penfluorobenzylation method was developed for derivatization of L-tryptophan and its acidic metabolites (L-kynurenine, kynurenic acid, anthranilic acid, xanthurenic acid, 3-hydroxyanthranilic acid, picolinic acid, quinolinic acid) present at trace levels in aqueous samples. This method employs lyophilization of aqueous samples in the presence of excess tetrabulammonium hydrogen sulfate, followed by base-catalyzed anhydrous penfluorobenzylation. A comparison with other published methods shows the advantage of this modification for the derivatization of kynurenine metabolites. The derivatives were analyzed by gas chromatography/electron capture negative ion mass spectrometry (GC/ECM-MS) or liquid chromatography/particle beam/ECM-MS (LC/ECNI-MS). The detection limits for injected standards are in the femtogram range by GC/ECM-MS and in the low picogram range by LC/ECNI-MS. GC/ECNIMS is 3.6 (xanthurenic acid) to 66 (quinolinic acid) times more sensitive than LC/ECNI-MS. The simultaneous determination of two neuroactive metabolites, quinolinic and kynurenic acids, in culture medium is presented. The minimum measurable concentrations of these metabolites in 100 pL of culture medium are 0.11 nM for quinolinic acid and 0.21 nM for kynurenic acid