PENTAFLUOROBENZYLATION METHOD FOR QUANTIFICATION OF ACIDIC TRYPTOPHAN-METABOLITES USING ELECTRON-CAPTURE NEGATIVE-ION MASS-SPECTROMETRY

An improved pentafluorobenzylation method was developed for derivatiza tion of L-tryptophan and its acidic metabolites (L-kynurenine, kynuren ic acid, anthranilic acid, xanthurenic acid, 3-hydroxyanlranilic acid, picolinic acid, quinolinic acid) present at trace levels in aqueous s amples. This method employs lyophilization of aqueous samples in the p resence of excess tetrabutylammonium hydrogen sulfate, followed by bas e-catalyzed anhydrous pentafluorobenzylation. A comparison with other published methods shows the advantage of this modification for the der ivatization of kynurenine metabolites. The derivatives were analyzed b y gas chromatography/electron capture negative ion mass spectrometry ( GC/ECNI-MS) or liquid chromatography/particle beam/ ECNI-MS (LC/ECNI-M S). The detection limits for injected standards are in the femtogram r ange by GC/ECNI-MS and in the low picogram range by LC/ECNI-MS. GC/ EC NIMS is 3.6 (xanthurenic acid) to 66 (quinolinic acid) times more sens itive than LC/ECNI-MS. The simultaneous determination of two neuroacti ve metabolites, quinolinic and kynurenic acids, in culture medium is p resented. The minimum measurable concentrations of these metabolites i n 100 mu L of culture medium are 0.11 nM for quinolinic acid and 0.21 nM for kynurenic acid.