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Date of Award


Access Type

Open Access Dissertation

Document type


Degree Name

Doctor of Philosophy (PhD)

Degree Program


First Advisor

Sidney Siggia

Second Advisor

Peter C. Uden

Third Advisor

Anthony M. Gawienowski


The analysis of the kynurenine metabolites of tryptophan by chromatographic methods has been investigated. The use of derivatizing reagents for gas chromatographic analysis has been explored. The feasibility of an HPLC fluorescence method has also been studied.

The use of silylating reagents has allowed the successful determination of anthranilic acid, quinaldic acid and quinolinic acid by gas chromatography. The utility of the direct current plasma emission spectrometer as a gas chromatographic detector has also been demonstrated in the silylation work. Surface activity of the GC-DCP inter­face as well as irreproducible silylations of other metabo­lites prevented the continuation of this work.

A preliminary investigation of the TFA-,PFP- and HFB-n-butyl esters of anthranilic acid, 3-hydroxy anthranilic acid, quinaldic acid, quinolinic acid, xanthurenic acid, kynurenic acid, kynurenine and 3-hydroxykynurenine has shown that a single derivatization procedure, employing butanolic HCl (6.0N) and N-methyl-bis-trifluoroacetamide is suitable for the analysis of most of the aforementioned metabolites. Quantitation by GC-FID on a QF-1 packed column resulted in detection limits for these compounds in the range of l.0ng to 26.0ng.

Derivative identification was performed by packed column gas chromatography-mass spectrometry. The electron impact (70eV) mass fragmentation patterns of the N(0)­trifluoroacetyl-n-butyl esters and the N(0)-heptafluoro­butryl-n-butyl esters of the kynurenine metabolites were recorded. Ion fragment identification was made by comparison of published mass spectra of the similarly derivatized 20 main amino acids.

The possibility of using HPLC with fluorescence detection for the analysis of these metabolites was investi­gated. Employing a filter fluorescence detector, a feasability study using kynurenine as a model compound resulted in detection limits of l.0ng. A study of the excitation and emission properties of kynurenine in the mobile phase of choice revealed that a sensitivity increase of approximately 10 fold could be achieved using the proper excitation wave­length in the fluorescence detector.