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Quantification of L ‐tryptophan and L ‐kynurenine by liquid chromatography/electron capture negative ion chemical ionization mass spectrometry
Author(s) -
Boni R. L.,
Simpson J. T.,
Naritsin D. B.,
Saito K.,
Markey S. P.
Publication year - 1994
Publication title -
biological mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1052-9306
DOI - 10.1002/bms.1200230106
Subject(s) - chemistry , kynurenine , chromatography , tryptophan , analyte , mass spectrometry , kynurenine pathway , metabolite , biochemistry , amino acid
In a number of infectious and inflammatory diseases, stimulation of the immune system can lead to increased accumulation of tryptophan metabolites via induction of kynurenine pathway enzymes in extrahepatic tissues. We developed a liquid chromatographic/mass spectrometric (LC/MS) method suitable for tracing the disposition of 13 C isotopomers of L‐tryptophan and L‐kynurenine in various cultured cell, tissue slice, and whole animal model systems used to investigate tryptophan flux through the kynurenine pathway. The method employs extractive derivatization of the analytes and their 2 H internal standards with pentafluorobenzyl bromide in order to enhance the negative ion chemical ionization (NICI) mass spectrometric response. Normal‐phase liquid chromatographic separation of derivatized analytes was optimized using a silica column with organic solvents, followed by particle beam transfer and NICI‐MS. Standard curves were linear over the range 1–250 ng per sample. Particle beam and mass spectrometric operating parameters were optimized with direct flow injections of 1‐(methylamino) anthra‐quinone, which is an ideal test compound for the evaluation of LC/NICI‐MS. The developed method was used to quantify the conversion of ( 13 C 6 )L‐tryptophan to ( 13 C 6 )L‐kynurenine by human monocytes (THP‐1) stimulated with interferon‐γ, lung and brain tissue slices obtained from gerbils immune‐stimulated with pokeweed mitogen. The effect of whole body immune stimulation on the plasma levels of engogenous L‐kynurenine in mice stimulated with interferon‐γ was also quantified.