Quantitative profiling of tryptophan metabolites in serum, urine, and cell culture supernatants by liquid chromatography-tandem mass spectrometry |
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| Authors: | Zhu Wentao Stevens Axel P Dettmer Katja Gottfried Eva Hoves Sabine Kreutz Marina Holler Ernst Canelas André B Kema Ido Oefner Peter J |
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| Institution: | (1) Institute of Functional Genomics, University of Regensburg, Josef-Engert-Str. 9, 93053 Regensburg, Germany;(2) Present address: Analytical Department, Helmholtz Centre for Environmental Research UFZ, Permoserstr. 15, 04318 Leipzig, Germany;(3) Department of Hematology and Internal Oncology, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany;(4) Division of Clinical Pharmacology, Ludwig-Maximilian University of Munich, Ziemssenstr. 1, 80336 Munich, Germany;(5) Department of Biotechnology, Kluyver Centre for Genomics of Industrial Fermentation, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands;(6) Department of Laboratory Medicine, University Medical Center, University of Groningen, 9700 RB Groningen, The Netherlands |
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| Abstract: | A sensitive, selective, and comprehensive method for the quantitative determination of tryptophan and 18 of its key metabolites
in serum, urine, and cell culture supernatants was developed. The analytes were separated on a C18 silica column by reversed-phase
liquid chromatography and detected by electrospray ionization tandem mass spectrometry in positive ion multiple reaction monitoring
(MRM) mode, except for indoxyl sulfate which was measured in negative ion MRM mode in a separate run. The limits of detection
and lower limits of quantification were in the range of 0.1–50 and 0.5–100 nM, respectively. Fully 13C isotope-labeled and deuterated internal standards were used to achieve accurate quantification. The applicability of the
method to analyze serum, urine, and cell culture supernatants was demonstrated by recovery experiments and the evaluation
of matrix effects. Precision for the analysis of serum, urine, and cell culture supernatants ranged between 1.3% and 16.0%,
1.5% and 13.5%, and 1.0% and 17.4%, respectively. The method was applied to analyze changes in tryptophan metabolism in cell
culture supernatants from IFN-γ-treated monocytes and immature or mature dendritic cells. |
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