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Quantitative profiling of tryptophan metabolites in serum, urine, and cell culture supernatants by liquid chromatography–tandem mass spectrometry

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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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Acknowledgments

This project was funded in part by BayGene, the intramural ReForM program of the Faculty of Medicine at the University of Regensburg, and the DFG (KFO 262, DE 835/2-1). We kindly thank Dr. Sanford Markey (Laboratory of Neurotoxicology, National Institutes of Health, Bethesda, MD, USA) for supplying 2H3-QA.

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Author notes

  1. Axel P. Stevens

    Present address: Analytical Department, Helmholtz Centre for Environmental Research UFZ, Permoserstr. 15, 04318, Leipzig, Germany

Authors and Affiliations

  1. Institute of Functional Genomics, University of Regensburg, Josef-Engert-Str. 9, 93053, Regensburg, Germany

    Wentao Zhu, Axel P. Stevens, Katja Dettmer & Peter J. Oefner

  2. Department of Hematology and Internal Oncology, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, 93053, Regensburg, Germany

    Eva Gottfried, Marina Kreutz & Ernst Holler

  3. Division of Clinical Pharmacology, Ludwig-Maximilian University of Munich, Ziemssenstr. 1, 80336, Munich, Germany

    Sabine Hoves

  4. Department of Biotechnology, Kluyver Centre for Genomics of Industrial Fermentation, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands

    André B. Canelas

  5. Department of Laboratory Medicine, University Medical Center, University of Groningen, 9700 RB, Groningen, The Netherlands

    Ido Kema

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  1. Wentao Zhu
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Correspondence to Peter J. Oefner.

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Zhu, W., Stevens, A.P., Dettmer, K. et al. Quantitative profiling of tryptophan metabolites in serum, urine, and cell culture supernatants by liquid chromatography–tandem mass spectrometry. Anal Bioanal Chem 401, 3249–3261 (2011). https://doi.org/10.1007/s00216-011-5436-y

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  • DOI: https://doi.org/10.1007/s00216-011-5436-y

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