Determination of N-acetylation phenotype using caffeine as a metabolic probe and high-performance liquid chromatography with either ultraviolet detection or electrospray mass spectrometry |
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| Institution: | 1. Laboratory of Molecular and Structural Microbiology, Institut Pasteur de Montevideo, Montevideo, Uruguay;2. Pasteur International Unit, Integrative Microbiology of Zoonotic Agents, Institut Pasteur de Montevideo, Montevideo, Uruguay/Institut Pasteur, Paris, France;3. Institut Pasteur, Université Paris Cité, CNRS UMR 6047, Biology of Spirochetes Unit, Paris, France;4. Microbial Genomics Laboratory, Institut Pasteur Montevideo, Montevideo, Montevideo, 11400 Uruguay;5. Center for Integrative Biology, Universidad Mayor, Santiago de Chile, Chile;6. Wellcome Sanger Institute, Hinxton, UK;7. Institut Pasteur, Département de Microbiologie Paris, France |
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| Abstract: | A rapid, sensitive method using liquid chromatography–electrospray mass spectrometry (LC–ES-MS) was developed and evaluated for the simultaneous quantitative determination of caffeine metabolites 1U, 1X and AAMU in human urine. This method involved a simple dilution of urine samples. The chromatographic separation was achieved on a C18 reversed-phase column using a gradient of acetonitrile in 2 mM, pH 3.0 ammonium formate as mobile phase. After ionisation in an electrospray source, mass spectrometric detection was performed in the negative ion, selected ion monitoring mode. This method yielded acceptable accuracy and precision within the range 0.25–50 μg/ml. This analytical method was applied to investigate the N-acetylator phenotype of HIV-infected patients and compared with high-performance liquid chromatography with UV detection. Its specificity was better, which appeared to be absolutely necessary to prevent errors in metabolic ratios and phenotype interpretation. |
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