Institute of Functional Genomics, University of Regensburg, Regensburg, Germany.
Anal Bioanal Chem. 2011 Dec;401(10):3249-61. doi: 10.1007/s00216-011-5436-y. Epub 2011 Oct 8.
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 (13)C 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.
建立了一种灵敏、选择性好、全面的方法，用于定量测定血清、尿液和细胞培养上清液中的色氨酸和 18 种关键代谢物。采用反相硅胶柱，通过液相色谱分离，电喷雾串联质谱在正离子多反应监测（MRM）模式下检测，除了硫酸吲哚酚在单独的运行中以负离子 MRM 模式测量。检测限和定量下限分别在 0.1-50 和 0.5-100 nM 范围内。完全（13）C 同位素标记和氘代内标用于实现准确的定量。通过回收率实验和基质效应评价，证明了该方法在分析血清、尿液和细胞培养上清液中的适用性。血清、尿液和细胞培养上清液分析的精密度范围分别为 1.3%至 16.0%、1.5%至 13.5%和 1.0%至 17.4%。该方法应用于分析 IFN-γ处理的单核细胞和未成熟或成熟树突状细胞的细胞培养上清液中色氨酸代谢的变化。
