Quantitative profiling of tryptophan metabolites in serum, urine, and cell culture supernatants by liquid chromatography–tandem mass spectrometry
Quantitative profiling of tryptophan metabolites in serum, urine, and cell culture supernatants by liquid chromatography–tandem mass spectrometry
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DOI：
10.1007/s00216-011-5436-y
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发表时间：
2011-10
影响因子：
4.3
通讯作者：
Wentao Zhu;A. P. Stevens;K. Dettmer;E. Gottfried;S. Hoves;M. Kreutz;E. Holler;A. Canelas;I. Kem
中科院分区：
文献类型：
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作者：
Wentao Zhu;A. P. Stevens;K. Dettmer;E. Gottfried;S. Hoves;M. Kreutz;E. Holler;A. Canelas;I. Kem
建立了一种灵敏、选择性和全面的方法，用于定量测定血清、尿液和细胞培养上清液中色氨酸及其18种关键代谢产物。在C18硅胶柱上通过反相液相色谱法分离分析物，并通过电喷雾电离串联质谱法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运行中以负离子MRM模式测量。检测限和定量下限分别为0.1-50和0.5-100 nM。使用完全13 C同位素标记和氘代内标物实现准确定量。通过回收率实验和基质效应评价证明了该方法分析血清、尿液和细胞培养上清液的适用性。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%-16.0%、1.5%-13.5%和1.0%-17.4%。应用该方法分析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。
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. Fully13C 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.