Department of Chemistry, Georgia State University, Atlanta, GA, USA.
Department of Chemistry, Georgia State University, Atlanta, GA, USA.
J Chromatogr A. 2021 Aug 16;1651:462294. doi: 10.1016/j.chroma.2021.462294. Epub 2021 May 28.
Few articles are reported for the simultaneous separation and sensitive detection of the kynurenine pathway (KP) metabolites. This work describes a capillary electrochromatography-mass spectrometry (CEC-MS) method using acrylamido-2-methyl-1-propanesulfonic acid (AMPS) functionalized stationary phase. The AMPS column was prepared by first performing silanization of bare silica with gamma-maps, followed by polymerization with AMPS. The CEC-MS/MS methods were established for six upstream and three downstream KP metabolites. The simultaneous separation of all nine KP metabolites is achieved without derivatization for the first time in the open literature. Numerous parameters such as pH and the concentration of background electrolyte, the concentration of the polymerizable AMPS monomer, column length, field strength, and internal pressure were all tested to optimize the separation of multiple KP metabolites. A baseline separation of six upstream metabolites, namely tryptophan (TRP), kynurenine (KYN), 3-hydroxykynurenine (HKYN), kynurenic acid (KA), anthranilic acid (AA), and xanthurenic acid (XA), was possible at pH 9.25 within 26 min. Separation of six downstream and related metabolites, namely: tryptamine (TRPM), hydroxy‑tryptophan (HTRP), hydroxyindole-3 acetic acid (HIAA), 3-hydroxyanthranilic acid (3-HAA), picolinic acid (PA), and quinolinic acid (QA), was achieved at pH 9.75 in 30 min. However, the challenging simultaneous separation of all nine KP metabolites was only accomplished by increasing the column length and simultaneous application of internal pressure and voltage in 114 min. Quantitation of KP metabolites in commercial human plasma was carried out, and endogenous concentration of five KP metabolites was validated. The experimental limit of quantitation ranges from 100 to 10,000 nM (S/N = 8-832, respectively), whereas the experimental limit of detection ranges from 31 to 1000 nM (S/N = 2-16, respectively). Levels of five major KP metabolites, namely TRP, KYN, KA, AA, and QA, and their ratios in patient plasma samples previously screened for inflammatory biomarkers [C-reactive protein (CRP) and tumor necrosis factor-alpha (TNF-α)] was measured. Pairs of the level of metabolites with significant positive correlation were statistically evaluated.
鲜有文献报道用于同时分离和灵敏检测犬尿氨酸途径（KP）代谢物的方法。本工作描述了一种使用丙烯酰胺基-2-甲基-1-丙磺酸（AMPS）功能化固定相的毛细管电色谱-质谱（CEC-MS）方法。AMPS 柱通过先用γ-巯丙基三甲氧基硅烷对裸硅胶进行硅烷化，然后再用 AMPS 进行聚合来制备。建立了用于六种上游和三种下游 KP 代谢物的 CEC-MS/MS 方法。首次在开放文献中无需衍生化即可同时分离所有 9 种 KP 代谢物。优化多种 KP 代谢物分离的参数包括 pH 值和背景电解质的浓度、可聚合 AMPS 单体的浓度、柱长、场强和内压等。在 pH 9.25 下，可在 26 min 内实现六种上游代谢物，即色氨酸（TRP）、犬尿氨酸（KYN）、3-羟基犬尿氨酸（HKYN）、犬尿喹啉酸（KA）、邻氨基苯甲酸（AA）和黄尿酸（XA）的基线分离。在 pH 9.75 下，可在 30 min 内实现六种下游和相关代谢物，即色胺（TRPM）、羟基色氨酸（HTRP）、羟基吲哚-3-乙酸（HIAA）、3-羟基邻氨基苯甲酸（3-HAA）、吡啶酸（PA）和喹啉酸（QA）的分离。然而，通过增加柱长和同时施加内压和电压，仅在 114 min 内才实现了具有挑战性的所有 9 种 KP 代谢物的同时分离。对商业人血浆中的 KP 代谢物进行定量，并验证了 5 种 KP 代谢物的内源性浓度。实验定量下限范围为 100-10,000 nM（S/N 分别为 8-832），实验检测下限范围为 31-1000 nM（S/N 分别为 2-16）。测量了先前筛选炎症生物标志物[C 反应蛋白（CRP）和肿瘤坏死因子-α（TNF-α）]的患者血浆样品中 5 种主要 KP 代谢物，即 TRP、KYN、KA、AA 和 QA 的水平及其比率。对具有显著正相关的代谢物水平对进行了统计学评估。
