The simultaneous quantitative estimation of tryptophan (TRP) and its metabolites represents a great challenge because of their diverse chemical properties, e.g., presence of acidic, basic, and nonpolar functional groups and their immensely different concentrations in biological matrices. A short ultra high-performance liquid chromatography (UHPLC)-tandem mass spectrometry (MS/MS) method was validated for targeted analysis of TRP and its 11 most important metabolites derived via both kynurenine (KYN) and serotonin (SERO) pathways in human serum and cerebrospinal fluid (CSF): SERO, KYN, 3-hydroxyanthranilic acid, 5-hydroxyindoleacetic acid, anthranilic acid, kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), xanthurenic acid, melatonin, picolinic acid (PICA), and quinolinic acid (QUIN). After selecting the "best" reversed-phase column and organic modifier, DryLab®4 was used to optimize the gradient time and temperature in chromatographic separation. To achieve absolute quantification, deuterium-labeled internal standards were used. Among all compounds, 3 were analyzed in derivatized (butyl ester) forms (3-HK, PICA, and QUIN) and the remaining 9 in underivatized forms. Validation was performed in accordance with the ICH and FDA guidelines to determine the intraday and interday precision, accuracy, sensitivity, and recovery. To demonstrate the applicability of the developed UHPLC-MS/MS method, the aforementioned metabolites were analyzed in serum and CSF samples from patients with multiple sclerosis (multiple sclerosis group) and those with symptomatic or noninflammatory neurological diseases (control group). The concentration of QUIN dramatically increased, whereas that of KYNA slightly decreased in the multiple sclerosis group, resulting in a significantly increased QUIN/KYNA ratio and significantly decreased PICA/QUIN ratio.

中文翻译：

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一种经验证的色氨酸代谢物的UHPLC-MS方法：在多发性硬化症的诊断中的应用。

色氨酸（TRP）及其代谢物的同时定量估计代表着巨大的挑战，因为它们具有多种多样的化学特性，例如酸性，碱性和非极性官能团的存在以及它们在生物基质中的浓度极为不同。经过验证的短超高效液相色谱（UHPLC）-串联质谱（MS / MS）方法可用于人血清中通过尿嘧啶（KYN）和血清素（SERO）途径衍生的TRP及其11种最重要的代谢物的靶向分析和脑脊液（CSF）：SERO，KYN，3-羟基邻氨基苯甲酸，5-羟基吲哚乙酸，邻氨基苯甲酸，运动尿酸（KYNA），3-羟基犬尿氨酸（3-HK），黄嘌呤酸，褪黑激素，吡啶甲酸（PICA），和喹啉酸（QUIN）。选择“最佳”之后 反相色谱柱和有机改性剂DryLab®4用于优化色谱分离中的梯度时间和温度。为了实现绝对定量，使用了氘标记的内标。在所有化合物中，分析了3种衍生化的（丁酯）形式（3-HK，PICA和QUIN），其余9种衍生化了。根据ICH和FDA指南进行验证，以确定日间和日间精度，准确性，敏感性和恢复性。为了证明开发的UHPLC-MS / MS方法的适用性，对多发性硬化症患者（多发性硬化症组）和有症状或非炎性神经系统疾病患者（对照组）的血清和CSF样品中的上述代谢物进行了分析。QUIN的浓度急剧增加，