开发并验证了一种使用紫外/荧光检测的液相色谱方法，用于定量测定人尿液中犬尿氨酸途径代谢产物：应用于心力衰竭患者。
Development and validation of a liquid chromatography method using UV/fluorescence detection for the quantitative determination of metabolites of the kynurenine pathway in human urine: Application to patients with heart failure.
机构信息
CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, Rua Central de Gandra, 1317, 4585-116, Gandra PRD, Portugal.
CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, Rua Central de Gandra, 1317, 4585-116, Gandra PRD, Portugal; Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR), Universidade do Porto, Edifício do Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4050-208, Matosinhos, Portugal.
出版信息
J Pharm Biomed Anal. 2021 May 10;198:113997. doi: 10.1016/j.jpba.2021.113997. Epub 2021 Feb 26.
Recent evidence indicates the relevant role of the tryptophan (TRP) metabolites in the pathophysiology of cardiovascular diseases via inflammatory and oxidative stress mechanisms. Therefore, quantification of TRP and its metabolites in biological samples can be a powerful tool to elucidate the disease mechanisms. The aim of this work was to develop and validate a liquid chromatography with ultraviolet (UV) and fluorescence detection (FD) (LCUV/FD) method for the quantification of TRP and its metabolites (L-kynurenine (KYN) and kynurenic acid (KA)) in urine samples from heart failure (HF) patients. Biochemical parameters and inflammatory markers were quantified, and data correlated with urinary concentrations of TRP and its metabolites. Optimized chromatographic conditions were achieved using a Luna® 3 μm PFP(2) analytical column, a mobile phase of 20 mM of ammonium formate in ultra-pure water (with 0.01 % of formic acid), acetonitrile and ethanol (95/2/3, v/v/v), a flow rate of 0.7 mL/min and a column oven temperature set at 25 °C. The method was validated according to the European Medicines Agency (EMA) guidelines and showed to be linear (r >0.99), accurate (82-116%) and precise (%RSD below 15 %). The limits of quantification varied between 50 and 125 ng/mL. The method was applied to the quantification of TRP, KYN and KA in healthy volunteers and male HF patients. The results obtained through this pilot study (small group of patients) showed a relationship between biochemical parameters, inflammatory markers and changes in the concentration of TRP, KYN and KA. The KYN/TRP and KA/KYN ratios were calculated. Results support the hypothesis that KYN/TRP ratio is related with enzymatic activity and that KA/KYN ratio can be a good neuroprotection indicator. The potential of the LCUV/FD method for the monitoring of the selected compounds in cardiac patients was also demonstrated.
最近的证据表明，色氨酸（TRP）代谢物通过炎症和氧化应激机制在心血管疾病的病理生理学中起重要作用。因此，在生物样本中定量测定 TRP 及其代谢物可以成为阐明疾病机制的有力工具。本工作的目的是开发和验证一种用于定量测定心力衰竭（HF）患者尿液中 TRP 及其代谢物（L-犬尿氨酸（KYN）和犬尿喹啉酸（KA））的液相色谱-紫外（UV）和荧光检测（FD）（LCUV/FD）方法。测定了生化参数和炎症标志物，并将数据与 TRP 及其代谢物的尿液浓度相关联。通过使用 Luna® 3 μm PFP（2）分析柱、20 mM 甲酸铵的超纯水（含 0.01%甲酸）、乙腈和乙醇（95/2/3，v/v/v）作为流动相，流速为 0.7 mL/min 和柱温箱设定在 25°C，实现了优化的色谱条件。该方法按照欧洲药品管理局（EMA）的指南进行了验证，结果表明该方法具有良好的线性（r＞0.99）、准确性（82-116%）和精密度（%RSD 低于 15%）。定量下限在 50 至 125 ng/mL 之间变化。该方法应用于健康志愿者和男性 HF 患者中 TRP、KYN 和 KA 的定量。通过这项初步研究（小患者组）获得的结果表明，生化参数、炎症标志物与 TRP、KYN 和 KA 浓度的变化之间存在相关性。计算了 KYN/TRP 和 KA/KYN 比值。结果支持 KYN/TRP 比值与酶活性有关的假设，并且 KA/KYN 比值可以作为神经保护的良好指标。该 LCUV/FD 方法用于监测心脏患者选定化合物的潜力也得到了证明。
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