Department of Pharmaceutical Analysis and Nuclear Pharmacy, Faculty of Pharmacy, Comenius University in Bratislava, Odbojárov 10, SK-832 32 Bratislava, Slovak.
Toxicological and Antidoping center, Faculty of Pharmacy, Comenius University in Bratislava, Odbojárov 10, SK-832 32 Bratislava, Slovak.
Molecules. 2017 Oct 7;22(10):1668. doi: 10.3390/molecules22101668.
An advanced two-dimensional capillary electrophoresis method, based on on-line combination of capillary isotachophoresis and capillary zone electrophoresis with cyclodextrin additive in background electrolyte, was developed for effective determination of serotonin in human urine. Hydrodynamically closed separation system and large bore capillaries (300-800 µm) were chosen for the possibility to enhance the sample load capacity, and, by that, to decrease limit of detection. Isotachophoresis served for the sample preseparation, defined elimination of sample matrix constituents (sample clean up), and preconcentration of the analyte. Cyclodextrin separation environment enhanced separation selectivity of capillary zone electrophoresis. In this way, serotonin could be successfully separated from the rest of the sample matrix constituents migrating in capillary zone electrophoresis step so that human urine could be directly (i.e., without any external sample preparation) injected into the analyzer. The proposed method was successfully validated, showing favorable parameters of sensitivity (limit of detection for serotonin was 2.32 ng·mL), linearity (regression coefficient higher than 0.99), precision (repeatability of the migration time and peak area were in the range of 0.02-1.17% and 5.25-7.88%, respectively), and recovery (ranging in the interval of 90.0-93.6%). The developed method was applied for the assay of the human urine samples obtained from healthy volunteers. The determined concentrations of serotonin in such samples were in the range of 12.4-491.2 ng·mL that was in good agreement with literature data. This advanced method represents a highly effective, reliable, and low-cost alternative for the routine determination of serotonin as a biomarker in human urine.
建立了一种基于在线组合毛细管等速电泳和胶束电动毛细管色谱，并在背景电解质中添加环糊精的二维毛细管电泳方法，用于有效测定人尿中的 5-羟色胺。选择水力封闭分离系统和大口径毛细管（300-800μm），以提高样品负载能力，并降低检测限。等速电泳用于样品预分离、定义消除样品基质成分（样品净化）和分析物的预浓缩。环糊精分离环境增强了毛细管区带电泳的分离选择性。这样，5-羟色胺可以与人尿中其他在毛细管区带电泳步骤中迁移的基质成分成功分离，从而可以直接（即无需任何外部样品制备）将人尿注入分析仪。该方法已成功验证，具有良好的灵敏度参数（5-羟色胺的检测限为 2.32ng·mL）、线性（回归系数高于 0.99）、精密度（迁移时间和峰面积的重复性在 0.02-1.17%和 5.25-7.88%之间）和回收率（在 90.0-93.6%之间）。该方法用于测定来自健康志愿者的人尿样品。在这些样品中测定的 5-羟色胺浓度在 12.4-491.2ng·mL 范围内，与文献数据吻合良好。这种先进的方法代表了一种高效、可靠且低成本的替代方法，可用于常规测定人尿中的 5-羟色胺作为生物标志物。
