手性 α-环糊精、阳离子-β-环糊精及其协同作用与堆积富集在毛细管电泳中对犬尿氨酸对映体的测定。

Determination of Kynurenine Enantiomers by Alpha-Cyclodextrin, Cationic-βeta-Cyclodextrin and Their Synergy Complemented with Stacking Enrichment in Capillary Electrophoresis.

机构信息

Key Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, 5 South Zhongguancun Street, Beijing, 100081, China.

School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.

出版信息

J Chromatogr A. 2020 Jul 5;1622:461128. doi: 10.1016/j.chroma.2020.461128. Epub 2020 Apr 18.

DOI:10.1016/j.chroma.2020.461128

PMID:32331779

Abstract

We present high resolution fast, cost-effective and sensitive Capillary zone electrophoresis (CZE) methods for determination of enantiomeric compounds of Kynurenine pathway, i.e. D, L-Kynurenine (KYN), in human serum and urine samples by cationic-β-CD and its synergistic dual chiral selector system (SD-CSs) with α-CD in 50 mM borax borate buffer (pH 9.0) as BGE. Acid-mediated stacking enrichment by HCl delivered 15 nM limit of detection (LOD) and 50 nM limit of quantification (LOQ). The methods gave advantages of linearity in the concentration range of 50 nM-1000 nM, reproducibility (RSD ≤ 3.35), selectivity against interfering amino acids, and remarkable recoveries. SD-CSs delivered resolution of D, L-KYN twice that of individual chiral selectors (CSs) under similar conditions. The binding constants (Kb) and electrophoretic mobilities (µeff) of D, L-KYN with different concentrations of CSs were calculated to find the migration order of enantiomers. The chiral recognition mechanism was investigated by molecular docking and molecular mechanics, which revealed strong hydrogen bonding between Kynurenine enantiomers and the SD-CSs as compared to individual CS as the key player in binding, formation of stable complexes which led to the ultimate separation.

摘要

我们提出了一种高分辨率、快速、经济且灵敏的毛细管区带电泳（CZE）方法，用于测定人血清和尿液样品中犬尿氨酸途径的对映异构体化合物，即 D、L-犬尿氨酸（KYN）。该方法采用阳离子-β-CD 及其与 α-CD 的协同双重手性选择体系（SD-CSs）作为 BGE，在 50mM 硼砂硼酸缓冲液（pH9.0）中进行测定。HCl 介导的酸堆积富集可实现 15nM 的检测限（LOD）和 50nM 的定量限（LOQ）。该方法具有线性范围为 50nM-1000nM、重现性（RSD≤3.35%）、对干扰氨基酸的选择性以及显著回收率等优点。在相似条件下，SD-CSs 使 D、L-KYN 的分辨率提高了两倍，优于单个手性选择剂（CS）。通过计算不同浓度 CS 下 D、L-KYN 的结合常数（Kb）和电泳迁移率（µeff），确定了对映体的迁移顺序。通过分子对接和分子力学研究了手性识别机制，结果表明，与单个 CS 相比，犬尿氨酸对映体与 SD-CSs 之间具有更强的氢键相互作用，这是结合的关键因素，形成稳定的配合物，最终导致分离。