The potential of capillary electrophoresis (CE) with ultraviolet (UV)-excited fluorescence detection for sensitive chiral analysis of amino acids (AAs) was investigated. dl-AAs were derivatized with 9-fluorenylmethoxycarbonyl chloride (FMOC)-Cl to allow their fluorescence detection and enhance enantioseparation. Fluorescence detection was achieved employing optical fibers, leading UV excitation light (< 300 nm) from a Xe-Hg lamp to the capillary window, and fluorescence emission to a spectrograph equipped with a charge-coupled device (CCD). Signal averaging over time and emission wavelength intervals was carried out to improve the signal-to-noise ratio of the FMOC-AAs. A background electrolyte (BGE) of 40 mM sodium tetraborate (pH 9.5), containing 15% isopropanol (v/v), 30 mM sodium dodecyl sulfate (SDS), and 30 mM β-cyclodextrin (β-CD), was found optimal for AA chemo- and enantioseparation. Enantioresolutions of 1.0 or higher were achieved for 16 proteinogenic dl-AAs. Limits of detection (LODs) were in the 10–100-nM range (injected concentration) for the d-AA enantiomers, except for FMOC-d-tryptophan (536 nM) which showed intramolecular fluorescence quenching. Linearity (R² > 0.997) and repeatability for peak height (relative standard deviations (RSDs) < 7.0%; n = 5) and electrophoretic mobility (RSDs < 0.6%; n = 5) of individual AA enantiomers were established for chiral analysis of dl-AA mixtures. The applicability of the method was investigated by the analysis of cerebrospinal fluid (CSF). Next to l-AAs, endogenous levels of d-glutamine and d-aspartic acid could be measured in CSF revealing enantiomeric ratios of 0.35 and 19.6%, respectively. This indicates the method’s potential for the analysis of low concentrations of d-AAs in presence of abundant l-AAs.

中文翻译：

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手性毛细管电泳和紫外激发荧光检测，对9-芴基甲氧基羰基衍生的氨基酸进行对映选择性分析

研究了毛细管电泳（CE）和紫外（UV）激发荧光检测技术对氨基酸（AAs）的敏感手性分析的潜力。dl -AAs用9-芴基甲氧基羰基氯（FMOC）-Cl衍生化，以进行荧光检测并增强对映体分离。使用光纤，将来自Xe-Hg灯的UV激发光（<300 nm）引导到毛细管窗口以及将荧光发射到配备有电荷耦合器件（CCD）的光谱仪来实现荧光检测。对时间和发射波长间隔进行信号平均以提高FMOC-AA的信噪比。40 mM四硼酸钠（pH 9.5）的背景电解质（BGE），含15％异丙醇（v / v），30 mM十二烷基硫酸钠（SDS）和30 mMβ-环糊精（β-CD）被发现最适合AA化学和对映体分离。16种蛋白质生成的dl -AAs的对映体分辨率达到1.0或更高。d -AA对映异构体的检出限（LOD）在10-100nM范围内（注入浓度），但FMOC- d-色氨酸（536 nM）表现出分子内荧光猝灭。 建立了各个AA对映异构体的线性（R ²  > 0.997）和峰高重复性（相对标准偏差（RSDs <7.0％; n  = 5）和电泳迁移率（RSDs <0.6％; n = 5）用于手性分析dl-AA混合物。通过脑脊液（CSF）的分析研究了该方法的适用性。除了1- AAs，可以在CSF中测量内源性的d-谷氨酰胺和d-天冬氨酸的水平，其对映体比率分别为0.35和19.6％。这表明该方法在存在大量1 - AA的情况下分析低浓度d- AA的潜力。