Chiral capillary electrophoresis with UV-excited fluorescence detection for the enantioselective analysis of 9-fluorenylmethoxycarbonyl-derivatized amino acids

Abstract

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.

Recent (2018–2020) development in capillary electrophoresis

“…CE with UV-excited fluorescence detection for sensitive chiral analysis of amino acids (AAs) was studied by Prior et al [80], employing optical fibers to direct the UV light from a Xe-Hg lamp and the emitted fluorescence to a chargecoupled device (CCD). LODs for enantioseparation of D -AA were in the 10-100-nM range.…”

Past, present, and future developments in enantioselective analysis using capillary electromigration techniques

“…The majority of chiral EKC studies reported which separated derivatized enantiomers have combined the derivatization with MEKC analysis, with the aid of an additional chiral selector (i.e., CD), using both chiral [174,182,183] as well as achiral [184][185][186][187][188][189][190][191][192] derivatizing reagents. In an early study, Mechref et al derivatized herbicide compounds with fluorescent tags, followed by CE with CDs and added micellar phases [185].…”

“…In recent years, one of the most commonly used derivatizing reagents has been FMOC, a popular choice as it yields stable derivatives from rapid reaction with amino compounds. Chiral separations employing FMOC derivatization typically use β-CDs in combination with a surfactant, and have been applied extensively to separate amino acid mixtures [190,192]. Compatibility of derivatization with technological advances, for example, MEKC separations with plastic microchips [178] and integrated sample pretreatment with in-capillary derivatization for high-throughput screening [177], was also demonstrated.…”

A Novel Determination Method of Thirty-Seven<i> o</i>-Phthalaldehyde-Derivatized D/L-Amino Acids with Complementary Use of Two Chiral Thiols by High Performance Liquid Chromatography

“…In general, various methods such as high performance liquid chromatography (HPLC) [4][5][6][7][8][9], gas chromatography (GC) [10][11][12], and capillary electrophoresis (CE) [13][14][15] are used to separate D/L-amino acids. Currently, LC/MS [16][17][18][19] and multi-dimensional LC having two or more dimensions [20][21][22][23][24][25] are used for this purpose because it is difficult to separate the proteinogenic D/L-amino acids under a single separation mode.…”