Online chiral derivatization coupled with CE-MS for baseline separation of 16 proteinogenic amino acid enantiomers.

Amino acid (AA) enantiomers influence protein function through their distinct spatial structures, making their precise separation essential for elucidating physiological and pathological pathways. In this work, a novel method for the efficient separation of AA enantiomers based on on-line chiral derivatization with (S)-N-(4-nitrophenoxycarbonyl) phenylalanine methoxyethyl ester ((S)-NIFE) coupled with CE-MS has been developed. The method utilizes the ultrafast reaction kinetics of (S)-NIFE with AA enantiomers to facilitate their efficient separation. The online strategy eliminates time-consuming offline derivatization by permitting immediate injection upon simple mixing of derivatization reagent with AA enantiomers, enabling a streamlined analytical workflow. By optimizing experimental parameters, baseline separation of 16 chiral proteinogenic AA enantiomers has been achieved with resolutions ranging from 1.5 to 11.03. The relative standard deviations of migration time and peak area for intraday and interday measurements are less than 5%, and the limits of detection (LOD) and quantification (LOQ) are as low as 40 nM and 80 nM, respectively. Furthermore, the method was further applied to comparative analyses of AA enantiomers in serum samples from healthy controls and patients with non-alcoholic fatty liver disease and atrial fibrillation. The baseline separation of three AA enantiomers was consistently achieved. The LODs and LOQs for three ᴅ-AAs in complex matrix were as low as 180 nM and 600 nM. The intraday and interday precisions were both below 11%, and the average recoveries range from 87.60% to 95.51% with low standard deviations. All these results confirm the effectiveness of the approach in separating AA enantiomers in complex matrices.