Chiral Derivatization Enables High-Resolution Ion Mobility Spectrometry of 30 Amino Acid Enantiomers

Chiral analysis of amino acid enantiomers is essential due to their frequently divergent biological activities. However, ion mobility spectrometry-mass spectrometry (IMS-MS) cannot directly resolve underivatized amino acid enantiomers due to their identical physicochemical properties. This study developed a derivatization-based analytical method utilizing (S)-N-(4-nitrophenoxycarbonyl) phenylalanine methoxyethyl ester ((S)-NIFE) as a chiral derivatization agent to achieve the stereoselective modification of amino acids. The resulting diastereomers form metal ion adducts, which enhance collision cross section differences and enable separation via trapped ion mobility spectrometry and time-of-flight mass spectrometry (TIMS-MS). The differential impact of alkali metal ion adduction (Na⁺, K⁺, Rb⁺, and Cs⁺) on chiral separation was investigated, revealing that Na⁺ adducts provide optimal enantioselective recognition (average resolution, R_pp = 2.02). The established method achieved separation for 30 chiral amino acids, demonstrating faster speed, broader coverage, and superior resolution compared with existing approaches. When applied to rat blood samples from chlorfenapyr-induced toxicity models, the method successfully detected d-Arg, d-Ile, d-Leu, d-Phe, and d-Met, confirming its practical utility in toxicological diagnostics and biomarker discovery.