Rigid urea-based structures drive analysis of chiral amino acids

Chiral amino acids (AAs) serve as essential building blocks of proteins and play vital physiological roles in living organisms. To achieve accurate, rapid, and high-throughput analysis of chiral AAs, this work proposed a methylbenzyl isocyanate (MBIC) derivatization strategy coupled with ultra-high performance liquid chromatography-mass spectrometry or trapped ion mobility spectrometry-mass spectrometry. The integration of a chiral carbon atom with a rigid urea-based structure can significantly enhance the separation of chiral MBIC-labeled AA enantiomers. This phenomenon can be attributed to the labeled L-AAs allow the carboxyl group to form intramolecular hydrogen bonds with the amino group in the rigid urea-based structure, whereas labeled D-AAs are unable to form such bonds. The method based on MBIC derivatization coupled with ultra-performance liquid chromatography-tandem mass spectrometry achieved simultaneous separation of 19 pairs of chiral AAs using only a C18 column within 30 min, enabling quantitatively detect twelve types of chiral AAs in the serum of healthy humans and Parkinson's patients. The distribution of twenty-four chiral AAs is observed in mouse brain using MBIC labeling-based matrix-assisted laser desorption/ionization-trapped ion mobility spectrometry-mass spectrometry imaging without prior separation. Our work elucidates the principles governing the separation of chiral AAs using derivatization methods, providing valuable guidance for the separation of chiral compounds.