Chirality determination of unusual amino acids using precolumn derivatization and liquid chromatography–electrospray ionization mass spectrometry

Unusual amino acids such as β-methoxytyrosine (β-MeOTyr), allo-threonine (allo-Thr) and allo-isoleucine (allo-Ile) were derivatized with N-α-(2,4-dinitro-5-fluorophenyl)-l-alaninamide (FDAA), 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl isothiocyanate (GITC), (S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester (S-NIFE), or o-phthalaldehyde/isobutyryl-l-cysteine (OPA-IBLC), and then separated via reversed-phase high-performance chromatography followed by UV and electrospray ionization mass spectrometry detection. FDAA generally showed the highest enantioselectivity but the lowest sensitivity among the chiral derivatizing agents (CDAs) investigated. The detection limit of FDAA-derivatized amino acids was in the low picomolar range. Although the enantioselectivity of FDAA derivatives was generally quite high, its selectivity among β-MeOTyr isomers was poor. The best separation of β-MeOTyr stereoisomers was achieved with S-NIFE. Due to the complex relationships between the investigated CDAs, stereochemical analyses using a combination of two or more of the CDAs gave the most reliable results for a given separation problem. In general, the methods described are selective and reliable, and are being applied to the analysis of unusual amino acids as they occur in marine peptides.