Fluorescent chiral derivatization reagents for carboxylic acid enantiomers in high-performance liquid chromatography

Abstract

Six chiral derivatization reagents, viz., (+)-4-(N,N-dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (D-DBD-APy), (–)-4-(N,N-dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (L-DBD-APy), (+)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (D-NBD-APy), (–)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (L-NBD-APy), (+)-4-(aminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (D-ABD-APy) and (–)-4-(aminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (L-ABD-APy), have been synthesized to permit separation of carboxylic acid enantiomers by high-performance liquid chromatography (HPLC). The reagents react with carboxylic acids at room temperature in the presence of activation agents (2,2′-dipyridyl disulfide and triphenylphosphine). The maximum emission wavelengths of the diastereomeric amide derivatives formed from D-naproxen (Nap) with D-DBD-APy, D-ABD-APy and D-NBD-APy were approximately 580 nm (excitation at 470 nm), 585 nm (excitation at 470 nm) and 540 nm (excitation at 470 nm), respectively. The emission wavelengths of the derivatives shifted towards the blue, and the fluorescence intensities increased with increasing acetonitrile concentration in the medium. The fluorescence intensities of three derivatives at pH 2–11 were higher in neutral than in acidic and alkaline solutions. The diastereomers derived from anti-inflammatory drugs and N-acetylamino acids were efficiently resolved by a reversed-phase column (5 µ Inertsil ODS-2) with a water-acetonitrile mixture as mobile phase. The detection limits (S/N = 2) of DBD-APy-Nap, NBD-APy-Nap and ABD-APy-Nap on HPLC chromatograms were 10, 15 and 30 fmol, respectively.