Separation of amino acid and peptide stereoisomers by nonionic micelle-mediated capillary electrophoresis after chiral derivatization

Abstract

Enantiomers of amino acids and peptides were derivatized with a fluorescent chiral reagent, 4-(3-isothiocyanatopyrrolidin-1-yl)-7-nitro-2,1,3-benzoxadiazole [ R-(−)- or S-(+)-NBD-PyNCS] and the resulting diastereomeric derivatives separated by capillary electrophoresis (CE). The CE running buffer consisted of 25 m M acetate buffer (pH 4) and 10 m M of the nonionic surfactant Triton X-100. The excitation maximum of NBD-PyNCS at 480 nm matches the major Ar-ion emission line at 488 nm allowing sensitive laser-induced fluorescence detection with limits of detection around 50 n M. d-Proline and d-aspartate spiked (at 10 −4 M and 10 −5 M concentrations, respectively) into complex biological matrices (rabbit serum and homogenate of Aplysia californica buccal ganglion) are detected without matrix interferences. This method has also been applied to the determination of d- and l-amino acid residues in peptides after acid hydrolysis. Results from the chiral analysis of the naturally-occurring peptide, gramicidin D, are shown.