Enantiometric separation of di- and tri-peptides with chiral fluorescence labeling reagents by liquid chromatography

Stereoisomers of di- and tri-peptides were separated on C18 columns after their pre-column derivatization with the chiral fluorescence labeling reagent, R(—)-4-(N,N-dimethylaminosulfonyl)-7-(3-isothiocyanatopyrrolidin-1-y1)-2,1,3-benzoxadiazole (R(—)-DBD-PyNCS). The labeling reaction was usually carried out with 1 μg of peptide sample dissolved in water in basic solution (0.1% (vv) triethylamine) at 60 °C for 10 min. No observable racemization occurred during the derivatization. All of the four Stereoisomers of the peptides tested could be resolved with mixtures of water/acetonitrile/methanol containing 0.05% (vv) trifluoroacetic acid (TFA) as the mobile phases. The eluate was monitored by a fluorescence detector with the excitation and emission wavelengths set at 450 nm and 560 nm, respectively. This method is useful for enantiospecific quantitations and quality assurance tests of peptides, particularly in the cases where only minute amounts of samples are available.