Methylated Nepsilon-dansyl-L-lysine as a fluorogenic reagent for the chiral separation of carboxylic acids

Dansyl amino acids having a free amino group and an asymmetric carbon atom were examined as a labeling reagent for chiral compounds containing carboxylic moieties to realize enantiomeric separation as well as fluorimetric determination. We tested dansyllysine by reacting it with (+)- and (-)-ibuprofen as a model carboxylic enantiomer. As the intramolecular carboxylic moiety of the dansyl amino acid interfered in the condensation reaction to form an amide bond with the carboxylic acid, the moiety was masked by methylation with trimethylsilyldiazomethane before the reaction. These derivatives were reacted with (+)- and (-)-ibuprofen and better enantiomeric resolution was achieved with methylated dansyllysine on a reversed-phase column. The derivatisation reaction was facilitated by the use of catalysts that are commonly employed in peptides synthesis. The reaction was completed within 5 min at room temperature when diethyl phosphorocyanidate was used. Due to the dansyl moiety, methylated dansyl-lysine enables a sensitive determination of ibuprofen with a fluorescence detector, in addition to the capability of enantiomer resolution. In tests, the detection limits for (+) and (-)-ibuprofen were 4 pmol per injection (S/N=3) at an excitation wavelength of 340 nm and an emission wavelength of 523 nm. Linear responses for the determination of (+) and (-)-ibuprofen in human urine were also demonstrated (r > or = 0.998) in the range from 10 to 1000 ng/ml. The precision and accuracy for urine samples spiked with (+)- and (-)-ibuprofen at 10, 100 and 1000 ng/ml were <10.1 and <14.6% (n=4), respectively.