[Studies on difference of fluorescence activity and elution order of propranolol enantiomers by reversed-phase high...

A sensitive RP-HPLC method for the determination of propranolol enantiomers was developed. It involves the formation of the monoester of propranolol with optically pure (R,R)-O,O'-diacetyl tartaric acid anhydride (DATAAN) in a dry, aprotic solvent with an excess of trichloroacetic acid. The resulting diastereomeric monoesters were easily resolved on a Shim-pack CLC-ODS column with the mobile phase system consisting of different proportions of 2% aqueous acetic acid (adjusted with concentrated ammonia to pH 3.48) and 90% aqueous methanol. And the column effluent was monitored by a fluorescence detector (lambda(ex) 296 nm, lambda(em) 340 nm). In our experiment, a very strong difference of the fluorescence absorbance of the two diastereomers was observed. After having studied chromatographic separation conditions including pH value, ionic strength and the volume percent of organic modifier in mobile phase, we presented the different configurations of the diastereomers and an intra-molecular hydrogen bond which exists only in the (R,R,S) isomer. These could account for the obvious difference of fluorescence activity of two diastereomeric monoesters and their elution order on the reversed-phase column. Our studies show that the intra-molecular hydrogen bond plays an important role on the polarity of the diastereomeric derivatives which determines their elution order and the fluorescence absorbance of the two diastereomers.