Resolution of thyroxine hormone enantiomers by precolumn derivatization with a fluorescent chiral reagent using reversed-phase high performance liquid chromatography

A highly fluorescent chiral tagging reagent, R(-)-4-(N,N-dimethylaminosulfonyl)-7- (3-isothiocy-anatopyrrolidino)-2, 1, 3-benzoxadiazole (R(-)-DBD-PyNCS), was employed for the enantiomer separation of thyroxine hormone D L-3,5, 3',5'-tetraiodothyronine (T-4) and L-3,5,3'-triiodothyronine (T-3). The reaction of R(-)-DBD-PyNCS with the thyroxine enantiomers was carried out at 40 degrees C for 20 min under a basic medium surrounding to yield the corresponding pair of diastereomers. No racemization occurs during the tagging reaction under the optimized conditions. Various experimental parameters for the derivatization reaction including the concentration of tagging reagent, reaction temperature and reaction time have been studied in order to get the highest yield of T-4/T-3 derivatives. The structures of T-4/T-3 derivatives were identified based on high performance liquid chromatography-mass spectrometry (HPLC-MS) measurement in negative mode. The efficient separation of derivatives have been achieved by isocratic elution with a water-acetonitrile mobile phase containing 1% acetic acid in a reversed phase column utilizing a conventional fluorescence detector. The calibration curves of L-T-3, D,L-T-4 were linear over the concentration ranges of 0.006 7-0.22 mu g/mu L and 0.016-0.30 mu g/mu L, respectively. The limits of detection (S/N = 3) for L-T-3 and D,L-T-4 were 0. 85 mu g/mL and 0.02 mu g/mL, respectively. The proposed method has been successfully applied to the determination of T-3 and T-4 in clinical pharmaceutics.