Enantioselective resolution of thyroxine hormone by high-performance liquid chromatography utilizing a highly fluorescent chiral tagging reagent

A highly fluorescent chiral tagging reagent, 4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole, [R(-)-DBD-PyNCS], was employed to develop an indirect resolution method for efficient separation of thyroxine enantiomers,D-T-4 and L-T-4. The reaction of R(-)-DBD-PyNCS with the thyroxine enantiomers proceeds effectively at 40 degrees C for 20 min in the presence of basic medium to produce the corresponding pair of diastereomers. No racemization occurs during the tagging reaction under the optimized conditions. Various experimental parameters for derivatization reaction including the species of catalyst, the concentration of tagging reagent and reaction temperatures, have been examined to get a highest yield for T-4 derivatives. The structure of T4 derivatives was identified based on ESI-MS/MS measurements in negative mode. The efficient separation of D-, L-T-4 derivatives was achieved by isocratic elution with water-acetonitrile mobile phase containing 1% AcOH on a reversed phase column utilizing a conventional fluorescence detector. The resolution (Rs) value of the diastereomers derived from thyroxine was 5.1. The calibration curves of both the D-T4 and L-T4 were linear over the concentration range of 0.1-20 mu g/ml. The limits of detection (S/N = 3) for both D-T-4 and L-T-4 were 0.2 ng per injection. The proposed method was applied to the determination of D-T-4 and L-T-4 in pharmaceutical formulations and human serum samples.