Determination of thyroxine enantiomers in pharmaceutical formulation by high-performance liquid chromatography–mass spectrometry with precolumn derivatization

A sensitive and specific liquid chromatography–electrospray ionization–mass spectrometry (LC–ESI–MS) method for the separation and analysis of d- and l-thyroxine was developed using R(−)/S(+)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole, [R(−)/S(+)-DBD-PyNCS] as a chiral derivatization reagents. The T4 derivatives with R(−)-DBD-PyNCS were efficiently separated on a reversed-phase column with water-acetonitrile containing 0.1% formic acid (41:59, v/v) as the eluent and analyzed using ESI–MS with negative selected ion monitoring (SIM) mode. The calibration curves of both the d-T4 and l-T4 were linear over the concentration range of 0.13–13 μg/ml. The detection limits (S/N = 3) were 28 ng/ml for d-T4 and 40 ng/ml for l-T4, respectively. The relative standard deviations (RSD, n = 5) were less than 3.6% at 1.3 μg/ml for both T4 enantiomers. The proposed method was applied to the determination of l-T4 enantiomer in a pharmaceutical formulation.