Development of Ultra High-Performance Liquid Chromatography–Tandem Mass Spectrometry Method for Enantiomer Resolution of Thyroxine on a Chiral Crown Ether Derived Chiral Stationary Phase

Chiral discrimination of thyroxine (T 4 ) enantiomers was performed using ultra high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) on a chiral crown ether-derived ChiroSil SCA (-) column. The different composition of mobile phases and the effect of column oven temperatures were investigated and the optimum chromatographic separation with respect to resolution and analysis time was achieved using a mixture of 60% methanol/water (v/v) with 0.1% formic acid at 40 °C having a flow rate of 1.4 mL min −1 . The thermodynamic data from van’t Hoff plots of temperature experiments revealed that the enantioseparation was enthalpically favored process. The method was validated in the concentration range of 0.5–100 μg mL −1 for both enantiomers and proved to be rapid, precise, sensitive, and selective method for the enantiodiscrimination of T 4 under the optimized conditions. The calibration curves of both D- and L-T 4 showed an excellent linearity with coefficient of determination ( R 2 ) > 0.9997. The developed chiral method was successfully applied for a quantitative assay to check the enantiomeric purities of the six pharmaceutical formulations of levothyroxine sodium tablets and the enantiomeric impurities identified were in the range of 0.11–0.29%. This method could be applied for the determination of enantiomeric purity on pharmaceuticals and also for the monitoring of thyroid hormone levels. Graphical Abstract