Development of a Validated HPLC Method for the Simultaneous Determination of D- and L-Thyroxine in Human Plasma

摘要

L-Thyroxine (L-T4), the naturally occurring thyroid hormone has been used for the treatment of thyroid dysfunctions, while D-T4 is not active in the thyroid and is used for the treatment of hyperlipidemia. Therefore, a convenient and reliable enantiomer separation method has been of great interest in the biological and pharmacological research field. Several groups reported the chromatographic resolution of thyroxine enantiomers by chiral ligand exchange method using cupper solution on achiral column. However, only a few of results for direct enantiomer separation on chiral stationary phases (CSPs) have been reported. Among these reports, the separation of thyroxine enantiomers has been performed on a proline derived CSP (alpha = 1.90), a protein ovomucoid derived CSP (alpha = 1.16-1.32) under aqueous buffer solutions and on a CSP derived from (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18-C-6-TA) (alpha= 2.08-3.11), depending upon the used mobile phases. For resolution of thyroxine enantiomers, the crown ether derived CSP showed better enantiosepa-ration than the proline or ovomucoid derived CSP. However, the validated direct determination of thyroxine enantiomers in plasma on a CSP derived from 18-C-6-TA (CSP 1) has not been performed. In this study, a liquid chromatographic method for the simultaneous determination of D- and L-thyroxine in human plasma using the chiral crown ether derived CSP 1 is developed and validated. To our knowledge, the validated direct simultaneous determination of thyroxine enantiomers in plasma samples using the chiral HPLC column is the first report.