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

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 (α = 1.90), a protein ovomucoid derived CSP (α = 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) (α = 2.08-3.11), depending upon the used mobile phases. For resolution of thyroxine enantiomers, the crown ether derived CSP showed better enantioseparation 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 Dand Lthyroxine 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. For the optimum chromatographic results of specificity experiment of Dand L-T4 in plasma samples, several mobile phase conditions were used. As shown in Table 1, chromatographic parameters such as separation factors, retention times and resolution factors on CSP 1 are considerably influenced by the nature of mobile phases. Although Aboul-Enein and his co-works have shown the chromatographic results using the mobile phases of methanol/H2O containing H2SO4 as an additive, these chromatographic conditions were not effective for the specificity results of Dand L-T4 present in plasma samples. Therefore, the several mobile phases of ethanol/H2O containing H2SO4 as an additive were used and optimized in this study. When the mobile phase of 90% ethanol/H2O (V/V) containing 10 mM H2SO4 was used for the validation study of Dand L-T4 in plasma samples, no matrix peaks interfered with internal standard (IS) of L-phenylglycine, L-T4 and D-T4 at their retention times. Figure 1 shows representative chromato-