Determination of serum thyroxine enantiomers in patients by liquid chromatography with a chiral mobile phase

A chromatographic method for the separation and determination of d- and l-thyroxine enantiomers (d-, and l-T4) in human serum with a chiral ligand ion-exchange system using a chiral mobile phase additive and a silica column was established. An aqueous eluent containing l-proline (l-pro) sufficiently complexed copper II ions and triethylamine (TEA) was used. It was monitored with a UV detector. The separation was completed in 12 min. The method has acceptable sensitivity, precision and accuracy for analysis. The limit of detection and the limit of quantitation for both d- and l-T4 were 0.1 μg/ml and 0.8 μg/ml, respectively. Calibration curves were linear within 1–100 μg/ml; the mean correlation coefficients were rd-T4=0.9986 for d-T4 and rl-T4=0.9978 for l-T4. T4 enantiomers were separated on baseline under the optimum condition. l-T4 eluted before d-T4. The concentration of d-T4 and l-T4 in 45 thyroid patients serum (hyperthyroid, hypothyroid, thyroidectomy, goitre or thyroiditis) using HPLC was determined, those results showed that d,l-T4 concentration varied in different thyroid patient. Attention should be paid to this result in treating thyroid disease in the clinic.