Simultaneous determination of unbound thyroid hormones in human plasma using high performance frontal analysis with electrochemical detection

A direct injection HPLC method in combination with high-performance frontal analysis (HPFA) and electrochemical detection (ECD) was developed for the simultaneous and sensitive determination of unbound thyroid hormones (thyroxine, triiodothyronine, and reverse triiodothyronine) in human plasma. The present on-line HPLC/HPFA system consists of an HPFA column, an extraction column and an analytical HPLC column connected through a column-switching device, and the eluent from the analytical column was monitored by ECD. The calibration lines showed good linearity (rsq. > 0.999) within 7.4-148.2 pM for T4 and 1.5-74.1 pM for T3 and rT3. Unbound T4 and T3 concentrations determined by the present system were 16.4 ± 2.4 pM (n = 15) and 7.14 ± 1.04 pM (n = 15), which were in agreement with those determined by the EIA method. The unbound rT3 concentration was 2.30 ± 0.27 pM (n = 15). The CV% values of intra-day and inter-day assays (n = 15) were less than 14.9% for T4, 14.5% for T3 and 13.2% for rT3. The present system was also applied to a competitive binding study of these thyroid hormones in human plasma.