RAPID HPLC ANALYSIS OF THYROID GLAND HORMONES TRI-IODOTHYRONINE (T₃) AND THYROXINE (T₄) IN HUMAN BIOLOGICAL FLUIDS AFTER SPE

Abstract

A rapid, accurate, and sensitive method has been developed for the quantitative determination of iodoamino acids, namely thyroxine (3,5,3′,5′-tetra-iodothyronine, (T₄) and 3,5,3′-tri-iodothyronine (T₃). These compounds are essential indicators in the clinical diagnosis of thyroid gland diseases.

An Inertsil ODS-3, 150 × 4.0 mm, 5 μm analytical column was used with a mixture of CH₃OH-H₂O with 2% acetic acid, at a volume ratio 65:35, with a flow rate 1 mL/min. Detection was performed with a variable wavelength UV-visible detector at 240 nm, resulting in detection limits of 1 ng and 2 ng for T₃ and T₄, respectively, per 20 μL injection.

For the quantitative determination, anthraquinone was used as internal standard at a concentration of 1.0 ng/μL. A rectilinear relationship was observed up to 28 and 40 ng/μL for T₃ and T₄, respectively.

Analysis time was approximately 10 min (retention time of internal standard) while the two compounds are eluted within 5 min. The statistical evaluation of the method was examined, performing intra-day (n = 8) and inter-day calibration (n = 8) and was found to be satisfactory with high accuracy and precision results.

The method was applied to the analysis of the iodothyronines in biological fluids, blood serum and urine, after solid phase extraction for sample clean-up and analyte retention, using diol cartridges. Percentage recovery of iodothyronines in spiked samples ranged from 79.90 to 103.15 for T₃ and from 83.65 to 106.15 for T₄, over the range of 0.5–3 ng/μL.

No interferences were observed from endogenous compounds of human serum and urine.

Acknowledgments