Development of a validated HPLC method for the determination of iodotyrosines and iodothyronines in pharmaceuticals and biological samples using solid phase extraction

Identification, separation and quantitation of iodoaminoacids, is essential for the biological research and the clinical diagnosis of thyroid gland disease. Under this aspect a reversed-phase high-performance liquid chromatographic method was developed for the determination of thyroid gland hormones and some of their primary metabolites, 3,3′,5,5′-tetra-iodo-l-thyronine (l-thyroxine), 3,3′,5-tri-iodo-l-thyronine, 3,5-di-iodo-l-thyronine, l-thyronine, 3,5-di-iodo-l-tyrosine, 3-iodo-l-tyrosine and l-tyrosine. Analysis was performed on an Inertsil C18 column with photodiode-array detection, using a 25 min gradient scale program of a binary mobile phase consisted of 0.1% aqueous solution of trifluoroacetic acid at pH 3 as solvent A and acetonitrile as solvent B, at a flow rate of 1 mL/min. Quantitation was performed using were obtained using theophylline as internal standard. The method was applied to commercial pharmaceuticals and biological samples (serum, urine and tissue). Drug-free urine and serum samples were spiked with known concentrations of the analytes standards and pretreated by solid phase extraction to remove matrix interferences. C18 cartridges were used, yielding recoveries ranging from 87.1% to 107.6% for serum samples and from 92.1% to 98.7% for urine samples. With regard to total-T4 concentrations in serum samples, results are cross-validated with RIA and found to agree well.