IDENTIFICATION AND QUANTITATION OF IODOTYROSINES AND IODOTHYRONINES IN PROTEINS USING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY BY PHOTODIODE-ARRAY ULTRAVIOLET-VISIBLE DETECTION

We describe a new method for the separation, identification and quanti tation of iodotyrosines and iodothyronines [3-monoiodo-L-tyrosine (MIT ), 3,5-diiodo-L-tyrosine (DIT), L-thyronine (T-0), 3,5-diiodo-L-thyron ine (T-2), 3,5,3'-triiodo-L-thyronine (T-3), reverse 3,3',5'-triiodo-L -thyronine (rT(3)) and 3,3',5,5'-tetraiodo-L-thyronine (T-4)]. Reverse d-phase highperformance liquid chromatography (RP-HPLC) was performed on a Nucleosil C-8 column with photodiode-array UV-Vis detection. A cl early defined elution profile was obtained of each iodoamino acid (iod otyrosines and iodothyronines) using a linear gradient from 20 to 80% phase B (90% acetonitrile, 10% water, 0.1% TFA)I phase A (water, 0.1% TFA, pH 2.0) eluted over 40 min. Iodoamino acid composition was determ ined, taking into account retention times and spectral characteristics . Thyroid protein samples were digested enzymatically and the complex mixture of IAA was then injected onto the RP-HPLC system. A photodiode -array detector with a dynamic range in the UV-Vis region was used in the HPLC system to monitor the absorbance at different wavelengths con tinuously, collecting data which were compared with standard samples. Each IAA was quantitated using linear calibration curves obtained at 2 80 nm. This method allowed identification and quantitation of iodoamin o acids from diverse sources in the range 2-500 ng, avoiding the need to radiolabel samples. The technique was tested with in vitro iodinate d and non-iodinated human thyroglobulin and the recoveries ranged from 84 to 91%.