Improved chromatographic methods for the separation of thyroid hormones and their metabolites.

In order to analyze iodoamino acids, two elution systems were established for cation-exchange column chromatography on AG50W-X4. A rapid method suitable for the separation of iodide, monoiodotyrosine, diiodotyrosine, 3,3′,5-triiodothyronine, and thyroxine, permits those compounds to be analyzed within 1.5 h. In this system, the volume of the starting solution (0.04 m ammonium acetate, pH 4.7) is kept constant throughout chromatography by adding 0.04 m Tris(hydroxymethyl)aminomethane, whereby a convex gradient of Tris is obtained. Both solutions contain 30% ethanol. Another system is suitable for the analysis of metabolites of thyroxine as well as for iodotyrosines and is based on the use of simultaneous NH₄OH (0 → 0.7 m) and ethanol (30 → 0%) linear gradients. Furthermore, 1-butanol saturated with 0.2 m NH₄OH was found useful for the separation of iodothyronine sulfates by thin-layer chromatography. The R_f values of various iodothyronines, iodotyrosines, and sulfates in thin-layer chromatography on silica gel were measured in several solvent systems and certain correlations between iodothyronine structure and R_f values were found.