Radiochemical analysis of Tc-99m human serum albumin with high-pressure liquid chromatography: concise communication

High-pressure liquid chromatography (HPLC) can be performed with an aqueous size-exclusion column to separate proteins or other macromolecules on the basis of molecular size. An HPLC system with a Spherogel-TSK SW column was modified to detect simultaneously uv absorption and radioactivity. Characteristic retention times (RT) were determined for pure human serum albumin (HSA) (RT = 17 min) and pertechnetate (RT = 28.5 min). When analysis was performed on Tc-99m HSA preparations, Tc-99m radioactivity was resolved into five different peaks, with RT ranging from 10.2 to 28.5 min. Less than 2% radioactivity was associated with the pertechnetate peak, whereas the remaining Tc-99m was protein bound. Most of the activity (90%) corresponded to the albumin peak, and 7% was bound to contaminants of high molecular weight with RTs of 10.2 and 14 min. Rapid separation of various radiochemical components differing in molecular size provides an improved basis for understanding the biodistribution of a Tc-99m HSA preparation. This technique would be useful for the preparation and analysis of various radiolabeled macromolecules such as enzymes, immunoglobulins, and other proteins.