Observed binding of 99M-technetium radiopharmaceuticals to human serum protein as a function of internal surface reversed phase HPLC column length

Because of the ideal nuclear properties of 99m-Tc, it would be desirable to use 99m-Tc radiopharmaceuticals for diagnostic myocardial perfusion imaging. Cations are known to accumulate in heart tissue; several readily prepared 99m-Tc cationic complexes with phosphorus-containing ligands have been prepared and tested in humans and animals. The animal models showed promising results; however, the tests in humans failed due to slow blood clearance of the radiopharmaceuticals. Slow blood clearance can be related to binding of the radiopharmaceutical to a constituent in human serum. As indicated by size exclusion HPLC analysis, the 99m-Tc radiopharmaceuticals bind to a low molecular weight serum component found only in humans. To evaluate serum binding of a variety of technetium complexes, a more rapid method of analysis was developed using Internal Surface Reverse Phase (ISRP) HPLC. ISRP HPLC columns allow direct injection of serum. Large proteins are excluded from the internal surfaces and are not retained by the external surfaces; they therefore pass through ISRP columns without retention. The 99m-Tc radiopharmaceuticals diffuse to the internal surfaces and are there retained and separated. Studies conducted with three different phosphorus-containing cationic 99m-Tc complexes in serum using a range of ISRP column length show that observed protein binding decreases with increasing ISRP column length. This is due to a shift in the equilibrium between the serum bound and unbound portions of the radiopharmaceutical during the separation. From these data a relative binding constant governing interaction between the cationic complex and serum protein can bedetermined