The separation of 99mTc(Sn)EHDP complexes by HPLC and GPC

For the characterization of the multi-component bone-scan agent 99mTc(Sn)EHDP we have analysed the complex mixture with reversed phase ion pair chromatography (IPC) and soft gel permeation chromatography (GPC). With IPC five major complexes were found within a separation-time of 40 min. To avoid decomposition of the complexes during separation, the concentrations of EHDP and the reductant Sn(II) in the eluent had to be identical to the EHDP and Sn(II) concentrations used for the preparation of the complexes. To investigate the stability of the complexes we applied separation by IPC, followed by re-analysis of the fractions within 1 h and after 21-25 h. It appears that there is a state of equilibrium between the five complexes. Within 1 h after isolation the separated complexes were still more than 90% in their original form while after 20 h considerable amounts of the other complexes are found. The two complexes with the largest retention time with IPC are the most stable ones. When the total mixture was re-analysed after 26 h all five components appeared to be still present, but the relative amount of the most stable component had increased. Using GPC for the separation of the complex mixture, we found four major peaks within a separation time of 14 h. The elution orders of the complexes with the two separation methods are opposite.