Determination of dissociation constants of 99mTechnetium radiopharmaceuticals by capillary electrophoresis

Capillary electrophoresis was applied to investigate pKa values of 99mTc radiotracers used in nuclear medicine. Therefore, the protonation equilibria of the carboxyl groups of 99mTc-mercaptoacetylglycylglycylglycine (99mTc-MAG3) and 99mTc-ethylenecysteine dimer (99mTc-EC) were studied by pH-dependent determination of electrophoretical velocities. 99mTc-ethylenecysteine dimer diethyl ester (99mTc-ECD) was used as a non-protonable standard. The capillary electrophoresis system was equipped with a radioactivity detector. Measurements were performed using a pressure-driven capillary zone electrophoresis which allowed runs even in the low pH range. For the determination of pKa values, the electrophoretical velocities of the analytes were referred to the electrophoretical velocities of tetraphenyle arsonium chloride as a positively charged marker. Calculation of pKa values was accomplished by non-linear curve fitting of both structure-based equilibria equations and sigmoidal decay functions to the experimental data. 99mTc-MAG3 was shown to have a carboxyl group pKa value of 4.22. The value for the carboxyl groups of 99mTc-EC is 2.90 (determined by structure-based equilibria equations), which represents a common value for both carboxyl groups. By the use of sigmoidal functions, similar values were elucidated. As expected, 99mTc-ECD shows no protonation step.