Rapid solid-phase extraction method to quantify [11C]-verapamil, and its [11C]-metabolites, in human and macaque plasma

Abstract

P-glycoprotein (P-gp), an efflux transporter, is a significant barrier to drug entry into the brain and the fetus. The positron emission tomography (PET) ligand, [(11)C]-verapamil, has been used to measure in vivo P-gp activity at various tissue-blood barriers of humans and animals. Since verapamil is extensively metabolized in vivo, it is important to quantify the extent of verapamil metabolism in order to interpret such P-gp activity. Therefore, we developed a rapid solid-phase extraction (SPE) method to separate, and then quantify, verapamil and its radiolabeled metabolites in plasma. Using high-performance liquid chromatography (HPLC), we established that the major identifiable circulating radioactive metabolite of [(11)C]-verapamil in plasma of humans and the nonhuman primate, Macaca nemestrina, was [(11)C]-D-617/717. Using sequential and differential pH elution on C(8) SPE cartridges, we developed a rapid method to separate [(11)C]-verapamil and [(11)C]-D-617/717. Recovery was measured by spiking the samples with the corresponding nonradioactive compounds and assaying these compounds by HPLC. Verapamil and D-617/717 recovery with the SPE method was >85%. When the method was applied to PET studies in humans and nonhuman primates, significant plasma concentration of D-617/717 and unknown polar metabolite(s) were observed. The SPE and the HPLC methods were not significantly different in the quantification of verapamil and D-617/717. The SPE method simultaneously processes multiple samples in less than 5 min. Given the short half-life of [(11)C], this method provides a valuable tool to rapidly determine the concentration of [(11)C]-verapamil and its [(11)C]-metabolites in human and nonhuman primate plasma.