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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Unadkat
Jashvant D
JD
Department of Pharmaceutics, University of Washington, Box 357610, Seattle, WA 98195, USA. jash@u.washington.edu
Chung
Francisco
F
Sasongko
Lucy
L
Whittington
Dale
D
Eyal
Sara
S
Mankoff
David
D
Collier
Ann C
AC
Muzi
Mark
M
Link
Jeanne
J
D000818:Animals; D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid; D005260:Female; D006801:Humans; D008254:Macaca nemestrina; D049268:Positron-Emission Tomography; D052616:Solid Phase Extraction / Q000379:methods*; D014700:Verapamil / Q000097:blood* / Q000378:metabolism
D002250:Carbon Radioisotopes; D014700:Verapamil