Accelerating the drug optimization process: identification, structure elucidation, and quantification of in vivo...

Most preclinical leads exhibit poor ADME/PK properties and require optimizing to increase the likelihood of becoming successful pharmaceuticals. As a means of accelerating the evaluation of these leads in vivo, we assessed the use of LC/MS with the chemiluminescent-nitrogen detector (CLND) and a stable isotope to identify and quantify in vivo metabolites and to measure excretion. A 14C-labeled preclinical lead that also contained two chlorine atoms was administered orally to rats, and samples of bile, urine, and plasma were collected and analyzed by LC with radiodetection and by LC/MS-CLND with the chlorine atoms used as tracers. Both methods identified seven metabolites in bile and two metabolites in urine. The amount and abundance of each metabolite was measured, and the results were equivalent for the two methods. Material balance was measured by liquid scintillation counting of the starting samples, by LC/radiodetection, and by LC/MS-CLND. All three methods yielded the same results and showed that the primary route of clearance was metabolism followed by immediate excretion. This study demonstrates that LC/MS-CLND with a stable isotope is a method that can efficiently track and accurately quantify metabolites, making it possible to rapidly study ADME/PK in vivo without radiolabeling.