Overcoming interference with the detection of a stable isotopically labeled microtracer in the evaluation of beclabuvir absolute bioavailability using a concomitant microtracer approach

The oral absolute bioavailability of beclabuvir in healthy subjects was determined using a microdose (100 μg) of the stable isotopically labeled tracer via intravenous (IV) infusion started after oral dosing of beclabuvir (150 mg). To simultaneously analyze the concentrations of the IV microtracer ([13C6]beclabuvir) and beclabuvir in plasma samples, a liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) method was initially developed. Surprisingly beclabuvir significantly interfered with the IV microtracer detection when using the selected reaction monitoring (SRM) in the assay. An interfering component from the drug substance was observed using a high resolution mass spectrometer (HRMS). The mass-to-charge (m/z) of the interfering component was −32 ppm different from the nominal value for the IV microtracer and thus could not be differentiated in the SRM assay by the unit mass resolution. To overcome this interference, we evaluated two approaches by either monitoring an alternative product ion using the SRM assay or isolating the interfering component using the parallel reaction monitoring (PRM) assay on the HRMS. This case study has demonstrated two practical approaches for overcoming interferences with the detection of stable isotopically labeled IV microtracers in the evaluation of absolute bioavailability, which provides users the flexibility in using either LC-MS/MS or HRMS to mitigate unpredicted interferences in the assay to support microtracer absolute bioavailability studies.