A Quantitative LC-MS/MS Method for the Determination of Tissue Brincidofovir and Cidofovir Diphosphate in a MuPyV Infected Mouse Model.

Guzman, Bryan B; Schauer, Amanda P; Dunn, John A; Cottrell, Mackenzie L; Sykes, Craig

Brincidofovir (BCV) is an investigational lipid conjugate of the nucleotide analog cidofovir (CDV), which is being developed as a medical countermeasure for the treatment of smallpox. BCV is active against double-stranded DNA viruses including BK and JC virus. Here, we validated procedures for quantifying BCV and its pharmacologically active moiety cidofovir diphosphate (CDV-PP) in mouse kidney, brain, and spleen tissue homogenates. Following homogenization, BCV and CDV-PP were extracted from the tissues by protein precipitation with their stable, isotopically labeled internal standards, BCV-d6 and 13 C3 15 N2 -CDV-PP. Then, samples were analyzed for BCV by reverse phase chromatography on a Waters Xterra MS C18 (50 x 2.1mm, 3.5μm particle size) column while CDV-PP was analyzed on a Thermo BioBasic AX (50 x 2.1 mm, 5μm particle size) column using anion exchange chromatography. Detection was achieved by electrospray ionization in positive ion mode on an AB Sciex API-5000 triple quadrupole mass spectrometer. The calibration curves were linear over a range of 1.00 - 1000 ng/mL homogenate and 0.050 - 50.0 ng/mL homogenate for BCV and CDV-PP, respectively. These methods were validated according to FDA guidance for industry and may be used to characterize the tissue pharmacology of both analytes to advance its preclinical development.