A Validated Liquid Chromatography–Tandem Mass Spectrometry Assay for the Determination of NX‐5948 in Beagle Dog Plasma: Application to a Pharmacokinetic Study

ABSTRACT Proteolysis targeting chimera (PROTAC) has been developed and currently enjoys widespread interest among the field of pharmaceutical manufacturing in recent decades. NX‐5948 is an orally active PROTAC Bruton tyrosine kinase degrader, which shows anti‐inflammatory and antitumor activities. In this study, a simple and fast bioanalytical method for the quantification of NX‐5948 in beagle dog plasma was developed using liquid chromatography–tandem mass spectrometry (LC‐MS/MS). A full method validation was performed according to regulatory guidelines. For the quantification, [M + H] + was formed using an electrospray ionization (ESI) source in the positive ion mode, and selective reaction monitoring (SRM) was employed using a triple quadrupole mass spectrometry. The monitored precursor‐to‐product transitions were m/z 807.5 > 790.5 for NX‐5948 and m/z 812.4 > 452.1 for internal standard. A linear response was obtained at the concentration range of 0.5–200 ng/mL, with correlation coefficient > 0.99. The interday accuracy ranged from −9.03% to 5.99% (RSD < 7.84%), and the intraday accuracy from −5.15% to 8.56% (RSD < 6.90%). NX‐5948 was demonstrated to be stable under the present storage conditions. After validation, the method was successfully applied for the quantification of NX‐5948 in beagle dog plasma after oral and intravenous administration of the NX‐5948.