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

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.