Simultaneous measurement of acalabrutinib, ibrutinib, and their metabolites in beagle dog plasma by UPLC-MS/MS and its application to a pharmacokinetic study.

In our present experiment, the aim of this paper was to develop and fully validate an accurate and simple ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for simultaneous quantification of acalabrutinib, ibrutinib, and their metabolites (ACP-5862 and PCI-45227) in beagle dog plasma and to survey the pharmacokinetic study of all analytes in beagle dogs. After a quick protein precipitation with acetonitrile, the chromatographic separation of acalabrutinib, ACP-5862, ibrutinib, PCI-45227 and bosutinib (internal standard, IS) were finished on an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 μm) column and their detections were conducted by a Xevo TQ-S triple quadrupole tandem mass spectrometer in the positive ion mode. The assay displayed excellent linearity in the calibration range of 0.5-300 ng/mL for acalabrutinib, 0.5-30 ng/mL for ACP-5862, and 0.5-200 ng/mL for both ibrutinib and PCI-45227, respectively. Notably, the lower limit of quantification (LLOQ) for each compound was determined to be 0.5 ng/mL. The accuracy of all analytes for intra- and inter-day ranged from -4.3% to 14.6%, while precision were ≤9.0%. The recovery of each substance was > 81.0%, and no significant matrix effects were observed. The stabilities of all analytes under different conditions met all requirements for the quantitation in plasma samples. In addition, our developed UPLC-MS/MS method could also be employed to measure the pharmacokinetic profiles of acalabrutinib, ACP-5862, ibrutinib, and PCI-45227 in beagle dog plasma.