A rapid and sensitive UPLC-MS/MS method for the determination of zanubrutinib in beagle plasma and its application in pharmacokinetics.

A reliable and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of zanubrutinib in the plasma of beagle dogs. The column used was an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 µm), maintained at 40°C with an injection volume of 2 µl. The gradient elution program was as follows: 0-1 min, 10-10% A; 1-1.1 min, 10-90% A; 1.1-2.1 min, 90-90% A; 2.1-2.2 min, 90-10% A; 2.2-3.0 min, 10-10% A. Mobile phase A was 0.1% formic acid, B was acetonitrile, and the total analysis time was 3 min. The mass spectrometry was performed in positive ion mode, and the scanning mode was multi-reaction monitoring mode with electrospray ionization as the ion source; m/z 472.2 → 455.01 for zanubrutinib and m/z 441.03 → 137.99 for ibrutinib (internal standard). The plasma samples were processed by protein precipitation. The standard curve showed good linearity (r2 = 0.999 8) in the range of 1.0-1,000 ng/ml (zanubrutinib) with a low limit of quantification of 1 ng/ml. Also, the intra-day and inter-day precision (RSD) was <5.88% and the accuracy (RE) ranged from -1.56 to 1.08%; the recoveries of zanubrutinib in beagle plasma ranged from 90.12 to 93.53% (RSD 1.67-6.42%) and the ME values of zanubrutinib were 98.70-101.06% (RSD 5.37-8.49%, n = 6). All values meet US Food and Drug Administration requirements. A rapid, highly selective and sensitive method for the determination of zanubrutinib concentration in plasma by UPLC-MS/MS was successfully developed. This method is suitable for pharmacokinetic studies in beagle dogs by following oral administration of zanubrutinib.

Effect of Chaihu Shugan Pills on the Pharmacokinetics of Duloxetine and its Metabolite 4-Hydroxyduloxetine in Beagle Dogs: A Herb-Drug Interaction Study.

The effect of Chaihu Shugan pills (CHSG) on the pharmacokinetics of duloxetine and its metabolite 4-hydroxyduloxetine in beagle dogs was investigated by establishing an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to simultaneously measure the concentrations of duloxetine and 4-hydroxyduloxetine in beagle dog plasma. Duloxetine and 4-hydroxyduloxetine were separated on the UPLC-C18 column after acetonitrile precipitation and detected by mass spectrometry with multireaction detection mode (MRM). Six adult healthy beagle dogs (weighing 7-9 kg, male and female) were randomly selected and examined for a single-dose administration of duloxetine hydrochloride (2 mg/kg, control group) and oral administration of CHSG (0.3 g/kg) twice daily for 15 consecutive days followed by a single-dose administration of duloxetine hydrochloride (2 mg/kg, experimental group) using the self-control method. All plasma samples were treated in the same way, and then the concentrations of duloxetine and 4-hydroxyduloxetine were determined using the established UPLC-MS/MS method. The obtained data were subjected to DAS 2.0 software to calculate the pharmacokinetic parameters, and SPSS 20.0 software was used to compare the differences between the two groups. Duloxetine and 4-hydroxyduloxetine had a good linear relationship in the ranges of 1-1000 ng/ml and 0.1-100 ng/ml, and the lower limits of quantification (LLOQ) were 1 ng/mL and 0.1 ng/ml, respectively. The precision, accuracy, extraction recovery, matrix effect, and stability meet the requirements of the guiding principles. After combination with CHSG, C max and AUC0⟶t of duloxetine decreased by 49.33% and 13.08%, respectively, and t 1/2 was shortened to 10.17 h; C max and AUC0⟶t of 4-hydroxyduloxetine decreased by 71.47% and 48.78%, respectively, and t 1/2 was shortened to 7.97 h. The UPLC-MS/MS method was fully developed to simultaneously measure the plasma concentration of duloxetine and its metabolite 4-hydroxyduloxetine in beagle dogs. CHSG could slow down the absorption of duloxetine, induce the metabolism of duloxetine and 4-hydroxyduloxetine in beagle dogs, and reduce plasma exposure.