UPLC-MS/MS analysis of axitinib and pharmacokinetic application in beagle dogs.

Objective: To develop a method for determining the concentration of axitinib in beagle dog plasma and utilize this method to investigate the pharmacokinetics of orally administered axitinib in beagle dogs.

Methods: Plasma samples were processed using acetonitrile precipitation and analyzed by UPLC-MS/MS with sunitinib as an internal standard (IS). Chromatographic separation was achieved on a Waters Acquisition UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 μm) with a gradient elution of acetonitrile and 0.1 % formic acid. Mass spectrometry uses an electrospray ion source for positive ion detection in a multiple reaction monitoring mode. The monitored ion transitions for axitinib and sunitinib were / 387 → 355.96 and / 399.3 → 282.96, respectively. Six beagle dogs were administered 0.33 mg/kg of axitinib orally, and venous blood samples were collected at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 h post-dose for pharmacokinetic analysis.

Results: The assay demonstrated a linear range of 0.5-100 ng/mL (r = 0.9992), and the lower limit of quantification was up to 0.5 ng/mL. Precision, as assessed by relative standard deviation (RSD), was within 8.64 % for both intraday and interday variability. The relative error (RE) for precision from -2.77 %-1.20 %. The recovery rate of the analytes exceeded 85.28 % and the matrix effect was approximately 100 %. Plasma samples maintained stability under various conditions, including room temperature storage for 12 h, processed on an automatic sampler at 4 °C for 6 h, three freeze-thaw cycles, and long-term storage at -80 °C for 60 days. Pharmacokinetic parameters were determined using DAS 2.0 software, revealing a half-life (T) of 6.05 h and an area under the curve (AUC ) of 97.13 ng h/mL for axitinib.

Conclusions: The UPLC-MS/MS method developed in this study offers high specificity, rapid analysis, high recovery, excellent linearity, and minimal plasma volume requirements, making it well-suited for pharmacokinetic and drug interaction studies in beagles dogs.