2.10.1. Administration Programme

All animal treatments were performed in accordance with the Regulations of the Administration of Affairs Concerning Experimental Animals and the study protocol was admitted by the Ethics Committee of China Pharmaceutical University (Approval No. 2018-0315). An open label, randomized, two-period crossover experiment design with one week wash-out period was used in this study. Six male beagle dogs (weight 8.7 ± 1.1 kg), fasted but free access to water for 12 h prior to the experiment, were used in the study. Pellets of the optimal formulation were filled into hard gelatin capsules. The immediate release tablet (Loxonin®, 60 mg anhydrous LXP) and the capsule of the optimal formulation (90 mg anhydrous LXP) were administered to beagles in the morning with 100 mL water. Then, 6 h after dosing, dogs were provided with standard food.

A total of 2 mL of the blood samples were withdrawn before and then 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0, 10.0, and 12.0 h after dosing via cannulated needle from front legs. Plasma was obtained by centrifuging the blood at 4000 rpm for 15 min, and was kept frozen at −20 ◦C before analysis.

### 2.10.2. Determination of LXP in Plasma

A stable and selective HPLC method, modified by previous papers [12,35], was applied for the analysis of LXP in dog plasma. Pretreatment was carried out by adding 50 μL of internal standard (100 μg/mL ketoprofen in acetonitrile), 50 μL of zinc sulfate solution (10%, *w*/*w*) and 750 μL acetonitrile into 500 μL plasma sample. After vortex-mixing for 1 min, sample was centrifuged at 8000 rmp for 15 min. Then 10 μL of the supernatant was injected into the HPLC system. The separation was performed on an Inertsil C18-ODS column (5 μm, 4.6 × 150 mm, Shimadzu, Japan) with a guard column (4.6 × 10 mm, 5 μm particle size, ANPEL Laboratory Technologies Inc, Shanghai, China) at a flow rate of 1 mL/min. Additionally, the mobile phase was a mixture of acetonitrile and 0.05 M monopotassium phosphate (35:65, *v*/*v*), adjusted to pH 3.0 with phosphoric acid. Chromatograms were recorded at 223 nm with a Shimadzu-SPD detector. The linear range of this method was 0.1–20.0 μg/mL with an *r*2 value of not less than 0.999. The lower limit of quantification (LLOQ) was 100 ng/mL and the extraction recoveries of high, middle, and low concentrations of LXP were 102.5 ± 2.0%, 97.1 ± 2.5% and 97.1 ± 5.9%, respectively. The R.S.D.s reflecting the intra-day and inter-day precision of LXP were less than 11.77%.
