In vivo pharmacokinetic studies

Rabbits (weighing between 2–2.5 kg) were used as the experiment animals. Ten rabbits were randomly divided into two groups with five animals in each group. Prior to the experiment, the rabbits were fasted for 12 h with free access to water. In the next day, the midazolam nanosuspension and the coarse midazolam suspension, 1 mg/kg body weight expressed as midazolam equivalents were administered to the buccal of rabbits under light anesthesia by ether in two groups. About 1 mL of blood samples was collected via the ear veins at 5–300 min after administration. Extraction of midazolam from blood samples was done in the following method and related recovery was calculated. The collected blood samples were placed in a heparinized tube, and then, separated immediately by centrifugation at 4000 rpm for 5 min and stored at − 40 °C until analysis. All the experiments were carried out in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals. The preparation of the plasma samples was as follows: 200 μL NaOH (0.1 M), 50 μL diazepam (as an internal standard), and 4 mL of diethyl ether were added to each 450 μL rabbit plasma sample. The mixtures were vortexed (MS3, IKA^®, Germany) for 10 min and centrifuged (H 2050R, Xiang Yi Centrifuge Instrument Co., Ltd., Hunan, China) at 3000 rpm for 5 min. The screw-capped tubes, containing the samples, were frozen at − 40 °C for about 35 min. The organic layer was decanted into conical glass centrifuge tubes and concentrated (at 40 °C) under a gentle stream of nitrogen. After evaporation, the residues were re-dissolved in 150 μL of the mobile phase; 50 μL was injected in the HPLC system (Guo et al. 2013; Jurica et al. 2007).