The injected eyes of five rabbits in each group were enucleated at 12, 24, 36, 48, 60, and 72 hours each for sampling of the drug. Eyes were frozen at −80°C immediately after enucleation. Whole vitreous was isolated from each eye by dissection of ocular tissue. The vitreous was mixed with 3 mL methanol, the mixture was centrifuged with 12,000 rpm for 10 minutes at 4°C, and the resultant supernatant was filtered with 0.22-μm micropore. Samples of 25 μL were analyzed using a liquid chromatograph-triple quadrupole mass spectrometer (6410 Triple Quadrupole LC/MS; Agilent Technologies, Palo Alto, CA). C-18 columns (Capcell Pak, 4.6 × 250 mm; Shisheido, Tokyo, Japan) were used. The flow rate used was 1 mL/min, and the mobile phase consisted of acetonitrile and water (0.1% formic acid).
Pharmacokinetic parameters such as half-life, area under curve (AUC), mean residence time (MRT), and clearance were determined (WinNonlin Pro version 5.2; Pharsight, Mountain View, CA).