Twenty-five eyes of 30 albino rabbits, weighing 2.0 to 2.5 kg each, were used. Only the right eye of each rabbit received the osmotic pump containing BP in phosphate-buffered saline (50 mg/mL), with or without 0.01% or 0.05% BAK. Animals were killed with an overdose of intravenous pentobarbital sodium at 1 week after implantation, and the eyes were enucleated. The eyes were immediately frozen at −85°C, and samples of ocular tissues (aqueous humor, vitreous, and retina-choroid) were retrieved. The ocular tissues were stored at −85°C until the BP concentration was determined by high-performance liquid chromatography (HPLC) with a C-18 reversed-phase column (150 × 6.0 mm inner diameter; YMC-Pack ODS-A312; YMC Co., Ltd., Kyoto, Japan). A pump (PU-980; Japan Spectroscopic Co., Ltd., Tokyo, Japan) was used at a constant flow rate of 1 mL/min. The mobile phase was a mixture of methanol and 50 mM potassium dihydrogenphosphate aqueous solution (55:45). The column oven (860-CO; Japan Spectroscopic Co., Ltd.) was equipped and set at 40°C. A spectrophotometer detector (L-4000; Hitachi, Ltd., Tokyo, Japan) was used at a wavelength of 240 nm. Fluorometholone (Wako Pure Chemical Industries, Osaka, Japan) was used as an internal standard.
Concentrations of BP and betamethasone (BM), a metabolite of BP, were determined in recovered ocular tissues (aqueous humor, vitreous, retina-choroid) by the described HPLC procedures. BP and BM were extracted from the tissues by the following process: One-tenth milliliter of internal standard solution (2.5 μg/mL) and 3.0 mL of 0.2 M HCl were added to each tissue sample. The mixture was homogenized and centrifuged at 3000 rpm for 15 minutes (KN-70; Kubota, Tokyo, Japan). The supernatant was collected, and BM was extracted twice with 3.0 mL of ethyl acetate. Ethyl acetate phases were then dried under reduced pressure with a centrifugal concentrator (VC-960; Taitec Co., Saitama, Japan). The residue was dissolved with 0.2 mL mobile phase. One hundred microliters of this solution was injected into the HPLC column, as described. Under these conditions, the detection limits for BP and BM were 100 ng/g in the aqueous humor and retina-choroid and 10 ng/g in the vitreous. The BP concentrations in ocular tissues were represented as the total BP and BM molecules per gram of wet tissue.