Ocular pharmacokinetic analysis of drugs is useful for evaluation of efficacy of novel ophthalmic formulations. A simple and sensitive ultra performance liquid chromatographic (UPLC) method with ultraviolet (UV) detection was developed and validated for the quantification of dorzolamide (DZA) in rabbit aqueous humor. The aqueous humor samples were deproteinized with acetonitrile prior to UPLC analysis. A chromatographic separation was achieved on a Waters Acquity BEH C18 (100 mm mm, 1.7 μm) column with a mobile phase consisting of 0.1% triethylamine (TEA) in water and acetonitrile (50:50, v/v) at a flow rate of 0.25 ml min-1. Detection of DZA was carried out at 254 nm. The method developed was linear (r2 = 0.9996) over the concentration range of 5 - 800 ng mL-1. Accuracy ranged from 98.23 to 99.53% and precision was between 0.12% and 0.36%. Limit of detection and quantitation of DZA in aqueous humor were 5 and 10 ng mL-1, respectively. The method was applied to determine pharmacokinetics of DZA in aqueous humor, following topical instillation of DZA-solution (DZA-SOL) and DZA nanoparticles (DZA-NPs) to rabbit eyes. The pharmacokinetic results showed that DZA-NPs enhances the ocular bioavailability of DZA by 6-fold compared to DZA-SOL.
Keywords: Aqueous humor, Dorzolamide, Nanoparticles, Ocular pharmacokinetics, Ultra performance liquid chromatography, Ultraviolet (UV) detection, Ocular bioavailability, Limit of detection, Limit of quantification, Assay validation
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