Abstract
New reversed-phase liquid chromatographic methods, with UV detection, were developed for the quantitative estimation of flurbiprofen in rabbit blood serum and aqueous humor. The mobile phase and other chromatographic conditions were optimized to minimize interference from biological matrix and at the same time provide sufficient sensitivity for the method to be adopted for in vivo studies of ophthalmic formulations of flurbiprofen. Acetonitrile was used to precipitate proteins from serum or aqueous humor during sample preparation. A mobile phase of methanol: acetonitrile: phosphate buffer pH 5.6 (40:20:40) was employed with UV detection at 248 nm for estimation of drug in both the biological matrix. The retention time and asymmetry factor for the proposed method of estimation in serum and aqueous humor was found to be 3.1312±0.0101 min and 1.1310±0.0091 respectively. The linear regression equations obtained by least square regression method, were Area (µV sec) = 52.27 × Conc. (in ng/ml)–1618.70 in serum and Area (µV sec) = 61.79 × Conc. (in ng/ml) − 783.24 in aqueous humor. The results of analysis were treated statistically, as per ICH guidelines for validation of analytical procedures, USP-2003, and by recovery studies. The results were found to be accurate, reproducible and free from interference. The developed methods were further used for estimation of flurbiprofen in rabbit serum and aqueous humor following single topical administration of in-house aqueous drop and market formulation to rabbit eye.
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