Development and Validation of HPTLC Method for Estimation of Nepafenac in Ophthalmic Suspension

A simple, accurate, precise and suitable High Performance Thin Layer Chromatography (HPTLC) method was developed and validated for estimation of Nepafenac in its marketed ophthalmic suspension formulation. Diclofenac sodium was used as Internal Standard (IS) in order to make the method more accurate and precise. Chromatographic separation of Nepafenac from suspension formulation and Diclofenac sodium was achieved on TLC Silica gel 60 F254 glass plates using the mobile phase comprising of Toluene: Ethyl acetate: Glacial acetic acid in the ratio of 65: 35: 0.2 % v/v/v. Densitometric detection and quantification was carried out at 280 nm. The Retention Factor (Rf) values of Diclofenac sodium and Nepafenac were found to be 0.453 and 0.268 respectively with good resolution and peak shapes. The method was validated in accordance with ICH guidelines for specificity, linearity, precision, recovery, sensitivity and robustness. The method was found to be linear in the concentration range of 50-500 ng/band with the Correlation coefficient value of 0.9991. Mean percent recovery of Nepafenac sample solutions was found to be 100.41 %. The Limit of Detection (LOD) and Limit of Quantitation (LOQ) values for Nepafenac were found to be 2.08 ng/band and 6.31 ng/band respectively. The proposed method was novel as no any HPTLC method was reported before for estimation of Nepafenac in eye drop and was applied successfully for the quantitative analysis of the same.