Stability indicating RP-HPLC method development and validation for determination of potential degradation impurities of tretinoin in tretinoin topical pharmaceutical formulation

Chinmoy Roy and Jitamanyu Chak

Abstract

A simple, specific, accurate and stability-indicating RP-HPLC method was developed and validated for determination of related substances in Tretinoin topical pharmaceutical formulation. Chromatographic separation was achieved on Hypersil BDS C18 250 × 4.6mm, 5μ column as stationary phase while mobile phase A was buffer, (Buffer=Water : Glacial acetic acid, 90:2 v/v) and mobile phase B as Methanol. Method was developed in gradient mode with 40 minutes, at flow rate of 1.2 mL/min. Effluents were monitored at 356 nm. The method was validated for specificity, linearity, accuracy, precision, limit of quantification, limit of detection, robustness and solution stability. The RRF (relative response factor) values of Isotretinoin impurity determined from linearity study were 0.91. Limit of quantification of Tretinoin and Isotretinoin was found to be 0.02 μg/mL and 0.02 μg/mL respectively. Recovery was found to be in the range of 95.0-105.0%. The method was proved to be robust with respect to changes in flow rate, buffer pH and column temperature. The proposed method was successfully applied for the quantitative determination of related substances in Tretinoin topical pharmaceutical formulation.