Development and Validation Of A Stability-Indicating Uplc Method For Simultaneous Estimation of Tretinoin And Mequinol in Topical Formulation

Abstract

Background: Tretinoin and mequinol are used in topical formulations and require reliable analytical procedures for their simultaneous quantitative determination. The present study aimed to develop and validate a simple, precise, accurate, and stability-indicating UPLC method for simultaneous estimation of tretinoin and mequinol in topical formulation.

Methods: Chromatographic separation was achieved using an Inertsil C18 column (250 × 4.6 mm, 5 µm) with methanol and phosphate buffer (pH 3.0) in the ratio of 70:30 (v/v) as the mobile phase. The flow rate was maintained at 1.0 mL/min, injection volume was 20 µL, and detection was performed at 325 nm under isocratic conditions. The method was validated for system suitability, specificity, linearity, precision, accuracy, sensitivity, robustness, and ruggedness. Linearity was evaluated over the concentration range of 10–50 µg/mL for both analytes. Forced degradation studies were performed under acidic, alkaline, oxidative, thermal, and photolytic stress conditions. The applicability of the method was further demonstrated through analysis of a marketed topical formulation.

Results: The developed method provided satisfactory chromatographic separation of mequinol and tretinoin, with mean retention times of approximately 2.9 and 5.7 min, respectively. The method demonstrated good linearity, with coefficients of determination (R²) of 0.9991 for mequinol and 0.9994 for tretinoin. System precision showed %RSD values of 0.114% and 0.133%, while method precision showed %RSD values of 0.456% and 0.703% for mequinol and tretinoin, respectively. The mean recoveries were approximately 100% for both analytes. The LOD/LOQ values were 0.586/0.943 µg/mL for mequinol and 0.215/0.892 µg/mL for tretinoin. The method remained unaffected by deliberate variation in flow rate. Forced degradation generated additional degradation-related peaks that were chromatographically separated from the principal analyte peaks. Tretinoin showed greater susceptibility to photolytic, oxidative, and alkaline stress, whereas mequinol exhibited comparatively lower degradation under the investigated conditions.

Conclusion: A reliable and stability-indicating UPLC method was successfully developed for simultaneous estimation of mequinol and tretinoin. The method exhibited satisfactory specificity, linearity, precision, accuracy, sensitivity, and robustness and was successfully applied to the analysis of a marketed topical formulation. The developed procedure can therefore be used for routine quality-control analysis and stability assessment of topical formulations containing mequinol and tretinoin.