DEVELOPMENT AND VALIDATION OF RELATED SUBSTANCE BY HPLC FOR DIPHENHYDRAMINE HYDROCHLORIDE SOFT GEL CAPSULE

Abstract

Objective: Many over-the-counter drug products does not have official compendial analytical methods for the analysis of the drug product. Thus, the United States Pharmacopeia (USP) are seeking to develop and validate new methods to establish analysis standards for the assessment of pharmaceutical over-the-counter drug products. One such method is the related substances by high-performance liquid chromatographic (HPLC) of diphenhydramine hydrochloride capsule.

Methods: A reversed-phase high-performance liquid chromatographic method for related substances of diphenhydramine hydrochloride capsule was developed comprising of YMC pack pro C8 column 250×4.6 mm, S-5µm, 12 nm HPLC column using waters Alliance 2690 HPLC system with a mobile phase of 65% of pH 3 monobasic potassium phosphate buffer and 35% of acetonitrile. The flow rate was 1.2 ml/min, 10 µl injection volume, at 220 nm wavelength with the column temperature of 25 °C. The method was validated as per International Council for Harmonisation of Technical Requirements for Pharmaceuticals (ICH) Q2R2 guidelines for specificity, precision (system precision, method precision), linearity, accuracy, solution stability and robustness (filter study).

Results: The method found to be specific without any interference from placebo and spiked sample with the retention time of 5.2 min, 5.5 min, 7.0 min, 23.2 min and 46.1 min for Diphenhydramine Related Compound-A (DRC-A), diphenhydramine hydrochloride, Diphenhydramine N-Oxide (DNO), benzhydrol and benzophenone respectively. The % Relative Standard Deviation (RSD) for DRC-A, DNO, benzhydrol and benzophenone was observed to be as 6.2, 5.9, 6.3 and 5.5, respectively. The accuracy ensures the detection and quantification at a very low level for all impurities [Limit Of Quantification (LOQ) 0.03%] with coefficient correlation of 0.9999, 0.9977, 0.9992, 0.9995,0.9990 and % RSD for LOQ precision was determined as 0.9, 2.1, 0.8, 1.9 and 8.0 for DRC-A, diphenhydramine hydrochloride, DNO, benzhydrol and benzophenone respectively. Since, DNO impurity is degradation impurity and vital in drug products and required to address its consistency and assurance towards the safety of patient, this method ensures to quantitate it at low level with precision and accuracy. Thus, the consistent, linear, precise, accurate and stability indicating results makes it more suitable for quantification of related substances in finished product and stability analysis.

Conclusion: The study findings shows that the method is quite simple and applicable for release of finished product for the related substance analysis of diphenhydramine hydrochloride soft gel capsule.