Stability-Indicating High-Performance Thin-Layer Chromatographic Method for the Estimation of Ambroxol Hydrochloride and Doxofylline in a Pharmaceutical Formulation Using Experimental Design in Robustness Study

Summary

A sensitive, specific, and accurate stability-indicating high-performance thin-layer chromatographic method for the analysis of ambroxol hydrochloride and doxofylline, for both bulk drug and formulation, was developed and validated according to the International Conference on Harmonization (ICH) guidelines. The densitometric analysis of both drugs was carried out in the absorbance mode at 276 nm using diethyl ether—n-butanol—ammonia (9:0.9:0.1, v/v) as the solvent system. This system was found to give compact spots for ambroxol hydrochloride at R_F of 0.74 ± 0.01 and doxofylline at 0.41 ± 0.01. Moreover, both drugs were subjected to acid and alkali hydrolysis, oxidation, and photodegradation. Also, the degraded products were well resolved from the pure drug with different R_F values. Linearity was found in the range of 20–100 and 100–500 ng band⁻¹ for ambroxol hydrochloride and doxofylline. The limit of detection (LOD) and limit of quantitation (LOQ) for ambroxol hydrochloride and doxofylline were 1.17 and 3.57 ng band⁻¹ and 30.68 and 92.97 ng band⁻¹, respectively. “Bartlett’s test” and “Lack of fit” applied on peak area for linearity additionally proved validity of the linearity of the developed method. Good accuracy and precision were obtained as revealed from percent relative standard deviation (% RSD) less than 2. Moreover, robustness testing was performed applying fractional factorial design, 2^4-1. All the four factors: volume of diethyl ether, volume of n-butanol, solvent front, and chamber saturation time, evaluated in the robustness testing, were found to have an insignificant effect on the retention factor. Similarly, no interference was observed from common excipients in tablet formulation as well as degradation product, indicating specificity of the method. As the method could effectively separate both drugs from their degradation products, it can be used as a stability-indicating method.