Design of Experimental Approach to Analytical Robustness Study for UHPLC Method Developed for Separation and Quantification of Spironolactone and its Impurities in Drug Substances

Abstract

The present work demonstrates the development of an optimal, robust, validated UHPLC method for quantification of related impurities and assay determination of spironolactone. Design of experiment procedure, in combination with statistical evaluation of the data was used to test the robustness of developed method. A stability indicating method was established by forced degradation experiments. Analytical robustness was determined using design of experiment approach. The chromatographic separation was achieved with Agilent SB-C18 RRHD column using gradient elution with mobile phase-A consists of a mixture of 0.1 % each formic acid and ammonia in water and methanol as mobile phase-B respectively. The developed method is exhaustively validated for parameters like precision, accuracy, linearity, LOD, LOQ, ruggedness and robustness. The stability tests were also performed on drug substances as per ICH norms. Base line separation was achieved for all impurities, degradation products and the API. All impurities were eluted within 12 min, there was a remarkable 3.5-fold decrease in runtime and a clear baseline separation between all peaks in comparison with Ph.Eur monograph. A multi-dimensional design space was built to study the robustness of developed method using design expert software. Significant parameters such as effect of flow rate, buffer strength and mobile phase compositions were optimized at three levels. Plackett-Burman design was applied for screening of chromatographic conditions and factorial design was applied for optimization of essential factors in robustness studies.