Is a DoE-based approach superior to OFAT for HPLC robustness assessment under ICH Q14? A practical case study

Method robustness is a critical aspect of analytical development, particularly in pharmaceutical research. This work investigates whether a Design of Experiments (DoE) approach provides advantages over traditional one-factor-at-a-time (OFAT) strategy for assessing robustness. Robustness was assessed for an assay/degradation liquid chromatography method by modifying key parameters such as flow rate, temperature, and mobile phase composition. This side-by-side comparison of DoE and OFAT addresses a gap in applied analytical methodology by examining how multivariate experimental design influences robustness outcomes. The DoE strategy enabled a systematic and multivariate analysis of the factors. For this analytical method, organic solvent content and pH were the main factors affecting the responses. Furthermore, DoE revealed factor-factor interactions that were not detectable through traditional methods. In contrast, the OFAT approach provided limited insight, was restricted to univariate parameter modifications, and required more experimental runs, time, effort, and costs to achieve comparable understanding. While the OFAT approach confirmed the method robustness across most ranges (except the higher pH), the DoE approach revealed additional conditions where the method may not be robust due to factor-factor interactions. DoE also enabled the definition of robust design space, providing greater flexibility for method changes with reduced need for regulatory interaction. Overall, the results demonstrate that DoE is indeed advantageous for robustness evaluation, enhancing analytical understanding, efficiency and regulatory alignment. These findings strongly support the integration of DoE into routine robustness assessment to strengthen the method reliability and compliance under modern pharmaceutical guidelines.