Application of quality by design concept to develop a dual gradient elution stability-indicating method for cloxacillin forced degradation studies using combined mixture-process variable models

- The development of stability-indicating method for cloxacillin has been described in terms of several key concepts of the AQbD framework.
- Flexible applications of different DoE methods were illustrated to meet different objectives.
- Mixture-process variable design and response surface methodology were conducted to achieve the optimal experiment parameters.
- The simultaneous existence of two design spaces became feasible by using a dual gradient elution.
- Our approach can be a powerful tool for developing stability-indicating methods for other easily degradable drugs.

Penicillins are typical of complex ionic samples which likely contain large number of degradation-related impurities (DRIs) with different polarities and charge properties. It is often a challenge to develop selective and robust high performance liquid chromatography (HPLC) methods for the efficient separation of all DRIs. In this study, an analytical quality by design (AQbD) approach was proposed for stability-indicating method development of cloxacillin. The structures, retention and UV characteristics rules of penicillins and their impurities were summarized and served as useful prior knowledge. Through quality risk assessment and screen design, 3 critical process parameters (CPPs) were defined, including 2 mixture variables (MVs) and 1 process variable (PV). A combined mixture-process variable (MPV) design was conducted to evaluate the 3 CPPs simultaneously and a response surface methodology (RSM) was used to achieve the optimal experiment parameters. A dual gradient elution was performed to change buffer pH, mobile-phase type and strength simultaneously. The design spaces (DSs) was evaluated using Monte Carlo simulation to give their possibility of meeting the specifications of CQAs. A Plackett-Burman design was performed to test the robustness around the working points and to decide the normal operating ranges (NORs). Finally, validation was performed following International Conference on Harmonisation (ICH) guidelines. To our knowledge, this is the first study of using MPV design and dual gradient elution to develop HPLC methods and improve separations for complex ionic samples.