Ultra high-performance liquid chromatography method development for separation of formoterol, budesonide, and related substances using an analytical quality by design approach.

The application of the Quality by Design (QbD) principles in developing a new ultra high performance liquid chromatography method for the analysis of formoterol/budesonide and related substances using Fusion QbD® software is explored. The effect of various chromatographic parameters including, column stationary phase, pH, temperature, flow rate, and gradient time on separations were systematically investigated. Results show that optimal separations of these compounds in a standard solution can be achieved using a BEH C column (2.1 × 1.7 μm × 10 cm) applying a pH of 8.2, a temperature of 35 °C, a flow rate of 0.35 mL min and a gradient time of 25 min. Furthermore, the results show that the main parameters affecting the performance of the method were the mobile phase pH, gradient time, and the temperature. For example, the most important factor for peak tailing was the pH of the mobile phase and the critical factors affecting resolution of the analytes were the gradient time and the temperature. As an application, the method was further used to analyze budesonide and formoterol in a sample obtained from a Symbicort® metered dose inhaler and it was found to provide similar separations to those obtained with the standard solution. These findings indicate that applying the QbD principles in analytical method development can be very advantageous not only in obtaining deep understanding of the effect of input parameters but also potential regulatory flexibility.