Analytical Quality by Design approach in the development of a green reversed-phase ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry method for the simultaneous analysis of synthetic antimicrobial and hypot

• AQbD approach used for peptide separation method development. • Core-shell columns offer superior performance for simultaneous peptide analysis. • Mobile phase composition is the most impactful variable on resolution and run time. • The defined design space offers a flexible, robust method. • Method scored positively in greenness. The growing therapeutic interest in peptides from Tityus stigmurus scorpion venom and their analogues highlights the need for the development of robust analytical methods for these compounds. However, traditional method development is often inefficient, failing to account for the complex interactions between chromatographic parameters that influence analytical quality. This study aimed to apply a Analytical Quality by Design (AQbD) approach to develop a highly efficient and robust method for the simultaneous separation of four synthetic peptides (Stigmurin, two analogs and TistH). The AQbD framework was used, employing risk assessment to identify critical variables and attributes. Two experimental designs— two two-level screening and a three-level optimization—evaluated the impact of column type, mobile phase composition, and flow rate. Statistical analysis generated models and response surface plots to define an optimal operating region. Core-shell particle columns demonstrated superior performance, achieving baseline resolution for all peptides. Statistical models confirmed that mobile phase composition was the most influential factor. The defined design space identified a region for optimal operation: 40-43% (v/v) organic mobile phase, 20-40°C, and a flow rate of 0.25-0.4 mL/min. Greenness analysis confirmed that it can be considered a green analytical method. The AQbD approach successfully created a reliable ultra-high performance liquid chromatography with time-of-flight mass spectrometry (UHPLC-ToF-MS) green and applicable method for peptide analysis, providing a flexible and high-performance tool for this application. This methodology serves as a valuable blueprint for developing robust analytical methods for other peptide mixtures, ensuring quality and repeatability.