AQbD-driven development of a green RP-HPLC method for nirogacestat with LC-MS structural elucidation

Area of Science:

• Pharmaceutical Analysis
• Analytical Chemistry
• Green Chemistry

Background:

• Nirogacestat (NGT), a γ-secretase inhibitor for desmoid tumors, requires robust analytical methods for impurity profiling.
• Current methods may lack the sensitivity, sustainability, or regulatory compliance needed for NGT analysis.
• Developing a green and stability-indicating method is crucial for quality control.

Purpose of the Study:

• To develop and validate a green, stability-indicating High-Performance Liquid Chromatography (HPLC) method for Nirogacestat (NGT) impurity profiling.
• To apply an Analytical Quality by Design (AQbD) approach for method optimization and establishment of a design space.
• To ensure the method is sensitive, specific, accurate, precise, robust, and sustainable.

Main Methods:

• Utilized an Analytical Quality by Design (AQbD) approach to optimize critical method parameters (ethanol percentage, buffer concentration, flow rate).
• Employed a central composite design for risk assessment and design space establishment.
• Validated the method according to ICH Q2(R2) guidelines, including specificity, linearity, accuracy, precision, and robustness testing.
• Performed forced-degradation studies to assess stability-indicating capability and peak purity.
• Evaluated method greenness using multiple assessment tools (AGREE, AGSA, CACI, CaFRI).

Main Results:

• Developed a robust HPLC method using an ethanol-ammonium formate mobile phase, achieving baseline separation of NGT, impurities, and degradation products.
• Method validation confirmed excellent specificity, linearity (R² > 0.999), accuracy (99.4-101.2%), precision (%RSD ≤ 1.8%), and robustness.
• Achieved high sensitivity with low Limit of Detection (LOD) and Limit of Quantitation (LOQ) values for NGT and impurities.
• Forced-degradation studies demonstrated good mass balance (95-105%) and confirmed the stability-indicating nature of the method.
• The method exhibited significant greenness, with an overall whiteness index of 74%.

Conclusions:

• The developed ethanol-based, AQbD-optimized HPLC method is reliable, sustainable, and regulatory-compliant for NGT impurity profiling.
• This method provides a robust platform for stability testing of Nirogacestat, ensuring drug quality and safety.
• The application of AQbD and green chemistry principles enhances the analytical process for pharmaceutical development.