Quality by Design‐Based Stability‐Indicating RP‐HPLC Method for Diminazene Diaceturate in Veterinary Formulation Supported by LC–MS Characterization of Degradation Products

ABSTRACT This study focuses on the development and validation of the Quality by Design RP‐HPLC method for diminazene diaceturate with fragmentation study by LC–MS/MS. The central composite design was employed to optimize critical factors: mobile phase ratio, flow rate, and wavelength. Analysis was conducted on Xterra C18 column with mobile phase composed of 0.1% triethylamine buffer: methanol (80:20% v/v) and flow rate 0.8 mL/min at 255 nm, with retention at 4.34 min. ICH Q1A(R2) guidelines, acid stress degradation study showed maximum degradation of 16.03%. The fragmentation pathway exhibited prominent product ions at m/z 518, 284, 285, and 268. Linearity was observed over the concentration range (50–100 μg/mL) with a regression coefficient of 0.9997. Limit of detection and limit of quantification values were observed to be 7.772 and 23.566 μg/mL, respectively; % RSD of each parameter was found to be less than 2. The validated method proved suitable for assay of marketed veterinary formulation, yielding recovery of 93.22%, with clear separation of the stabilizer phenazone. This study focuses on the development of a stability indicating RP‐HPLC method using a Quality by Design approach with LC–MS compatible triethylamine buffer, enabling degradation product characterization while maintaining a regulatory‐compliant RP‐HPLC method suitable for routine and stability testing of diminazene diaceturate in pharmaceutical laboratories.