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

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.