RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR STABILITY-INDICATING ANALYSIS OF FLURALANER AND PROCESS-RELATED IMPURITIES IN DRUG SUBSTANCE

Abstract

Objective: To develop and validate a stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method for simultaneous quantification of Fluralaner and its process-related impurities in active pharmaceutical ingredient (API).

Methods: We employed a Phenyl hexyl column (250 × 4.6 mm, 5µm) with gradient elution using 0.1% perchloric acid, acetonitrile, methanol, and isopropanol as mobile phase components. Detection was performed at 210 nm wavelength with 1.0 mL/min flow rate. The method was validated according to ICH Q2(R1) guidelines [1] encompassing linearity, specificity, accuracy, precision, detection limits, and robustness parameters.

Results: Our method demonstrated excellent analytical performance with correlation coefficients (r²) of 0.9999 for Fluralaner and all four process impurities across the concentration range of 0.05-200%. Recovery studies yielded values between 88.88-108.28% with relative standard deviation (RSD) values ≤1.69%. Forced degradation studies revealed significant degradation under acidic conditions (26.70%) and minimal alkali degradation (3.83%), while oxidative, thermal, and humidity conditions showed no degradation.

Conclusions: This validated RP-HPLC method provides a robust, sensitive, and specific analytical tool for routine quality control analysis of Fluralaner drug substance. The method successfully separates Fluralaner from its four process-related impurities with baseline resolution and demonstrates stability-indicating capability suitable for pharmaceutical manufacturing and regulatory compliance