Development of sensitive stability indicating HPLC method for quantification of process related impurities of Fluphenazine; LC–MS/MS elucidation of their degradation products and degradation pathway

Abstract

This study focused on the development of a simple and sensitive HPLC method for the resolution and quantification of process related impurities of fluphenazine and further assessment of the forced degradation behavior of fluphenazine. Chromatographic separation was achieved on phenomenex™ Gemini C18 column (250×4.6mm, 5.0μm) at room temperature using acetonitrile, methanol and TFA in 65:30:05 (v/v) at pH 4.2 as the mobile phase was pumped isocratically at a 0.9 mL/min flow rate. The detection wavelength was selected as 255 nm. In the proposed conditions, the retention time of the analytes was noticed to be 6.23 min for fluphenazine and 4.57, 5.30, 9.01 and 8.29 min for impurity 1-4 respectively. The method produces sensitive detection limits of 0.003 μg/mL, 0.013 μg/mL, 0.018 μg/mL and 0.022 μg/mL for impurity 1 to 4 respectively with calibration range of 75-450 μg/mL for fluphenazine and 0.075 - 0.45 μg/mL for impurities. The other validation parameters were noticed to be within the acceptable levels for fluphenazine and its impurities. The drug was exposed to different stress conditions (acid, base, peroxide, thermal and UV light) according to the ICH Q1A (R2) guidelines. The DPs formed during the stress study were identified and characterized by LCMS/MS in the ESI positive mode.