Development and validation of a novel stability indicating RP–UPLC method for the estimation of Rezafungin and characterization of its degradation products by UPLC–MS/MS

A simple, novel, straight forward, isocratic, linear, precise, specific, accurate, robust and validated stability indicating reverse phase chromatographic technique has been established for the analysis of Rezafungin and its degradation products in both bulk and pharmaceutical dosage forms by means of UPLC–MS. As per our observation, until now no chromatographic technique has been published in the literature for the analysis of Rezafungin and its degradants. The proposed chromatographic technique has been designed using a Water’s Acquity BEH C18 Column (50 × 2.1 mm, 1.7 µm) and an isocratic mobile phase consist of of 0.1% TFA: ACN in “70:30 v/v” ratio at a rate of flow 0.3 mL/min at room temperature. The analyte was monitored at 228 nm employing a photo diode array detector and Empower-2.0 software. The retention time of Rezafungin was observed to be 0.925 min. The established reverse phase UPLC technique was statistically validated in terms of linearity, LOD, LOQ, precision, specificity, robustness and accuracy in compliance with ICH guidelines and the outcomes were observed to be within the prescribed limits. The described method exhibited brilliant linearity throughout the concentration range of 5–30 µg/mL, and the relevant regression equation was y = 122416.4x + 11606.43 (R2 = 0.9999). The LOD and LOQ of Rezafungin were observed to be 0.6 µg/mL (s/n = 3) and 2 µg/mL (s/n = 10) respectively. Rezafungin’s mean percentage recoveries were ranged between 100.1 and 101.1%. The RSD values of all the validation parameters were observed to be below 2%. The forced degradation studies have been executed in compliance with ICHQ2(R) recommendations and the degradants were characterized by employing UPLC–MS. The chromatographic peaks of degradation products were effectively resolved from the peaks of pure pharmaceutical drug with notable differences in their retention times with adequate tailing and resolution. This forced degradation studies provide the knowledge about the circumstances under which the Rezfungin was unstable to evade the potential instabilities and appropriate precautions were often taken during the formulation process. The described RP–UPLC stability indicating method plays a vital role in numerous pharmaceutical applications such as stability testing, quality control, shelf-life assessment, packaging integrity and optimization of packaging design.