Quantitative Estimation of Process-Related Impurities in Zavegepant Hydrochloride Api: Method Development and Validation of a Novel Stability-Indicating RP-HPLC Approach

Objective: This study describes the development and comprehensive validation of a novel, robust, and stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method for the quantitative determination of process-related impurities and degradation products in Zavegepant hydrochloride API. The proposed method demonstrates superior selectivity, sensitivity, and accuracy, providing a reliable analytical tool for quality evaluation of the active pharmaceutical ingredient (API). Methods: Chromatographic separation of Zavegepant HCl and its four specified process impurities was achieved on a YMC-Triart C18 column (150 mm × 4.6 mm, 3 µm, 12 nm) under gradient elution using a binary mobile phase system: Mobile Phase A consisting of potassium dihydrogen phosphate buffer (pH 8.0±0.05) and acetonitrile (95:5, v/v), and Mobile Phase B comprising acetonitrile, methanol, and ultra-pure water (70:20:10, v/v/v) at a flow rate of 0.8 ml/min. UV detection was monitored at 215 nm, yielding complete chromatographic resolution (R_s>1.5) among all target analytes. The stability-indicating capability of the method was established through stress studies under hydrolytic, oxidative, photolytic, thermal, and humidity conditions as per ICH protocols. The analytical method was fully validated according to current ICH Q2(R2) guidelines for specificity, LOD, LOQ, linearity, accuracy, precision, ruggedness, and robustness. Results: Complete chromatographic separation of Zavegepant HCl and its specified impurities (quinoline, acid, N-methyl, and N-oxide) was achieved within a 65 min run time. The method exhibited high accuracy, with mean recovery rates ranging from 95% to 115% for all specified impurities. Linearity was demonstrated with correlation coefficients (r>0.99) over concentration ranges of 0.12–3.0 µg/ml for the specified impurities and 0.12–2.0 µg/ml for Zavegepant HCl. This validated analytical technique provides a robust and reliable method for the quantification of both known and unknown impurities in drug substances, making it highly suitable for routine quality control (QC) testing and drug regulatory affairs (DRA) submissions. Conclusion: The proposed stability-indicating method was fully validated in accordance with ICH guidelines, demonstrating superior specificity and baseline separation of process-and degradation-related impurities in Zavegepant hydrochloride API. Consequently, this robust technique provides a reliable solution for routine quality control (QC) release testing and drug regulatory affairs (DRA) submissions.