Development and Validation of an Analytical Method for Related Substances in N-acetyl–L-cysteine Effervescent Tablets by RP-HPLC

Abstract

Background: The reported chromatographic methods for N-acetyl cysteine [NAC] are reverse phase HPLC and ion pair chromatography [IPC] for related substances test in bulk and in formulations. No reported stability indicating methods for the estimation of related substances in NAC effervescent formulation was found in literature.Objective: The present work was aimed at developing a selective, sensitive and reproducible stability indicating high-performance liquid chromatographic method for the quantitative determination of known, unknown impurities, degradation impurities and processrelated impurities of NAC effervescent formulation.Method: A reversed phase ion pair chromatographic method was developed employing Cadenza C18 column as the stationary phase and 0.01M octane sulphonate [pH 2.20], methanol and acetonitrile in the ratio 90:8:2 as the mobile phase. A gradient programme was followed with a run time of 55 minutes. 0.3 M hydrochloric acid was selected as the optimum diluent. The performance of the method was validated according to the ICHQ2R1 guidelines.Results: The method was found to be linear from 1.5 to 25μg/ml for impurities A, C and D and from 2.0 to 25 μg/ml for impurity B. The official impurities C and D were mapped in all stress conditions. Additionally, impurity B was also seen in acidic conditions.Conclusion: The results from the study demonstrate that the method is suitable for evaluating the stability of NAC effervescent tablet.

Keywords: N-acetyl cysteine; Reverse phase HPLC; Effervescent formulation; Ion pair; chromatography; Related substances; Validation