Abstract

In this study, we synthesized and characterized N-nitrosochlordiazepoxide, a potential genotoxic impurity originating from chlordiazepoxide hydrochloride, assessing its toxicity via quantitative structure-activity relationship (QSAR) analysis. Characterization techniques including UV spectroscopy and HPLC demonstrated purity at 97.0%, complemented by Fourier transform infrared spectroscopy analysis indicating characteristic peaks at 1,500 cm-1 (nitroso group) and 945 cm-1 (N-O bond), as well as mass spectrometry (m/z 329.2 [M+H]+) and NMR. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, utilizing an HPLC octyldecylsilane column, revealed ion transitions from (Q1) m/z 329.20 to (Q3) m/z 281.90, 299.25, and 240.80. Method validation confirmed linearity (0.18–3 ppm) following The International Conference of Harmonization Q2 (R1) guidelines, with LOD and LOQ determined at 0.18 ppm and 0.375 ppm, respectively. Robustness studies showed

Key words: N-Nitrosochlordiazepoxide, Potential genotoxic impurity, Synthesis, Characterization, LC-MS/MS, method Validation.