Stability-indicating and liquid chromatography–mass spectrometry compatible ultra high performance liquid chromatography method was developed for the degradation and drug substances related impurities of Prothionamide. Forced degradation of Prothionamide was carried out under acidic, basic, thermal, oxidative, and photolytic stress conditions. The impurities separation was achieved on Acquity UPLC BEH-C18 (50 mm × 2.1 mm, 1.7 μm) with the mobile phase of 10 mm ammonium acetate pH 6.0 and Acetonitrile in a time gradient mode. Related substances by ultra-performance liquid chromatography method was validated according to ICH tripartite guidelines. Degradation products were isolated by Column chromatography and characterized by liquid chromatography–mass spectrometry, ¹H, and ¹³C nuclear magnetic resonance spectroscopy. The developed related substances method showed adequate specificity, sensitivity, accuracy, linearity (0.4–1.5 μg/mL), precision, and robustness in line with ICH tripartite guidelines for validation of analytical procedures. Limits of detection and quantitation were 0.1 and 0.4 μg/mL, respectively, for Prothionamide and all the impurities. The method was found to be linear with a correlation coefficient > 0.99, precise (%RSD < 5.0), robust and accurate (%recovery 85–115%).

中文翻译：

---

通过质谱、核磁共振光谱和超高效液相色谱方法开发鉴定和表征丙硫磷降解杂质

开发了稳定性指示和液相色谱-质谱兼容的超高效液相色谱方法，用于检测丙硫磷的降解和原料药相关杂质。Prothionamide 的强制降解在酸性、碱性、热、氧化和光解胁迫条件下进行。杂质分离是在 Acquity UPLC BEH-C18（50 mm × 2.1 mm，1.7 μm）上实现的，流动相为 10 mm 醋酸铵 pH 6.0 和乙腈，以时间梯度模式进行。超高效液相色谱法相关物质根据ICH三方指南进行验证。降解产物通过柱色谱分离，并通过液相色谱-质谱、¹ H 和¹³C 核磁共振波谱。开发的相关物质方法显示出足够的特异性、灵敏度、准确度、线性 (0.4–1.5 μg/mL)、精密度和稳健性，符合 ICH 三方分析程序验证指南。Prothionamide 和所有杂质的检测限和定量限分别为 0.1 和 0.4 μg/mL。发现该方法呈线性，相关系数 > 0.99、精确 (%RSD < 5.0)、稳健且准确（% 回收率 85–115%）。