液相色谱结合液质联用法测定酮康唑原料药及复方酮康唑软膏中的有关物质

摘要：目的 建立一种复方酮康唑软膏中有关物质的液相色谱及液相色谱质谱测定方法。方法 采用Agilent HPLC，选择 ZORBAX Eclipse Plus C18色谱柱(250mm×4.6mm，5mm)，以10mmol/L四丁基硫酸氢铵溶液-乙腈(85:15)为流动相A，10mmol/L 四丁基硫酸氢铵溶液-乙腈(20:80)为流动相B，流速为1mL/min，检测波长为220nm。经优化流动相比例确定了线性梯度洗脱， 开展了专属性、线性、准确度、精密度、重复性、稳定性和耐用性等方法学研究。并将该方法应用于酮康唑原料药及复方酮康 唑软膏中有关物质的检测。在液相色谱-四级杆飞行时间质谱方法中，选择0.1%甲酸水溶液为流动相A，乙腈为流动相B，梯度 洗脱，通过二级质谱数据推导了酮康唑的裂解规律。结果 通过检测酮康唑原料药、复方酮康唑软膏并结合降解实验结果发 现，酮康唑与相邻杂质均可实现基线分离。根据确定的HPLC方法对比了不同来源复方酮康唑软膏有关物质的差异。建立了基 于高分辨质谱的酮康唑及其杂质的结构解析方法，修正了酮康唑及杂质E的特征子离子的产生过程及结构，并确认了酮康唑的 主要降解杂质为杂质D。结论 本方法灵敏度较高、重复性较好，为复方酮康唑软膏的质量控制提供了一种可行的分析方法， 适用于该产品的质量控制。基于高分辨质谱的结构解析方法也为酮康唑中其他杂质的结构推断提供了研究基础。

Analysis of the related substances in ketoconazole bulk sample and compound ketoconazole cream by HPLC and HPLC-MS

Objective A suitable HPLC and HPLC-MS method were developed to analyze the related substances in ketoconazole bulk sample and compound ketoconazole cream. Methods The separation was performed on Agilent HPLC with a ZORBAX Eclipse Plus C18 (250mm×4.6mm, 5mm) using 0.34% (W/V) tetrabutylammonium hydrogen sulfate-acetonitrile (85:15) as mobile phase A and 0.34% (W/V) tetrabutylammonium hydrogen sulfate acetonitrile (20:80) as mobile phase B at a flow rate of 1mL/min under gradient elution. The detection wavelength was set at 220nm. The HPLC method was optimized by adjusting the ratio of mobile phases. In addition, the specificity, linearity, accuracy, precision, repeatability, stability, and durability were studied. The HPLC method was applied to the determination of related substances in ketoconazole bulk sample and compound ketoconazole cream. The method of HPLC-Q/TOF-MS with 0.1% formic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B under gradient elution was used to confirm the structures of ketoconazole and the main degradation impurities. Results The established HPLC method verified by the methodology was suitable for analyzing the related substances in ketoconazole bulk sample and compound ketoconazole cream. Baseline separations between the adjacent impurities and ketoconazole were achieved. The content of the maximum impurity in different batches of ketoconazole was compared. Based on high-resolution mass spectrometry, a fragment pathway was established for the structure elucidation of ketoconazole and its impurities. The characteristic ions of ketoconazole and impurity E were modified. The main degradation impurity of ketoconazole was confirmed as impurity D. Conclusion The sensitive HPLC method was suitable for analyzing the related substances in ketoconazole and provided a valuable idea for the quality control of the product. The structure elucidation method based on high-resolution mass spectrometry also provided the basis for the structure inference of other impurities in ketoconazole bulk sample and compound ketoconazole cream.