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目的建立同时测定灯盏乙素乙酯原料药主成分及其有关物质含量的反向高效液相色谱法,并采用UPLC-MS对灯盏乙素乙酯的主要有关物质进行结构鉴定。方法采用Kromasil C18色谱柱(4.6 mm×250 mm,5μm)对主成分及其有关物质进行含量测定,柱温25℃,以甲醇-0.2%甲酸水溶液为流动相,梯度洗脱,流速1.0 m L·min-1,检测波长为335 nm;采用高分辨液质联用仪对其主要有关物质进行结构鉴定。结果灯盏乙素乙酯与有关物质及其降解产物的分离度良好;灯盏乙素乙酯质量浓度在5.005~1 001 mg·L-1内与峰面积呈良好线性关系,r2=0.999 9,平均回收率为99.6%;灯盏乙素乙酯原料药中的主要有关物质为灯盏乙素,含量为0.89%,杂质总量为1.14%。结论该方法操作简便、快速、准确、灵敏度高,可用于灯盏乙素乙酯原料药的含量测定及有关物质的检查。
OBJECTIVE To establish a reversed-phase high performance liquid chromatography (HPLC) method for the simultaneous determination of the major components of scutellarin ethyl ester and its related substances, and to identify the main related substances of ethyl scutellarein by UPLC-MS. Methods The main components and their related substances were determined by Kromasil C18 column (4.6 mm × 250 mm, 5 μm). The column temperature was 25 ℃ and the mobile phase consisted of methanol-0.2% formic acid as mobile phase with a gradient of 1.0 mL · Min-1, the detection wavelength was 335 nm. The structure of the main related substances was identified by high-resolution liquid chromatography-mass spectrometry. Results The separation of ethyl acetate from related substances and their degradation products was good. The mass concentration of ethyl acetate from 5.005 to 1 001 mg · L-1 showed a good linear relationship with the peak area, r2 = 0.999 9, the average The recovery rate was 99.6%. The main related substance in the scutellarin ethyl ester raw material was scutellarin with a content of 0.89% and a total amount of impurities of 1.14%. Conclusion The method is simple, rapid, accurate and sensitive. It can be used for the determination of scutellarin and its related substances.