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目的:建立测定恩替卡韦有关物质的梯度洗脱反相高效液相色谱法。方法:采用Waters C18色谱柱(5μm,4.6mm×250mm),以乙腈-水(3∶97)为流动相A,乙腈为流动相B;梯度洗脱条件:0～10min,A从100%→90%;10～15min,A为90%;15～25min,A从90%→60%;25～28min,A从60%→100%;以1.0mL·min-1的流速进行梯度洗脱,检测波长为254nm。结果:在上述色谱条件下,恩替卡韦与各中间体杂质及降解杂质均能有效分离,分离度大于2.0;检测限为0.4ng,精密度良好(RSD为0.8%)。结论:本方法操作简便,专属性强,灵敏度高,可用于恩替卡韦有关物质的测定。
Objective: To establish a gradient elution RP-HPLC method for the determination of entecavir related substances. METHODS: Waters C18 column (5μm, 4.6mm × 250mm), acetonitrile-water (3:97) was used as mobile phase A and acetonitrile was used as mobile phase B. The gradient elution conditions were 0-10min, A ranged from 100% → A was 90%; A was 90%; A was from 90% to 60%; A was from 60% to 100%; A was eluted with a gradient of 1.0 mL · min -1 from 15 to 25 min, Detection wavelength of 254nm. Results: Under the above chromatographic conditions, the entecavir and each intermediate impurity and degradable impurity could be effectively separated with the resolution greater than 2.0. The detection limit was 0.4ng and the precision was good (RSD was 0.8%). Conclusion: The method is simple, specific, sensitive and can be used for the determination of entecavir related substances.