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目的:建立高效液相色谱法测定利伐沙班中苯胺类潜在基因毒性杂质4-(4-氨基苯基)-3-吗啉酮。方法:采用Hedera~(TM)苯基硅烷键合相色谱柱(250mm×4.6mm,5μm),流动相A为乙腈,流动相B为乙腈-10 mmol·L~(-1)磷酸二氢钠溶液(pH 3.5)(10:90),梯度洗脱,流速1.2mL·min~(-1),柱温30℃,检测波长242 nm。结果:杂质4-(4-氨基苯基)-3-吗啉酮质量浓度在20.12~201.2 ng·mL~(-1)范围内线性关系良好(r=0.998 1),检测限为10.10 ng·mL~(-1),定量限为30.30 ng·mL~(-1),平均回收率为97.4%(RSD=2.8%,n=9),重复性良好(RSD=2.0%,n=6)。3批原料药中未检出4-(4-氨基苯基)-3-吗啉酮。结论经方法学验证,本法适用于利伐沙班中4-(4-氨基苯基)-3-吗啉酮的测定。
Objective: To establish a method for the determination of 4- (4-aminophenyl) -3-morpholinone, an aniline potential genotoxic impurity in rivaroxaban by high performance liquid chromatography. METHODS: Hedera ~ (TM) phenylsilane bonded phase (250 mm × 4.6 mm, 5 μm) was used. The mobile phase A was acetonitrile and the mobile phase B was acetonitrile-10 mmol·L -1 sodium dihydrogen phosphate (PH 3.5) (10:90). The gradient elution was carried out at a flow rate of 1.2 mL · min -1. The column temperature was 30 ℃ and the detection wavelength was 242 nm. Results: The linear range of the impurity 4- (4-aminophenyl) -3-morpholinone ranged from 20.12 to 201.2 ng · mL -1 (r = 0.998 1) with a detection limit of 10.10 ng · The average recovery was 97.4% (RSD = 2.8%, n = 9) with good reproducibility (RSD = 2.0%, n = 6) . No 4- (4-aminophenyl) -3-morpholinone was detected in 3 batches of APIs. Conclusion This method is validated by the method of law and is applicable to the determination of 4- (4-aminophenyl) -3-morpholinone in rivaroxaban.