HPLC-MS/MS法检测利伐沙班中痕量基因毒性杂质

Detection of trace genotoxic impurities in rivaroxaban by HPLC-MS/MS

目的:建立HPLC-MS/MS法检测利伐沙班中基因毒性杂质(S)-5-氯-N-(3-氯-2-羟丙基)噻吩-2-酰胺和4-(3-氧代-4-吗啉基)硝基苯的方法,系统考察影响杂质分离和质谱响应的因素。方法:采用ZORBAX SB-C_(18)(150 mm×2.1 mm, 3.5μm)色谱柱,以10 mmol·L^(-1)甲酸铵溶液-甲醇为流动相,梯度洗脱,流速0.4 mL·min^(-1);电喷雾正离子化MRM检测。结果:(S)-5-氯-N-(3-氯-2-羟丙基)噻吩-2-酰胺和4-(3-氧代-4-吗啉基)硝基苯质量浓度分别在0.008~3.4 ng·mL^(-1)和0.2~3.4 ng·mL^(-1)范围内线性关系良好,平均回收率(n=9)分别为97.9%和98.9%,RSD分别为0.94%和0.68%;定量限分别为0.008 ng·mL^(-1)和0.2 ng·mL^(-1);15批利伐沙班样品中基因毒性杂质残留量检测结果均远低于限度值。结论:该方法灵敏度高,专属性强,可有效检测和控制利伐沙班中痕量基因毒性杂质的含量。

Objective:To establish an HPLC-MS/MS method for the determination of the genotoxic impurities(S)-5-chloro-N-(3-chloro-2-hydroxypropyl) thiophene-2-amide and 4-(3-oxy-4-morpholine) nitrobenzene in rivaroxaban. The factors affecting impurities separation and the response of mass spectrometry were systematically investigated. Methods: A ZORBAX SB-C_(18) column(150 mm×2.1 mm, 3.5 μm) was used with 10 mmol·L^(-1 )ammonium formate solution-methanol as mobile phase at flow rate of 0.4 mL·min^(-1). And MRM detection by electrospray positive ionization was used. Results:(S)-5-chloro-N-(3-chloro-2-hydroxypropyl) thiophene-2-amide and 4-(3-oxy-4-morpholine) nitrobenzene had good linear relationships in the range of 0.008-3.4 ng·mL^(-1) and 0.2-3.4 ng·mL^(-1), respectively. The average recoveries(n=9) were 97.9% and 98.9%, RSD were 0.94% and 0.68% respectively. The limits of quantitation were 0.008 ng·mL^(-1) and 0.2 ng·mL^(-1) respectively. The results of genotoxic impurity residue determination in 15 batches of rivaroxaban samples were far below the limit value. Conclusion: The method is sensitive and specific, and can effectively detect trace genotoxic impurities in rivaroxaban.