高效液相色谱质谱联用法测定达比加群酯中间体2个基因毒性杂质

目的： 建立高效液相色谱质谱联用法测定达比加群酯中间体中具有基因毒性警示结构的杂质2-氨基吡啶和吡啶-2-氨基-丙酸乙酯含量。方法： 采用色谱柱为Zorbax Eclipse XDB C₁₈（4.6 mm×250 mm，5 μm）；以20 mmol·L^-1乙酸铵水溶液（乙酸调节pH至5.5）为流动相A，乙腈为流动相B，梯度洗脱；柱温40℃；流速1.0 mL·min^-1，分流进样；进样量20 μL。MS采用MRM（多反应监测）阳离子模式进行测定；气帘气压力为0.11 MPa；电压为5.5 kV；离子源温度为400.0℃；碰撞电压为30.00 V。结果： 在该色谱条件下，2-氨基吡啶和吡啶-2-氨基-丙酸乙酯与相邻杂质峰之间的分离度大于1.5；2-氨基吡啶和吡啶-2-氨基-丙酸乙酯的检测下限和定量下限分别均为0.000 002 5%、0.000 005%；通过添加标准回收试验，2-氨基吡啶和吡啶-2-氨基-丙酸乙酯的回收率分别为99.0%（RSD=2.7%）、99.0%（RSD=2.5%）（n=9）。结论： 该法简便、快速，灵敏，适用于达比加群酯中间体中具有基因毒性警示结构的杂质2-氨基吡啶和吡啶-2-氨基-丙酸乙酯的测定。

Objective: To establish a method for the determination of two genotoxic impurities of dabigatran etexilate intermediate-2-aminopyridine and pyridine-2-amino-ethyl propionate by high performance liquid chromatography-mass spectrometry. Methods: Gradient elution was used for analysis. A Zorbax Eclipse XDB C₁₈(4.6 mm×250 mm,5 μm)column was used. 20 mmol·L^-1 ammonium acetate solution was used as the mobile phase A,acetonitrile as the mobile phase B. The column temperature was 40℃,the flow rate was 1.0 mL·min^-1. Split injection was used and the injection volume was 20 μL. MS was measured by MRM(multi-reaction monitoring)cation mode. The air curtain pressure was 0.11 MPa,the voltage was 5.5 kV,the temperature of ion source was 400.0℃,and the collision voltage was 30.0 V. Results: Under this condition,the resolution factors between 2-aminopyridine,pyridine-2-amino-ethyl propionate and their adjacent peaks were more than 1.5. The lower detection limit and quantitative limit of 2-aminopyridine and pyridine-2-amino-ethyl propionate were 0.000 002 5% and 0.000 005% respectively. The recovery rate of 2-aminopyridine and pyridine-2-amino-ethyl propionate were 99.0%(RSD=2.7%)and 99.0%(RSD=2.5%)(n=9)respectively by standard adding recovery test. Conclusion: The method is simple,rapid and sensitive for the determination of 2-aminopyridine and pyridine-2-amino-ethyl propionate in dabigatran etexilate intermediate.

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