目的: 建立HPLC-MS/MS法测定吲哚布芬中的4个基因毒性杂质:2-(4-硝基苯基)丁酸、2-(4-氨基苯基)丁酸、2-(3-氨基苯基)丁酸、2-(2-氨基苯基)丁酸。方法: 采用Agilent Eclipse Plus C18(4.6 mm×100 mm,3.5 μm)色谱柱,0.1%甲酸-5 mmol·L-1甲酸铵为流动相A,0.1%甲酸-甲醇为流动相B,梯度洗脱,流速为0.55 mL·min-1,进样体积为10.0 μL,柱温35 ℃;采用电子喷雾源ESI,负离子模式采集分析,多反应监测(MRM),离子源温度为550 ℃。结果: 4个基因毒性杂质的质量浓度在31.8~211.7、30.6~204.0、30.0~199.8、29.9 ~199.2 ng·mL-1范围内,r均大于0.990 0,峰面积与浓度线性关系良好;检测下限和定量下限的范围分别为1.06~10.2 ng·mL-1和12 ~20.4 ng·mL-1;平均加样回收率为94.2%~108.4%、94.7%~106.6%、96.4%~108.9%、83.2%~100.3%(n=3),RSD均不超过7.6%;重复性为3.2%~7.6%(n=6);中间精密度为3.4%~8.2%(n=12);对照品溶液和样品加标溶液在8 h内稳定性良好。实际样品中4个基因毒性杂质均未检出。结论: 该法简便、快速,灵敏高,专属性好,适用于吲哚布芬中基因毒性杂质的测定。
Objective: To establish an HPLC-MS/MS method for the determination of 4 genotoxic impurities in indobufe:2-(4-nitrophenyl) butyric acid, 2-(4-aminophenyl) butyric acid,2-(3-aminophenyl) butyric acid,2-(2-aminophenyl) butyric acid. Methods: The separation was performed on an Agilent Eclipse Plus C18 (4.6 mm×100 mm, 3.5 μm) column using the mobile phase consisting of 0.01% formic acid-5 mmol·L-1 ammonium formate (mobile phase A) and 0.01% formic acid-methanol (mobile phase B) by gradient elution at a flow rate of 0.55 mL·min-1. The column temperature was 35 ℃ and the injection volume was 10.0 μL. The electrospray was operated with the source ESI under negative ionization scanning mode. The temperature of ion source was 550 ℃. Results: The linearity ranges of four genotoxic impurities were 31.8-211.7,30.6-204.0, 30.0-199.8,29.9-199.2 ng·mL-1(r>0.990 0), respectively. The LODs and LOQs for the compounds ranged from 1.06 to 10.2 ng·mL-1 and from 2.12 to 20.4 ng·mL-1, respectively. The average recoveries were 94.2%-108.4%,94.7%-106.6%,96.4%-108.9%,83.2%-100.3%(n=3), respectively, with RSD not more than 7.6%. The repeatability and intermediate precision were between 3.2% and 7.6%(n=6) and 3.4% and 8.2%(n=6), respectively. The control solution and the labeled sample solution were stable after 8 h. None of the four genotoxic impurities were detected in the samples. Conclusion: The method is simple, rapid, sensitive, selective. It can be used to detect genotoxic impurities in indobufen.
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