HPLC-MS/MS法分析吉非替尼中痕量基因毒性杂质

目的：建立液相色谱-串联质谱法测定吉非替尼中基因毒性杂质的含量。方法：采用Accucore C₁₈色谱柱（2.1 mm&#215;100 mm，2.6 μm），以0.1%甲酸水溶液-0.1%甲酸乙腈溶液（70：30）为流动相，流速0.3 mL&#183;min^-1，柱温30 ℃；采用ESI离子源正离子模式，多反应离子监测（MRM）模式下选择离子对m/z130.1→83.1（3，4-二氟苯胺）和m/z146.0→111.0（4-氯-3-氟苯胺）进行测定。结果：基因毒性杂质3，4-二氟苯胺和4-氯-3-氟苯胺的线性浓度范围为1~24 ng&#183;mL^-1，且线性关系良好（r=0.999 4、r=0.999 2）；检测限为0.2 mg&#183;kg^-1，定量限为0.5 mg&#183;kg^-1；3，4-二氟苯胺和4-氯-3-氟苯胺的精密度试验的RSD（n=6）分别为2.3%和2.0%；杂质3，4-二氟苯胺低、中、高浓度的加标回收率（n=3）分别为99.4% 、97.0% 、105.5% ，RSD=8.2% 、5.1% 、1.7%；杂质4-氯-3-氟苯胺低、中、高浓度的加标回收率（n=3）分别为102.0% 、98.6% 、106.0% ，RSD分别为3.9% 、7.9% 、2.4%。经检测，3批吉非替尼供试品中3，4-二氟苯胺均未检出，批号2吉非替尼样品中4-氯-3-氟苯胺有少量检出，但低于样品的定量限（<0.5mg·kg^{-1）。结论：本方法操作简便，结果可靠，经方法学验证，可用于吉非替尼药物中基因毒性杂质3，4-二氟苯胺和4-氯-3-氟苯胺含量的同时测定。}

Objective: To establish an HPLC-MS/MS analytical method for the determination of genotoxic impurity in Gefitinib. Methods: The analytical column was ThermoAccucore C₁₈(2.1 mm&#215;100 mm, 2.6 μm), the mobile phase was 0.1% formic acidsolution-0.1% formic acid acetonitrile(70: 30), and the flow rate was 0.3mL&#183;min^-1. The column temperature was set at 30 ℃. The detection was achieved in ESI positive ion mode with m/z 130.1→83.1 for the determination of 3, 4-difluoroaniline and m/z 146.0→111.0 for 4-chloro-3-fluoroaniline. Results: The calibration curve of 3, 4-difluoroaniline and 4-chloro-3-fluoroaniline were in a good linearity over the range of 1-24 ng&#183;mL^-1(r=0.999 4 and 0.999 2, respectively). The limit of detection was 0.2 mg&#183;kg^-1, and the limit of quantification was 0.5 mg&#183;kg^-1. Repetition of genotoxic impurity was fine, with RSD(n=6)of 2.3% and 2.0%, respectively. The recoveries(n=3)of low, middle and high adding concentrations of 3, 4-difluoroaniline were 99.4%, 97.0%, 105.5%, respectively, and inter-day RSD were 8.2%, 5.1%, 1.7%, respectively. The recoveries(n=3)of low, middle and high adding concentrations of 4-chloro-3-fluoroaniline were 102.0%, 98.6%, 106.0%, respectively, and inter-day RSD were 3.9%, 7.9%, 2.4%, respectively. 3, 4-Difluoroaniline was not detected in 3 batches of samples and 4-chloro-3-fluoroaniline in batch number 2 was detected in trace amount, lower than the limit of quantification(<0.5 mg&#183;kg^-1). Conclusion: The established method is simple, rapid and reliable, which is applicable for both quantification of 3, 4-difluoroaniline and 4-chloro-3-fluoroaniline in Gefitinib.