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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×100 mm,2.6μm),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·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·mL~(-1),且线性关系良好(r=0.999 4、r=0.999 2);检测限为0.2 mg·kg~(-1),定量限为0.5 mg·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(<0.5 mg·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 a method for the determination of genotoxic impurities in gefitinib by liquid chromatography-tandem mass spectrometry. Methods: The mobile phase consisted of a mobile phase of Accucore C18 (2.1 mm × 100 mm, 2.6 μm) with 0.1% formic acid in water and 0.1% formic acid in acetonitrile (70:30) at a flow rate of 0.3 mL · min -1 Temperature was 30 ℃. The ion pair m / z130.1 → 83.1 (3,4-difluoroaniline) and m / z146.0 → 111.0 (4) were selected by using ESI ion source positive mode and MRM mode -chloro-3-fluoroaniline). Results: The linear range of genotoxic impurities 3,4-difluoroaniline and 4-chloro-3-fluoroaniline ranged from 1 ng / mL to 24 ng · mL -1 with good linearity (r = 0.999 4, r = 0.999 2). The detection limit was 0.2 mg · kg -1 and the limit of quantification was 0.5 mg · kg -1. The precision test of 3,4-difluoroaniline and 4-chloro-3-fluoroaniline RSD (n = 6) were 2.3% and 2.0%, respectively. The recoveries (n = 3) were 99.4%, 97.0% and 105.5% for the 3,4- RSD was 8.2%, 5.1% and 1.7% respectively. The recoveries (n = 3) were 102.0%, 98.6% and 106.0% for the impurity 4-chloro-3- 3.9%, 7.9%, 2.4%. After testing, 3 batches of gefitinib were not detected 3,4-difluoroaniline in the test sample, batch 2 gefitinib samples 4-chloro-3-fluoroaniline a small amount of detected, but lower than the sample Of the limit of quantitation (<0.5 mg · kg -1). Conclusion: The method is simple, reliable and can be applied to the simultaneous determination of genotoxic impurities 3,4-difluoroaniline and 4-chloro-3-fluoroaniline in gefitinib.