高效液相色谱-电喷雾电离串联质谱法测定克唑替尼原料药中的基因毒性杂质

doi:10.3724/SP.J.1123.2025.04017

摘要/Abstract

摘要：

建立了一种高效液相色谱-电喷雾电离串联质谱（HPLC-ESI-MS/MS）方法，用于测定克唑替尼原料药中潜在基因毒性杂质（WHT1408-Q2H）的含量。实验中利用Agilent Eclipse XDB C8色谱柱（150 mm×4.6 mm，3.5 µm），以0.1%甲酸水-0.1%甲酸乙腈为流动相，流速为0.4 mL/min，梯度洗脱，柱温为40 ℃，进样量5 μL；质谱部分采用电喷雾正离子（ESI⁺）多反应监测（MRM）扫描模式，杂质WHT1408-Q2H的［M+H］⁺母离子为m/z 205.3，碎片离子为m/z 121.0。结果显示，所建立的方法专属性良好，WHT1408-Q2H在2~40 ng/mL范围内呈现出良好的线性关系，相关系数（r）为0.999 9；检出限为0.396 9 ng/mL，定量限为1.984 6 ng/mL；在低、中、高3个水平下的回收率为95.6%~102.7%，RSD为0.4%~0.7%。应用该方法对3批克唑替尼原料药样品中的WHT1408-Q2H进行测定，3批样品中均未检出WHT1408-Q2H，表明当前生产工艺可有效控制该基因毒性杂质的含量。该方法专属性强，灵敏度高，操作简单，适用于克唑替尼原料药中基因毒性杂质WHT1408-Q2H的严格质量控制。依据M7基因毒性杂质指导原则，该方法能够准确定量测定痕量的遗传毒性杂质，将进一步确保药物符合监管要求并保障药物安全。未来该分析方法将扩展到其他治疗药物中类似的遗传毒性杂质评估，从而推进药物杂质分析和控制。

关键词: 非小细胞肺癌, 克唑替尼原料药, 基因毒性杂质, 高效液相色谱-电喷雾电离串联质谱

Abstract:

A method based on high performance liquid chromatography-electrospray ionization tandem mass spectrometry （HPLC-ESI-MS/MS） was developed for the determination of the potential genotoxic impurity （WHT1408-Q2H） in the bulk drug of crizotinib. An Agilent Eclipse XDB C8 chromatographic column （150 mm×4.6 mm， 3.5 µm） was used for chromatographic separation. The mobile phases were 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile solution at a flow rate of 0.4 mL/min. The column temperature was maintained at 40 ℃ and the sample size was 5 μL. In the mass spectrometry section， the electrospray positive ion （ESI⁺） mode with multiple reaction monitoring （MRM） scanning was adopted. The accurate mass of the ［M+H］⁺ parent ion of WHT1408-Q2H was m/z 205.3， and the accurate mass of the extracted fragment ion was m/z 121.0. The results of methodological validation demonstrated that the established method exhibited excellent specificity. The peak area and mass concentration of WHT1408-Q2H exhibited a good linear relationship within the range of 2-40 ng/mL， with a correlation coefficient （r） of 0.999 9. The limit of detection （LOD） and limit of quantitation （LOQ） for WHT1408-Q2H were 0.396 9 ng/mL and 1.984 6 ng/mL， respectively. The recoveries of WHT1408-Q2H at low， medium， and high levels were in the range of 95.6%-102.7%， while the relative standard deviations （RSDs） were between 0.4% and 0.7%. Finally， the proposed method was successfully applied to analyze three independent batches of the bulk drug of crizotinib. The results revealed that WHT1408-Q2H was not detected in all samples， indicating that the current production process can effectively control the content of this genotoxic impurity. In conclusion， the developed HPLC-ESI-MS/MS method is highly specific， sensitive， and simple， making it suitable for the stringent quality control of WHT1408-Q2H in the bulk drug of crizotinib. According to the M7 guideline on genotoxic impurities， this method is capable of accurately quantifying trace amounts of genotoxic impurities and will further ensure compliance with regulatory requirements and safeguard drug safety. Future applications may extend this analytical framework to similar genotoxic impurities assessment in other therapeutic compounds， thereby advancing the field of pharmaceutical impurity profiling and control.

Key words: non-small cell lung cancer, the bulk drug of crizotinib, genotoxic impurity, high performance liquid chromatography-electrospray ionization tandem mass spectrometry （HPLC-ESI-MS/MS）

中图分类号:

O658

占小兵, 秦怡, 颜瀅, 周婕, 赵龙山. 高效液相色谱-电喷雾电离串联质谱法测定克唑替尼原料药中的基因毒性杂质[J]. 色谱, 2025, 43(11): 1262-1267.

ZHAN Xiaobing, QIN Yi, YAN Ying, ZHOU Jie, ZHAO Longshan. Determination of genotoxic impurity in the bulk drug of crizotinib by high performance liquid chromatography-electrospray ionization tandem mass spectrometry[J]. Chinese Journal of Chromatography, 2025, 43(11): 1262-1267.

图/表 7

图1 基因毒性杂质WHT1408-Q2H的警示结构

Fig. 1 Warning structure of genotoxic impurity WHT1408-Q2H

图2 以（a）0.8 mL/min与（b）0.4 mL/min作为流速时对照品溶液的TIC色谱图

Fig. 2 TIC chromatograms of a control solution at flow rates of （a） 0.8 mL/min and （b） 0.4 mL/minColumn： Agilent Eclipse XDB C8 column （150 mm×4.6 mm， 3.5 μm）； mobile phase： 0.1% formic acid aqueous solution （A）-0.1% formic acid acetonitrile solution （B）. Gradient elution： 0-5.0 min， 30%B-95%B； 5-7 min， 95%B； 7-7.01 min， 95%B-30%B； 7.01-11.0 min， 30%B. The column temperature was set at 40 ℃， and the injection volume was 5 μL.

图3 优化条件下WHT1408-Q2H对照品溶液的TIC色谱图

Fig. 3 TIC chromatogram of WHT1408-Q2H control solution under optimized conditionsGradient elution： 0-10.0 min， 10%B-95%B； 10.0-10.1 min， 95%B-10%B； 10.1-15.0 min， 10%B. Other conditions are the same as in Fig. 2.

图4 优化条件下供试品溶液的TIC色谱图

Fig. 4 TIC chromatogram of test solution under optimized conditionsThe chromatogram conditions are the same as in Fig. 3.

图5 WHT1408-Q2H的二级质谱图

Fig. 5 MS2 spectrum of WHT1408-Q2H

图6 （a）空白溶剂、（b）对照品溶液和（c）供试品溶液的TIC图

Fig. 6 TIC chromatograms of （a） a blank solvent，（b） control solution and （c） test solutionConditions are the same as in Fig. 3.

表1 克唑替尼中杂质WHT1408-Q2H的加标回收率（n=3）

Table 1 Spiked recoveries of the impurity WHT1408-Q2H in crizotinib （n=3）

Added/（ng/mL）	Recovery/%	RSD/%
9.9231	95.6	0.7
19.8462	100.2	0.4
29.7693	102.7	0.4

[1]	孙春艳, 纪颖鹤, 秦昆明, 高珣, 赵龙山. 液相色谱-串联质谱法测定吉非替尼中4种基因毒性杂质[J]. 色谱, 2019, 37(12): 1297-1304.
[2]	刘雪薇, 厉程, 韩海云, 张文鹏, 陈东英. 药物中磺酸酯类基因毒性杂质研究进展[J]. 色谱, 2018, 36(10): 952-961.
[3]	杨华梅, 杭莉. 超高效液相色谱-串联质谱法同时测定食品中4种常用香精[J]. 色谱, 2015, 33(3): 250-255.
[4]	陈婷, 温裕云, 欧延, 弓振斌. 固相萃取净化及超高效液相色谱-串联质谱法测定橡胶制品中的13种N-亚硝胺[J]. 色谱, 2014, 32(1): 89-94.