Determination of genotoxic impurity in the bulk drug of crizotinib by high performance liquid chromatography-electrospray ionization tandem mass spectrometry

doi:10.3724/SP.J.1123.2025.04017

Abstract

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）

CLC Number:

O658

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.

Figures/Tables 7

Fig. 1 Warning structure of genotoxic impurity WHT1408-Q2H

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.

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.

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

Fig. 5 MS2 spectrum of WHT1408-Q2H

Fig. 6 TIC chromatograms of （a） a blank solvent，（b） control solution and （c） test solutionConditions are the same as in Fig. 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]	SUN Chunyan, JI Yinghe, QIN Kunming, GAO Xun, ZHAO Longshan. A high-performance liquid chromatography-tandem mass spectrometry method for the quantitative determination of four genotoxic impurities in gefitinib [J]. Chinese Journal of Chromatography, 2019, 37(12): 1297-1304.
[2]	LIU Xuewei, LI Cheng, HAN Haiyun, ZHANG Wenpeng, CHEN Dongying. Advances on genotoxic impurities of sulfonate esters in pharmaceuticals [J]. Chinese Journal of Chromatography, 2018, 36(10): 952-961.
[3]	YUE Lijun, WEI Yuxia, CHEN Jia, LIN Nini, WANG Rui, YANG Xi, ZHANG Yajiao, XIE Jianwei*. Biomonitoring method of dermal exposure to sulfur mustard based on DNA adduct in lung of SD rats by high performance liquid chromatography-electrospray ionization tandem mass spectrometry [J]. Chinese Journal of Chromatography, 2011, 29(04): 325-329.