Citation: QU Min-Min, CHEN Jia, XU Bin, ZHANG Ya-Jiao, XU Hua, XIE Jian-Wei. A Toxic Effect-Directed Screening Method for Genotoxic Impurities in Drugs Based on Mass Spectrometry Quantitative Analysis of Phosphorylated Histone H2AX[J]. Chinese Journal of Analytical Chemistry, ;2021, 49(9): 1531-1539. doi: 10.19756/j.issn.0253-3820.211096 shu

A Toxic Effect-Directed Screening Method for Genotoxic Impurities in Drugs Based on Mass Spectrometry Quantitative Analysis of Phosphorylated Histone H2AX

State Key Laboratory of Toxicology and Medical Countermeasures and Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China

Corresponding author: XU Hua, huarxu@163.com
Received Date: 31 January 2021
Revised Date: 6 April 2021

Fund Project: Supported by the National Key R&D Program of China (No.2018YFC1602600) and the National Natural Science Foundation of China(No.21974151).

Inspired by the fact that phosphorylated histone H2AX (γ-H2AX) has emerged as a useful biomarker for break of double-strand DNA. In this study, an isotope dilution-based liquid chromatography-tandem mass spectrometry screening method for genotoxic impurities (GTI) was established. The detection limits for H2AX and γ-H2AX target peptides were 1 ng/mL and 2 ng/mL, respectively. The accuracy and precision could meet the methodological requirement for biological samples analysis. This study focused on two kinds of important genotoxic impurities, ethylmethylsulfone (EMS) and N-nitrosodimethylamine (NDMA), in two human cell lines (HepG2 and HeLa) with different metabolic capabilities. The results showed that EMS had genotoxic characteristics in the two cell lines, while NDMA had genotoxicity only in HepG2 cells. The sensitivities of the method were 0.06 μg/g for EMS and 0.03 μg/g for NDMA, which satisfied the international detection limit standard (EMS:0.6 μg/g, NDMA:0.3 μg/g). The method was further applied to the screening of GTI in five commercially available tablets. The tablets were dissolved in water, followed by incubating with the cells to detect γ-H2AX level. The results showed that other unconcerned ingredients in these drugs did not interfere with the detection. This method was convenient, accurate and sensitive, and could be used for rapid detection of GTI in drugs.
- Mass spectrometry,
- Phosphorylated histone H2AX,
- Quantification,
- Genotoxic impurities,
- Screening
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