Analysis of Genotoxic Impurities

Genotoxic impurities can directly act on DNA in the human body, causing DNA damage and having carcinogenic, teratogenic, or mutagenic properties. A small amount of genotoxic impurities can also cause great damage to the human body. The study of genotoxic impurities has attracted the wide attention of drug researchers.


  1. Quantitative analysis

Because genotoxic impurities are so dangerous, the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have established a toxicological concern threshold (TTC) that humans should not consume more than 1.5 μg per day of potentially toxic impurities for long-term use.


According to the above regulations, the daily dosage of the drug is 200mg, and the mass fraction of toxic impurities should not exceed 7.5-×10-6, so the quantitative analysis of genotoxic impurities requires trace analysis methods.


The commonly used UV detector can detect the minimum mass fraction of 5×10-4 impurities. However, with the invention of 2D-HPLC and the use of high-sensitivity UV detectors, trace impurities in drugs can also be accurately quantified by one-dimensional liquid phase analyzer separation, solid phase extraction technology enrichment, and two-dimensional liquid phase analyzer high-sensitivity detection.


However, this method requires multiple high-concentration injections and is not suitable for impurity content determination. Therefore, mass spectrometry detection technology is an important prerequisite for the establishment of quantitative analysis methods for genotoxic impurities. Kakasaheb et al. quantitatively determined the genotoxic impurities in the synthesis starting material of candesartan by GC-MS, and the methodological verification results could meet the requirements of ICH regulations. This method is suitable for the determination of the content of genotoxic impurities in marketed drugs.


In the process of establishing a method for trace impurities, the pre-treatment of the sample is very important, and the loss of trace impurities may have a great impact on the results. Devenport et al. has used mass spectrometry under atmospheric pressure to directly quantify the genotoxic impurities in simulated drugs. This method makes the sample determination more convenient and can realize the high-throughput detection of drugs.


  1. Determination of toxic impurities

In 2006, EMA issued guidelines for genotoxicity limits, and since then, ICH and FDA have also proposed research and control programs for genotoxic impurities. According to the relevant guidelines, the toxicity assessment of impurities is required even if the content of impurities is below the specified quantitative requirements.


Toxicity assessment of compounds is achieved through in vitro bacterial toxicity assays, and guidelines recommend the use of impurity monomers for studies. However, toxic impurities in drugs are present in trace or trace amounts, and obtaining impurity monomers by methods such as preparation is time-consuming and costly.


To this end, the FDA and other companies have jointly developed computer software to assess the toxicity of compounds. This software does not need to obtain monomers. After the structure of the impurity is identified, the toxicity assessment software can be used to evaluate the structure of the impurity for the presence of warning structures.


In terms of software, FDA recommends the use of MC4PC, MDL-QSAR, and Derk for Windows for evaluation. For accurate toxicity assessment, each software has a specific algorithm and scope of application.


The MC4PC software divides the structure to be evaluated into two to three (non-hydrogen) atom structural fragments, and then compares these fragments with the known toxic compound fragment database, makes the toxicity score of the fragments in the impurity structure, describes the molecular characteristics of the structure, and finally generates a comprehensive and professional report to evaluate the structural toxicity of the impurity.


Although MC4PC can assess the toxicity of drugs from the perspective of the fine structure of impurities, it can only search the same fragment in the existing database for comparison, and cannot comprehensively evaluate the structure of more than one unknown fragment.


The results of toxic compounds show that some electrophilic groups or structures can be activated to electrophilic groups in the structure of toxic compounds. MDL-QSAR toxicity assessment software is based on the correlation between the toxicity of compounds and the electrophilic ability of compounds. This software can calculate the quantitative relationship between the structure of compounds and their toxicity. It can also predict the results of in vitro bacterial toxicity experiments.


However, to achieve the most accurate assessment of the toxicity of impurities with known structure, it is necessary to find the most suitable model for calculation when using MDL-QSAR software. Therefore, since the proposed calculation software, there have been many reports on different data models and their evaluation.


Although some impurities are evaluated by the computer as potential toxic impurities with warning structure, the evaluation software will over-evaluate the toxicity of the impurities in the calculation process, so it is still necessary to conduct in vitro bacterial reverse mutation experiment (Ames experiment) or mammalian cell analysis to determine whether the drug impurity is toxic. FDA gives detailed instructions on the process of this experiment.


  1. Control methods

Following the EMA guidelines, all compounds present in the drug should pass a compound toxicity assessment or control method conforming to the guidelines, and Ames tests are required to determine whether a potentially toxic impurity is a toxic impurity.



Impurity research plays an important role in the process of drug development, which not only affects the speed of new drugs to market but also affects the safety of human medication. With the continuous development of analytical techniques, the strategies for impurity research have become more and more diverse. With the improvement of the resolution and sensitivity of the detector, the quantification of impurities has realized the leap from trace to trace, and the qualitative work of impurities has also realized the transformation from offline to online detection. However, the rapid development of new drugs, the continuous emergence of biological drugs, and the requirements for rapid qualitative and quantitative analysis will bring great challenges to researchers.


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