Genotoxic impurities, Analytical Method development & Validations

Recently, genotoxic impurities manifestation in active pharmaceutical ingredient (API), as well as products of drugs, received more attention by industries and various governing agencies because of their toxic effect on human health. Even lower concentration (<1 ppm) of genotoxic impurities causes hereditary transformations, chromosome disruptions and leads to oncological diseases. In general, the sources of organic genotoxic impurities in drugs are starting materials, reagents, intermediates, degradation products and by-products of the production of the drug. As per the recommendations of ICH, there is a need to remove the impurities or to reduce their content to a safe level. As per the guidelines of ICH M7, FDA and EMA, the acceptable level of genotoxic impurities is calculated based on the toxicological concern and is considered safe for lifetime use is 1.5 μg day −1. Hence there is a need for chemists to focus development of the manufacturing process of these drugs on minimizing such genotoxic impurities, as it is not possible to eliminate genotoxic impurities completely from the synthetic scheme.

Fluconazole is used for the treatment of systemic and superficial fungal infections, by inhibiting the action of fungal cytochrome
enzyme [5-7]. Fluconazole active pharmaceutical ingredient was commercially prepared by reacting its intermediate 1-[2-(2,4-
difluor-ophenyl)-2,3-epoxypropyl]-1H-1,2,4-triazole, genotoxic impurity] with triazole in the presence of a base [8]. The scheme of
the synthesis of fluconazole is presented in fig. 1. From the scheme given in fig. 1, an intermediate of the fluconazole was prepared from

1-(2,4-difluorophenyl)-

2-[1,2,4]triazol-1-yl-ethanone (precursor of intermediate, in the scheme it is represented as compound

3). As per the study of Muller et al. , this intermediate belongs to group 2 of genotoxic impurities and may cause the unwanted genotoxicity and carcinogenicity. According to the study of micronucleus assay by Fucic et al. fluconazole was caused genome damage in young animals and in new-born pups. So, quantitative determination of this genotoxic impurity and its precursor in fluconazole at lower levels is important for organic chemists to control them during the synthesis of fluconazole. Here onwards the impurities, 1-(2,4-difluorophenyl)- 2-[1,2,4]triazol-1-yl-ethanone (precursor of genotoxic impurity) and 1-[2-(2,4-difluorophenyl)-2,3-epoxypropyl]-1H-1,2,4-triazole (genotoxic impurity) are abbreviated as impurity A and impurity B in the manuscript.