A Universal Strategy for Evaluation and Quantification of Potential Genotoxic Impurities of Hydrazine Derivatives in Isoniazid Injection.

Isoniazid is an irreplaceable first-line anti-tuberculosis drug. Its synthesis requires hydrazine as a starting material, which is classified as a Class 2 genotoxic impurity according to the ICH M7 guideline. Consequently, there is a risk of introducing potential genotoxic impurities (PGIs) containing hydrazine-related alerting structural during production. This study employed a systematic approach to assess the risks of PGIs in isoniazid and developed a novel LC-MS/MS method for accurate control and quantification of these impurities. First, candidate impurities were preliminarily predicted using quantitative structure-activity relationship (QSAR) systems based on expert rule and statistics. Subsequently, acceptable impurity limits were established based on the prediction results. Finally, an LC-MS/MS method was developed to quantify the selected PGIs. The results indicated that the impurities were classified as Class 3 (3,5-bis(4-pyridyl)-4-amino-1,2,4-triazole), Class 4 (benzohydrazide, picolinohydrazide, and nicotinohydrazide), and Class 5 (3,6-di(4-pyridyl)-1,4-dihydro-1,2,4,5-tetrazine). According to the ICH Q3B guidelines and the threshold of toxicological concern (TTC) based on the duration of administration, the limits of Class 4 and 5 impurities were set at 0.1%, and the limit of Class 3 impurities was set at 0.0066%. The established method demonstrated excellent linearity (r > 0.999) within the range of 0.2-25 ng/mL. And good recoveries were observed in the range of 88.1%-113.0%. The method was successfully applied to quantify impurities in 77 batches of isoniazid injections, all of which complied with the established acceptance limits. This universal strategy enhances quality control of isoniazid formulations, ensuring clinical safety through robust impurity assessment and validated analytical methodology.