Innovative QSRR modeling approach for the development of an ultra-sensitive LC-MS/MS method for trace analysis of N-nitrosamines

To address regulatory concerns regarding N-nitrosamine contamination in pharmaceutical products, generic LC-MS/MS methods for determining N-nitrosamines were developed using an innovative in silico approach based on Quantitative Structure Retention Relationship modeling (QSRR). The development process included screening and optimization phases, offering flexibility in targeting N-nitrosamines and addressing the challenges related to the matrix effect. This methodology represents a significant advancement in method development. Among the developed methods, a highly sensitive and accurate LC-MS/MS method was successfully validated to simultaneously determine 5 small-molecule N-nitrosamine impurities in tablets, which was used in the present proof-of-concept study. The validation followed the ICH Q2 (R2) guidelines, employing a combined approach for accuracy and precision based on total error risk-based methodology. The method was validated to function as both an impurity limit test and a quantitative method. Validation results demonstrated adequate quantitative performance of the method, establishing a validated dosing range from 1 to 30 ng/mL for all N-nitrosamines. The estimated detection limit ranged from 0.75 pg/mL to 0.02 ng/mL. The detection and quantification limits for each N-nitrosamine met the EMA N-nitrosamine investigation approach requirements. Moreover, both are always below 10 % of their respective acceptable limit in the studied finished product formulation. This proposed method is suitable for investigating small-molecule N-nitrosamines in pharmaceutical products and also provides a starting point for further method development, particularly for the determination of newly identified small-molecule N-nitrosamines and drug-substance-related N-nitrosamines.