Valsartan—Mutagenic Nitroso Drug Substance Related Impurity, Structural Evaluation, Carcinogenic Potency Categorization Assessment; a Novel and Extremely Sensitive Quantification Method for Impurity in Drug Substances Using LC‐MS/MS

ABSTRACT Nitrosamines and nitroso‐drug substance‐related impurities are known for their direct carcinogenic and mutagenic potential in humans, prompting global regulatory agencies to enforce stringent control strategies. Valsartan, an angiotensin II receptor antagonist, has been identified as a drug compound with potential for nitroso‐drug substance‐related impurity formation due to its chemical structure. This study aimed to evaluate the structural characteristics of valsartan for potential impurity formation called as N‐Nitroso Desvaleryl Valsartan and establish acceptable intake limits using carcinogenic potency categorization assessment. In addition, the study focused on the development and validation of a highly sensitive and reliable LC‐MS/MS method for quantifying N‐Nitroso Desvaleryl Valsartan. Structural analysis revealed that valsartan degrades under acidic conditions, losing its valeric acid moiety to form a desvaleryl impurity. This impurity contains a secondary amine functional group, which can undergo nitrosation to yield N‐Nitroso Desvaleryl Valsartan. Based on carcinogenic potency categorization assessment, the impurity falls into carcinogenicity Category 5, with a permitted daily intake of 1500 ng/day. However, method validation was conducted considering a stricter limit of 96 ng/day as per recent regulatory guidance. The developed LC‐MS/MS method utilized a gradient elution with formic acid and acetonitrile on an Agilent's, Zorbax SB‐C18 column (150 mm × 4.6 mm, 3 µm). The method exhibited excellent linearity ( r = 0.999), precision, and recovery within 80%–120%. In conclusion, this study establishes a robust analytical approach to detect trace levels of impurity in valsartan, ensuring regulatory compliance and patient safety. The validated method demonstrated high sensitivity, precision, and reliability, making it suitable for routine quality control applications.