Abstract
ABSTRACT
Nitrosamine drug substance–related impurities (NDSRIs) pose a significant safety risk due to their mutagenic and carcinogenic potential, prompting strict regulatory oversight under ICH M7. The propensity for in situ formation of NDSRIs during analytical procedures, particularly in active pharmaceutical ingredients containing reactive amines, emphasizes the need for highly accurate and scrupulously validated methodologies. Melatonin, containing a vulnerable amine, was chosen as a model drug product. A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for quantifying N‐Nitroso melatonin. To prevent in situ formation, a scavenger was incorporated during sample preparation. Validation followed regulatory standards, assessing specificity, linearity, accuracy, precision, sensitivity, and robustness. The method showed excellent linearity (1.8–22.5 ng/mL,
R
2
= 0.9983), sensitivity with LOD of 0.9 ng/mL and LOQ of 1.8 ng/mL, and accuracy with recoveries between 85.3% and 95.9%. Precision was confirmed with %RSD below 2%, whereas robustness testing demonstrated consistent performance under varied conditions. Crucially, the scavenger eliminated false positives by suppressing in situ nitrosamine formation, ensuring data integrity. This validated approach strengthens analytical accuracy and provides a transferable framework for monitoring NDSRIs in other drug products containing reactive amines, supporting regulatory compliance and safeguarding patient health.