Ultra‐Sensitive and Green LC–MS/MS Method for Trace‐Level Determination of Nitrosamine Drug Substance‐Related Impurities in the Anti‐Diuretic Drug Furosemide

ABSTRACT In recent years, the international pharmaceutical community has expressed heightened concern about the occurrence of nitrosamine impurities and nitrosamine drug substance‐related impurities (NDSRI), as these compounds are well recognized for their strong mutagenic activity in humans. Among the drugs of interest, furosemide, an anti‐diuretic drug, is one. Structural assessments of furosemide have revealed the potential for the formation of an NDSRI, specifically N‐nitroso‐furosemide (N‐FUR). According to the Carcinogenic Potency Categorization Approach, this impurity is classified as a Category 4 carcinogen, with an acceptable daily intake limit of 1500 ng/day. Given the elevated cancer risk associated with such impurities, the development of highly sensitive and selective quantitative analytical methodologies is imperative. In response to this need, a robust liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was established for the trace‐level quantification of N‐FUR. Chromatographic separation was achieved using a Kinetex C 18 (100 mm × 3.0 mm, 2.6 µm), maintained at 45°C. The mobile phase consisted of water and methanol, in a 50:50 (%, v/v) ratio with 0.1% trifluoroacetic acid, delivered at a flow rate of 0.5 mL/min. Detection was performed using a multiple reaction monitoring approach in electro spray ionization, Negative mode, transitions monitored at m / z 358.0 → 284. The method was rigorously validated in accordance with international regulatory guidelines and demonstrated excellent performance characteristics, confirming its suitability for routine analysis. This validated LC–MS/MS method has been successfully transferred to the quality control unit, enabling routine monitoring of nitrosamine impurities in furosemide formulations.