A sensitive LC-MS/MS method for quantification and conformer resolution of N-nitroso cinacalcet in cinacalcet drug products

Nitrosamine drug substance-related impurities (NDSRIs) are a cohort of concern due to their potential mutagenic and carcinogenic effects. Strict guidelines from regulatory authorities have been implemented for their identification and quantification in pharmaceutical formulations to reduce health-related risks. N-Nitroso cinacalcet (NCIN), which is a type of high-risk NDSRI related to cinacalcet is classified under potency class three by United States food and drug administration (USFDA) having daily intake limit of 400 ng/day. In this study, an liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of NCIN in cinacalcet tablet formulations. Chromatographic separation was achieved on an Eclipse Plus C18 RRHD column (100 × 2.1 mm, 1.8 µm) using a gradient elution program consisting of 10 mM ammonium acetate buffer (pH 3.8, adjusted with 0.1% formic acid) and methanol at a flow rate of 0.4 mL/min. Under the optimized conditions, NCIN was resolved into two chromatographic peaks at retention times of 11.86 and 12.39 min, corresponding to anti and syn conformational isomers generated by restricted rotation around the nitrosamine N–N bond. The method exhibited excellent linearity (r2 > 0.99) over 1–200 ng/mL with limit of detection (LOD) and limit of quantification (LOQ) values of 0.5 and 1.0 ng/mL, respectively, demonstrating suitability for regulatory compliance and pharmaceutical safety monitoring.