Trace-level quantitation of N-((2-isopropylthiazol-4-yl)methyl)-N-methylnitrous amide by liquid chromatography-tandem...

Nitrosamines, a class of N-nitroso compounds, have raised significant health concerns due to their status as highly potent mutagenic carcinogens. Recently, the potential occurrence of the nitrosamine drug substance-related impurity, N-((2-isopropylthiazol-4-yl)methyl)-N-methylnitrous amide (NITMA), also known as N-nitroso-2,4-thiazole amine (NNTA), has been reported in some finished drug products containing ritonavir. Stringent acceptable intake (AI) limits, as low as 18 ng/day, have been recommended for NITMA by various regulatory agencies, requiring a need for highly sensitive quantitative methods. In this study, a versatile and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the detection and quantitation of NITMA in ritonavir drug substance and test formulation blends of ritonavir. NITMA was ionized and quantified in positive mode APCI using multiple reaction monitoring. The formation of NITMA from its precursor, 1-(2-isopropylthiazol-4-yl)-N-methylmethanamine (ITMA), was studied under accelerated conditions to ultimately prevent its artifactual formation during sample preparation and analysis. Adequate peak selectivity for NITMA was also studied to demonstrate the versatility of the method in the presence of high concentrations of APIs, impurities, degradation products, and excipients. The method was successfully validated as per ICH Q2(R2) guidelines over a wide concentration range (0.0225-0.27 ng/mL). The validated quantitation limit (0.0225 ng/mL) aligns with current regulatory requirements for NITMA. Overall, the newly developed method was found suitable to support risk assessment strategies and regulatory compliance.