68TWENTYFOURSEVENBIOPHARMA Issue 1 / March 2026 mass spectrometry (MS)-based techniques that struggled to concretely determine the presence of nitrosamines in certain types of NDSRIs. According to Dr. Fanelli, the analytical assessment of complex NDSRIs can be particularly challenging because structurally related species may generate similar mass signals, making it difficult to distinguish nitrosamines from closely related impurities on the basis of MS data alone. In such cases, Dipharma’s approach relies on orthogonal analytical strategies combining targeted sample preparation, chromatographic separation, and confirmatory MS evaluation to improve specificity and support reliable identification. “15N-NMR overcomes these ambiguities in a very direct way: it allows us to observe the nitrogen atom of the nitroso group itself,” said Dr. Fanelli. “Nitrosamines possess a characteristic chemical shift in a very narrow diagnostic region (120–190 ppm), and no other nitrogen-containing group interferes with this signature. By conducting the NAP test with 15N-enriched nitrite, any nitroso group formed during the reaction carries a unique isotopic label, making its detection both direct and unequivocal. If a signal appears in that region, a nitrosamine has formed; if the spectrum remains silent, the molecule is not susceptible to nitrosation under the tested conditions.” Notably, the technique is capable of distinguishing between subtle structural variations, including cistrans isomers or conformational rotamers, which traditional mass spectrometry cannot detect. This is especially important for families of APIs that tend to form multiple nitrosated species, such as nortriptyline, protriptyline, vortioxetine, and other nitrogen-rich APIs, according to Dr. Fanelli. “As regulators increasingly expect companies to demonstrate not only control, but also an understanding of the mechanistic basis of nitrosamine formation, approaches capable of distinguishing nitrosation from oxidation, rearrangement, or other pathways become essential,” said Dr. Fanelli. “Techniques that provide structural certainty — rather than probabilistic interpretation — are likely to become even more relevant in the regulatory discourse.” GRANT PLAYTER Editorial team 24/7 BIOPHARMA DIPHARMA
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