Development and validation of a sensitive LC-MS/MS method for quantification of N-Nitroso-norcodeine in codeine phosphate with fragmentation pathway characterization

A sensitive and reliable LC–MS/MS method was developed and validated for the quantification of N-Nitroso-Norcodeine in Codeine Phosphate. N-Nitroso-Norcodeine is a structurally related nitrosamine drug substance-related impurity that may form in codeine phosphate by nitrosation of the opioid amine functionality. Owing to the potential mutagenic and carcinogenic liability of nitrosamine impurities, sensitive sub-ppm analytical control is required to support regulatory compliance and patient safety. This study describes the development and validation of a LC-MS/MS method for trace quantification of N-Nitroso-Norcodeine in codeine phosphate drug substance. Detection was performed in multiple reaction monitoring mode using positive electrospray ionization. The method was linear over the calibration range 0.104–1.559 ng/mL. The limits of detection and quantification were 0.052 ng/mL, and 0.104 ng/mL, corresponding to 0.41 ppm relative to codeine phosphate and meeting the regulatory target derived from the 1500 ng/day acceptable intake limit. Recovery ranged from 94.5% to 105.9%, with percentage relative standard deviation values ≤1.0%, and robustness was demonstrated under deliberate chromatographic and mass spectrometric variations. The fragmentation behavior of N-Nitroso-Norcodeine was interpreted using established morphinan collision-induced-dissociation (CID) pathways. Overall, the validated method provides a sensitive, selective, and regulatory-aligned platform for routine monitoring of N-Nitroso-Norcodeine in codeine phosphate drug substance.