In-situ formation and evaluation of N-nitrosamine drug substance related impurities in glycopeptides implying orbitrap mass spectrometry.

Nitrosamines, a class of N-nitroso compounds, have raised significant health concerns due to their established carcinogenicity. ICH M7 enlisted N-nitroso compounds in the so called cohorts of concern due to their carcinogenic effects. Glycopeptides (GPs) are complex molecules composed of peptide and glycan moieties. GPs serve as the last resort for the mitigation of bacterial infections, particularly against gram-positive bacteria. GPs are susceptible to nitrosamine drug substance related impurities (NDSRIs) contamination due to the presence of secondary amine in their core structure. The incidence of formation of NDSRI impurities in GPs could be either during their semi-synthetic route or storage. However, till date, no studies have been reported on the occurrence of NDSRI in GPs which is a pre-requisite. Hence, the current study investigates the plausible mechanisms and detection methods for the NDSRI in GPs. In-situ studies were performed to evaluate the possible formation of NDSRI in GPs. In the current study, GPs of different generations were screened for their potential of forming NDSRI impurities implying nitrosating agent. LC and LCHRMS/MS studies were performed to identify the in-situ generated impurities. Interestingly, the formation of NDSRIs is evident in all the selected GPs. The molecular mechanisms and pathways for individual GPs and respective NDSRIs were elucidated. Interestingly, isomeric NDSRIs were also identified during in-situ generated samples. Using CPCA the potency scores and acceptable intakes for the NDSRIs were evaluated. Thus, this work aims to enhance the safety and efficacy of GPs ensuring compliance with the regulatory standards by advancing the understanding of NDSRIs in GPs.