Development and Validation of a Sensitive LC-MS/MS Method for Quantifying N-Nitroso-Ketamine in Ketamine Hydrochloride Injections
Abstract
<title>Abstract</title> <p>N-Nitrosamine drug substance–related impurities (NDSRIs) are important quality aspects in medicines due to their mutagenesis and carcinogenic potential, requiring strict toxicological assessment and sensitive analytical monitoring. This research examines the toxicology and analysis of N-nitroso-ketamine (NNK), a nitrosamine impurity generated by ketamine hydrochloride nitrosation. To classify toxic substances, the FDA-aligned Carcinogenic Potency Categorization Approach (CPCA) was used, evaluating structural factors such α-hydrogen availability, steric hindrance, and metabolic activation. Ketamine is a nitrosatable secondary amine, but its NNK structure is sterically protected around the α-carbon, limiting cytochrome P450-mediated α-hydroxylation and reducing DNA-reactive intermediates. These structural factors placed NNK in CPCA Category 5, allowing 1500 ng/day of intake. A highly sensitive and selective LC-MS/MS method was developed and validated for trace-level NNK detection in ketamine hydrochloride drug substance and injectable formulations. The chromatographic separation was done on an Acquity HSS T3 column utilizing gradient settings and electrospray ionization in multiple reaction monitoring mode (m/z 267.0 → 207.0). The method met regulatory expectations with strong linearity (r > 0.999), accuracy (95-104%), precision (RSD ≤ 5%), and low quantification limit (∼139 ppb). This paper proposes a thorough and structure-guided approach to pharmaceutical NDSRI monitoring and management.</p>
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