UPLC-TQ MS Method Development and Validation for N-Nitroso-N-Desmethyl Methadone Detection in Methadone Hydrochloride Tablets: A Comprehensive Analysis

The detection and stringent control of N-nitrosamine impurities in pharmaceutical products is a critical imperative for regulatory agencies and the global pharmaceutical industry, driven by the potent genotoxic and carcinogenic potential inherent to these compounds. This research work describes the development and rigorous validation of a highly sensitive Ultra-Performance Liquid Chromatography-Triple Quadrupole Mass Spectrometry (UPLC-TQ MS) method specifically designed for the quantification of N-Nitroso-N-Desmethyl Methadone in Methadone Hydrochloride (HCL) Tablets (10 mg). Chromatographic separation was successfully achieved utilizing an ACQUITY UPLC BEH Shield RP 18 stationary phase under an isocratic elution program composed of ammonium acetate buffer and acetonitrile. Mass spectrometric detection was executed via electrospray ionization in positive mode (ESI+) employing Multiple Reaction Monitoring (MRM) to guarantee maximal selectivity for the two isomeric peaks of the impurity. The analytical method was validated in strict conformity with USP < 1225 > and ICH Q2(R1) guidelines. Linearity was demonstrated over a concentration range of 0.33 ng/mL to 6.67 ng/mL, corresponding to 10% to 200% of the established specification limit. The limit of quantification (LOQ) was confirmed at 0.33 ng/mL (0.33 ppb), verifying the method's capacity to quantify trace levels significantly below the acceptable intake. Precision assessments yielded a cumulative relative standard deviation (RSD) of 3.7%, while accuracy studies demonstrated mean recoveries ranging from 87.5% to 93.5%. The proposed method represents a robust, specific, and reliable analytical tool for the routine quality control of Methadone HCL, ensuring patient safety through the effective monitoring of mutagenic impurities.