Validation and stability analysis of N-Nitroso Acebutolol in acebutolol hydrochloride capsules using UHPLC–MS/MS for regulatory compliance and patient safety

Nitrosamine drug substance‑related impurities (NDSRIs) remain a major regulatory concern due to their potential genotoxicity, necessitating highly sensitive and validated analytical methods for pharmaceutical quality control. Conventional High-Performance Liquid Chromatography (HPLC) methods, though widely used in routine testing, have critical drawbacks, including limited sensitivity and poor selectivity, making them inadequate for trace‑level nitrosamine detection in complex pharmaceutical matrices. To overcome these limitations, we developed and validated an Ultra‑High Performance Liquid Chromatography–Tandem Mass Spectrometry (UHPLC–MS/MS) method to quantify N-Nitroso Acebutolol (NNA) in Acebutolol Hydrochloride Capsules (AHC) formulation. The method design was guided by the European Medicines Agency (EMA) carcinogenic potency categorization approach (CPCA) threshold of 1.25 µg/g and the International Council for Harmonization (ICH) M7 (R2) Mutagenicity Classification. Validation in accordance with ICH Q2 (R2) demonstrated excellent performance, with % Relative Standard Deviation (RSD) ≤ 3.5%, correlation coefficient r² = 0.999, Limit of Quantitation (LOQ) = 0.646 µg/g, recoveries of 96–102%, and solution stability up to 10 h. Stability analysis of three expired batches of AHC further confirmed the method’s ability to detect degradation without interference from the placebo matrix. All results were within the specification limit of 1.25 µg/g, ranging from 0.210 to 0.509 µg/g, thereby ensuring regulatory compliance and patient safety. Although the LOQ (0.646 µg/g) is higher than ultra-trace Active Pharmaceutical Ingredient (API) reports, it remains well below the EMA, CPCA threshold (1.25 µg/g), ensuring regulatory compliance. This method is explicitly designed for routine release and stability sample analysis of finished dosage forms, not ultra-trace API analysis. This study presents the first validated UHPLC–MS/MS method for trace-level detection of NNA in AHC. Method design integrated toxicological thresholds from EMA, CPCA and ICH M7 (R2), with full ICH Q2(R2) compliance and alignment to United States Pharmacopeia (USP) < 736 > Mass Spectrometry standards. Further Stability analysis confirmed robustness, establishing suitability as a stability-indicating procedure. Benchmarking via the Multicolored Analytical Chemistry profile highlighted analytical excellence, sustainability, practicality, and Quality by Design (QbD) integration. Together, these attributes underscore the novelty of a reproducible, risk-based, and future-ready platform for NDSRI monitoring in AHC.