UHPLC–MS/MS Method Detects Trace Nitrosamine Impurity in Acebutolol Capsules

Nitrosamine impurities have become one of the most pressing quality-control challenges in modern pharmaceutical manufacturing, and a newly published study reports a validated analytical method designed to detect one of them in a widely used cardiovascular medicine. The research describes an Ultra-High Performance Liquid Chromatography–Tandem Mass Spectrometry (UHPLC–MS/MS) method for quantifying N-Nitroso Acebutolol (NNA), a nitrosamine drug substance-related impurity, in Acebutolol Hydrochloride Capsules. The work, published in BMC Chemistry, was carried out by Rajamani Dhanasekar, Vadivel Velmurugan, Sankham Devendran Rajendran and Viswas Raja Solomon.

Nitrosamine drug substance-related impurities, often abbreviated as NDSRIs, remain a major regulatory concern because of their potential genotoxicity. Conventional High-Performance Liquid Chromatography (HPLC) methods, though widely used in routine testing, have critical drawbacks for this class of contaminants, including limited sensitivity and poor selectivity, which make them inadequate for trace-level nitrosamine detection in complex pharmaceutical matrices such as capsule formulations.

To overcome these limitations, the research team developed and validated a UHPLC–MS/MS method specifically tailored to quantify NNA in the capsule formulation. The design of the method was guided by two toxicological frameworks: the European Medicines Agency’s carcinogenic potency categorization approach, which establishes a threshold of 1.25 µg/g for this impurity, and the International Council for Harmonization’s ICH M7 (R2) Mutagenicity Classification. By anchoring the analytical performance targets to these regulatory thresholds, the researchers ensured that the method could reliably distinguish acceptable from unacceptable impurity levels in the finished dosage form.

Validation was performed in accordance with ICH Q2 (R2), the current international guideline for analytical procedure validation. The method demonstrated strong performance across the key validation parameters. Relative standard deviation values were at or below 3.5 percent, indicating excellent precision across repeated measurements. The calibration curve showed a correlation coefficient of r² = 0.999, reflecting a highly linear response over the tested concentration range. The limit of quantitation was determined to be 0.646 µg/g, meaning the method can reliably quantify NNA at concentrations well below the 1.25 µg/g regulatory threshold. Recovery studies yielded values between 96 and 102 percent, confirming that the measurement is accurate across the analytical range.

Solution stability testing showed that prepared samples remained stable for up to 10 hours, an important practical consideration for laboratories that process batches of samples over extended analytical sessions. The researchers also applied the method to stability analysis of three expired batches of Acebutolol Hydrochloride Capsules. This testing confirmed the method’s ability to detect degradation without interference from the placebo matrix, a critical requirement for any stability-indicating procedure. The ability to analyse aged samples without matrix effects strengthens the case for the method’s use in routine quality-control laboratories.

All measured results in the study fell within the specification limit of 1.25 µg/g, with detected concentrations ranging from 0.210 to 0.509 µg/g across the tested batches. These findings support the conclusion that the analysed capsule batches met current regulatory expectations for NDSRI content. The authors note that although the limit of quantitation of 0.646 µg/g is higher than what has been reported for ultra-trace Active Pharmaceutical Ingredient analysis, it remains well below the EMA carcinogenic potency categorization threshold, ensuring regulatory compliance for the intended application.

The researchers are explicit about the scope of their method. It is designed for routine release and stability sample analysis of finished dosage forms, not for ultra-trace API analysis. This distinction matters because different analytical contexts demand different detection limits and sample preparation strategies. For finished capsule products, where the impurity is measured within a complex matrix of excipients and the regulatory threshold is 1.25 µg/g, a limit of quantitation below that threshold is sufficient for compliance testing. The authors position the method as the first validated UHPLC–MS/MS procedure for trace-level detection of NNA in Acebutolol Hydrochloride Capsules.

Beyond the core validation, the study integrated toxicological thresholds from EMA, the carcinogenic potency categorization approach and ICH M7 (R2) into the method design, with full ICH Q2 (R2) compliance and alignment to United States Pharmacopeia chapter <736> Mass Spectrometry standards. The stability analysis confirmed robustness and established the procedure’s suitability as a stability-indicating method, meaning it can detect changes in impurity levels over a product’s shelf life. Benchmarking through the Multicolored Analytical Chemistry profile highlighted analytical excellence, sustainability, practicality and Quality by Design integration, attributes the authors argue underscore the novelty of a reproducible, risk-based and future-ready platform for NDSRI monitoring in this capsule formulation.

The broader significance of this work lies in the growing regulatory demand for sensitive, validated methods capable of monitoring nitrosamine impurities in finished medicines. This study contributes a fully validated, stability-indicating UHPLC–MS/MS method that meets those demands for a specific cardiovascular capsule product, providing a template that may inform similar method development efforts for other nitrosamine drug substance-related impurities across the pharmaceutical industry.

Subject of Research: Development and validation of a UHPLC–MS/MS method for detecting the nitrosamine impurity N-Nitroso Acebutolol in acebutolol hydrochloride capsules

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

Article References: Dhanasekar, R., Velmurugan, V., Rajendran, S. D., & Solomon, V. R. (2026). Validation and stability analysis of N-Nitroso Acebutolol in acebutolol hydrochloride capsules using UHPLC–MS/MS for regulatory compliance and patient safety. BMC Chemistry. https://doi.org/10.1186/s13065-026-01930-x

Image Credits: AI Generated

DOI: 10.1186/s13065-026-01930-x

Keywords: UHPLC–MS/MS, N-Nitroso Acebutolol, nitrosamine impurities, NDSRI, acebutolol hydrochloride capsules, ICH Q2 (R2), ICH M7 (R2), EMA carcinogenic potency categorization, pharmaceutical quality control, stability-indicating method, genotoxic impurity, regulatory compliance