HILIC-SPE and LC–HRMS Method Used for N-Nitrosamine Detection in Pharmaceuticals

The 2018 discovery of N-nitrosodimethylamine (NDMA) in valsartan (1,2) changed the pharmaceutical landscape forever. The discovery of NDMA during routine quality control at an average level of 66.5 parts million forced the pharmaceutical industry to re-evaluate their manufacturing processes. Several global recalls have meant that this interest has continued, and nitrosamine drug substance-related impurities (NDSRIs) have been reported recently (3). A team of researchers from the University of Ljubljana in Slovenia, has introduced a method that joins hydrophilic interaction chromatography (HILIC)-based solid-phase extraction (SPE) with liquid chromatography–high-resolution mass spectrometry (LC–HRMS) (4) to determine N-nitrosamine (NA) in pharmaceuticals.

Assorted pharmaceutical medicine pills, tablets and capsules.Pills background: © klepach - stock.adobe.com

Potentially highly carcinogenic, NAs are formed during synthesis or degradation and pose significant health risks even at trace levels. Highly rigorous processes are therefore required to determine them. While existing methods address single active pharmaceutical ingredients (APIs), there is a lack of a versatile method to detect multiple NAs in a range of drug products. This new approach using a HILIC-based SPE step allows multiple APIs and polar excipients to be retained while NAs pass through. This clean-up step significantly mitigates matrix interference and removes excessive amounts of API.

The SPE process employs sequential elution steps optimized for different NAs. This ensures good recovery while maintaining high precision, even in complex matrices. After sample clean-up, LC–HRMS was used in the analysis and demonstrated linearity (R² > 0.999), accuracy (85–115%), and repeatability (RSD < 10%). Recoveries exceeded 80% and detection limits were as low as 42.5% of regulatory thresholds, underscoring the method's efficacy. These parameters were tested across 59 APIs and validated across 15 tested NAs, including multi-API formulations.

The researchers tested the method on a variety of commercially available and expired DPs. While no NAs exceeded limits in active DPs, expired ranitidine products contained NDMA levels up to 32 times higher than acceptable. These findings underscore the importance of proper storage and immediate sample preparation to prevent degradation-related NA formation.

The team concluded that the adaptability of the method is one of its best features. It is the first method to overcome the difficulties involved with analyzing such a class of compounds, such as the differing physicochemical properties of the NAs, the low concentrations relative to the API, and the complexity of the matrix (4). By modifying the SPE elution protocol, the method can accommodate newly identified NDSRIs and tailor detection for specific APIs. As the pharmaceutical landscape continues to evolve to address this, this offers continued flexibility in routine quality control and regulatory compliance.

References

(1) EMA, EMA Reviewing Medicines Containing Valsartan from Zhejiang Huahai Following Detection of an Impurity. European Medicines Agency; 2018. https://www.ema.europa.eu/en/news/ema-reviewing-medicines-containing-valsartan-zhejiang-huahai-following-detection-impurity-some (Accessed 2024-12-23).

(2) James, M.; Edge, T. Low-Level Determination of Mutagenic Nitrosamine Impurities in Drug Substances by LC–MS/MS. LCGC Eur. 2021, 34 (7), 267–276. DOI: 10.56530/lcgc.eu.dd3576s1

(3) Nudelman, R.; Kocks, G.; Mouton, B.; et al. The Nitrosamine “Saga”: Lessons Learned from Five Years of Scrutiny. OPR&D 2023, 27 (10), 1719–1735. DOI: 10.1021/acs.oprd.3c00100

(4) Planinsek, T. P.; Roskar, R. A Comprehensive Approach for N-nitrosamine Determination in Pharmaceuticals Using a Novel HILIC-based Solid Phase Extraction and LC-HRMS. Talanta 2025, 282, 126752. DOI: 10.1016/j.talanta.2024.126752