(W1030-09-58) Challenges in Nitrosamine Determination for Pharmaceutical Products: An Analytical Perspective

Purpose: In recent years, several instances of nitrosamine contamination in widely used medications have led to widespread recalls, drawing attention to the need for robust analytical methods to detect and quantify these impurities [1]. The presence of nitrosamines in pharmaceuticals can be attributed to various sources, including the synthesis process, raw materials, and storage conditions, making it imperative for manufacturers to develop stringent quality control measures. The determination of nitrosamines in pharmaceutical products predominantly relies on advanced analytical techniques, with mass spectrometry being the primary method of choice due to its exceptional sensitivity, selectivity, and capability to detect these compounds at trace levels. In this study, we employed state-of-the-art mass spectrometry-based approaches to address the challenges associated with nitrosamine determination in pharmaceutical formulations. This abstract highlights the challenges faced by analytical scientists in the quest to ensure the safety and quality of pharmaceutical products concerning nitrosamine determination. It addresses the complexities encountered during sample preparation, method validation, and sensitivity requirements to meet the ever-evolving regulatory guidelines. Moreover, it explores into the technological advancements and novel strategies that are being explored to tackle these challenges effectively.

Methods: A comparative study in terms of sensitivity, selectivity, robustness and also suitability for routine analysis was conducted. Three technologies have been employed, namely, gas chromatography coupled with a single quadrupole (Q) and liquid chromatography equipped with tandem mass spectrometry, namely, a quadrupole-time of flight (QTOF) mass spectrometer and a triple quadrupole (QqQ) mass spectrometer. Comparinson was also made between different types of ionization sources. For sample preparation, both liquid and solid phase extraction was used.

Results: Results were obtained using different nitrosamine impurities, namely N-Nitrosodimethylamine (NDMA), N-Nitrosodiethylamine (NDEA), N-Nitrosomethylphenylamine (NMPA), N-Nitrosodibutylamine (NDBA), N-Nitrosodiisopropylamine (NDIPA) and N-Nitroso-N-methyl-4-aminobutyric Acid (NMBA). Typical limit of quantitation of these methods must be at or below 0.03ppm. The analytical conditions for GC-MS and LC-MS/MS were optimized to assure maximum sensitivity and selectivity. Quantification of nitrosamines in the drug product samples was performed using external calibration curves. The results obtained has shown that LC-MS/MS is more comprehensive for the determination of the impurities under evaluation, this is especially true when atmospheric pressure chemical ionization (APCI) ion source was used together with the triple quadrupole mass analyser. When electrospray ionization (ESI) ion source was used, the ionization efficiency for low molecular weight nitrosamines, such as NDMA and NDEA was deeply affected and the target concentration of 0.03ppm was not achievable. The use of the QTOF equipment also showed good results and the simultaneous determination of the nitrosamine impurities under evaluation was also accomplished. Nonetheless, for this equipment, before sample analysis, an extensive equipment clean-up was required to assure sensitivity was improved. This fact makes this technology less suitable for routine analysis in the QC laboratories when compared to the triple quadrupole equipment. Likewise, when solid phase extraction and liquid extraction was employed, lower limits of quantitation was obtained, LOQ < 0.01ppm. Both SPE mechanism of separation selected, and liquid extraction solvents were made taking into consideration the physicochemical properties of the drug products used in this study. The use of sample preparation techniques allows the implementation of standard methodologies in the analytical laboratories, since sample matrix interferences are deeply reduced. However, in the QC laboratories, the introduction of clean-up and concentration strategies using these sample preparation methodologies may have impact on the lead time for this analysis. At the same time, this type of methodology also requires knowledgeable analyst in the QC labs and effective knowledge transfer between R&D and QC. In Figure 1, a typical chromatogram obtained with the LC-APCI-QqQ equipment is presented for NDMA, NDEA and NDBA at 0.03ppm. The results obtained using the GC-MS technology proved to be less comprehensive as it was not possible to achieve the required limit of quantitation for all the nitrosamines tested, namely, for NMBA. Despite, not being so comprehensive as the LC-MS/MS methodologies, the GC-MS presented some advantages. The use of higher concentration sample preparation was possible due to the dilution capacity of the diluent selected (dimethylacetamide). In addition, the employment of GC-MS methodologies in QC laboratories is quite straightforward and it does not required extensive knowledge or extensive care with the equipment.

Conclusion: Our findings highlighted the challenges faced for the quantification of nitrosamine impurities at trace levels. By utilizing mass spectrometry in combination with optimized sample preparation techniques, we achieved precise and reliable results using the three technologies under evaluation. The selection of each of these techniques must be done taking into consideration its suitability and practicality for the QC laboratories together with the sensitivity requirements for each drug product. Our findings demonstrated that the LC equipped with a triple quadrupole (QqQ) mass spectrometer is a valuable tool for comprehensive and simultaneous determination of several nitrosamine impurities.

References: 1 - FDA (US Food and Drug Administration). Recalls, Market Withdrawals, & Safety Alerts.; 2023 Jul. Available from: https://www.fda.gov/safety/recalls-market-withdrawals-safety-alerts.

Figure 1 – Extracted chromatogram obtained for NDMA, NDEA and NDBA at 0.03ppm using a LC-APCI-QqQ equipment, considering a sample target concentration of 30mg/mL. Figure caption: (a) NDMA, (b) NDEA and (c) NDBA