High-throughput Headspace Analysis of Volatile Nitrosamines and their Secondary Amine Precursors

Certain drug products are susceptible to contamination by trace concentrations of mutagenic volatile nitrosamine impurities, especially when secondary amines used in synthesis are, or have been, present together with nitrosating agents during manufacture. Although regulators have tended to focus on the determination of nitrosamine impurities, applying soft chemical ionization directly to sample headspace—in the absence of chromatographic separation—may enable simultaneous determination of amine precursors. By using selected ion flow tube mass spectrometry (SIFT-MS), this article demonstrates that there is potential for rapid screening of volatile nitrosamines and their amine precursors in a single run.

In 2018, the pharmaceutical industry was shaken by the discovery of N­-nitrosodimethylamine (NDMA) in sartan medications—a discovery that led to the identification of other N-nitrosamines in diverse drug products (1,2). The known or suspected mutagenicity of many N-nitrosamines means that their presence in any product to which humans are exposed is of concern.

Research has revealed that many N-nitrosamine issues arise from nitrosating agents used in synthesis—especially when secondary amines are present (1,2). Several volatile nitrosamines are summarized in Table I together with their precursor secondary amines. Volatile nitrosamines, such as NDMA, are conventionally analyzed using either liquid chromatography (LC) or gas chromatography (GC) coupled with mass spectrometry (MS), with four procedures listed in reference 3. Volatile amines are typically analyzed using GC, with a recent universal method for 14 volatile amines used in pharmaceutical manufacturing reviewing the challenges involved (4). These include additional sample preparation steps, peak tailing or loss due to adsorption on active sites within the inlet or column, and low throughputs (GC cycle time of 37 min [4]).

This article describes a feasibility study for the development of a direct mass spectrometry method that provides rapid screening of drug products for both volatile nitrosamines and their amine precursors. The selected ion flow tube mass spectrometry (SIFT-MS) technique applied here uses ultra-soft chemical ionization (5) to analyze diverse functionalities simultaneously in real time in the absence of chromatographic separation and following minimal sample preparation. SIFT-MS-based nitrosamine analysis for drug products has been demonstrated previously (6,7), and amines are readily detected (8). With further development, an extensible, rapid screening method for volatile nitrosamines and their precursors is feasible.

Experimental

The SIFT-MS technique is described in detail elsewhere (5,9). Briefly, rapidly switchable reagent ions (H₃O⁺, NO⁺, and O₂^+•) are utilized for gas-phase soft chemical ionization of volatile organic compounds (VOCs) in air and headspace. Real-time, high-throughput analysis is achieved because SIFT-MS is chromatography-free. Specificity is achieved through the use of multiple ionization mechanisms coupled with mass spectrometric detection. A commercial SIFT-MS instrument was used in this study (Voice200ultra; Syft Technologies).

Automated headspace-SIFT-MS analysis is best achieved using syringe-injection autosamplers (10). In this work, automated headspace analysis was carried out using a SIFT-MS instrument coupled with a multipurpose autosampler (MPS Robotic Pro; Gerstel). Samples (10 mL) were placed in 20-mL headspace vials and incubated for 20 min at 60 °C. Two aliquots of headspace were extracted in succession using a 2.5-mL headspace syringe, followed by steady injection at 50 μL s^-1 into a flow of zero-air make-up gas (approx. 10-fold sample dilution in the sample inlet). Figure 1 shows data obtained by the SIFT-MS instrument. The dual-injection approach has generally been utilized for analyses requiring positively and negatively charged reagent ions (5). Here, the first injection is used to prime the headspace syringe and SIFT-MS inlet system, providing improved repeatability for the amines. Sample analysis is conducted within 3.5 min.

The target nitrosamines and their precursor secondary amines are listed in Table I, together with the SIFT-MS reagent ion-product ion pairs utilized to detect and quantify them. The nitrosamines were utilized from a standard mixture (all analytes at 2000 ppm in methanol; Sigma Aldrich EPA8270 nitrosamines mix). A house-prepared standard was utilized for the amines, but with varying analyte concentrations due to disparate headspace partitioning. Dimethylamine(DMA), methylethylamine (MEA), and piperidine (PIP) are 5x higher and morpholine (MOR) and pyrrolidine (PYR) are 50x higher in solution concentration than diethylamine (DEA), dipropylamine (DPA), and dibutylamine (DBA), which have the same concentrations as the nitrosamines. Test samples contained all analytes and were prepared in 50% aqueous potassium carbonate to enhance headspace partitioning.

Results and Discussion

The present study focused on the specificity, linearity, system precision, and limit of quantitation (LOQ) to assess the feasibility of full method development. Note that where applicable, data are reported for all reagent ion-product ion pairs.

Specificity: As noted in reference 5, when chromatographic separation is not used, SIFT-MS attains specificity by employing at least one unique reagent ion–product ion pair for each analyte in the method. Table I shows that this is achieved for all ions used for this target compound list, albeit with a potential need for straightforward ¹³C isotopologue correction if ions lying one mass-to-charge ratio (m/z) lower are present at a similar intensity to the target ion.

Future method development needs to consider this suite in the context of residual solvents, see the universal headspace-GC amine and residual solvent method in reference 4. In general, the use of several product ions for each analyte in a SIFT-MS method enables matrix effects, such as those arising from residual solvents, to be overcome because multiple ions are not usually interfered with. For a given drug product, this will be confirmed during method validation.

Linearity: Figure 2 shows linear responses obtained for three nitrosamines and their corresponding secondary amine precursors, while Table II summarizes the results obtained for all analytes. Note, however, the reduced sensitivity for DMA and MOR compared to the other analytes due to poorer headspace partitioning. The data collected at notionally 1000 ppb in solution were not included here because the high methanol concentration introduced with the commercial nitrosamine standard was starting to overload the chromatography-free SIFT-MS instrument (5,11). The potassium carbonate added to the solution to enhance nitrosamine and amine headspace partitioning also enhances methanol partitioning. Note that the range can be extended by slowing the rate of headspace injection (12).

System Precision (Repeatability): Results obtained for six replicate measurements at three concentrations are summarized in Table II as percent relative standard deviation (%RSD). The concentrations shown in the table header are ppb in solution for all nitrosamines plus several amines (DEA, DPA, and DBA). For DMA, MEA, and PIP, the concentrations are fivefold higher, while for MOR and PYR, they are 50-fold higher. Only morpholine fails to meet typical acceptance criteria, with RSD>10%. More work is required to improve the repeatability of morpholine injections.

Limit of Quantitation: Repeatability performance can be used to estimate analyte LOQs in solution, given that empirical RSDs are often determined from the lowest test concentration for which RSD < 20%. Since an RSD less than 10% is achieved for all analytes except morpholine, LOQs are estimated as less than half the lowest concentration evaluated (Table II). Since additional work is required on morpholine repeatability, an LOQ is not estimated here.

Conclusion

This study has demonstrated the feasibility of automated headspace-SIFT-MS analysis for rapid, sensitive screening of eight volatile nitrosamines and their precursor secondary amines. SIFT-MS applies soft chemical ionization, enabling direct, chromatography-free analysis of nitrosamines and amines, circumventing the common chromatographic challenges for the amines. Nevertheless, preliminary recovery work has indicated that other sample matrix and headspace delivery effects need to be addressed, as described by You et al. (4) for their GC–flame ionization detection (FID) method. Subsequent research will focus on improving recovery—perhaps with the help of an efficient standard additions workflow—and evaluate the method’s specificity in the presence of a panel of common residual solvents.

References

(1) European Medicines Agency (EMA), Assessment Report: Procedure under Article 5(3) of Regulation EC (No) 726/2004. Nitrosamine Impurities in Human Medicinal Products. Procedure Number: EMEA/H/A-5(3)/1490, EMA/369136/2020.https://www.ema.europa.eu/en/documents/opinion-any-scientific-matter/nitrosamines-emea-h-a53-1490-assessment-report_en.pdf(accessed 2025-10-15).

(2) United States Food and Drug Administration, Guidance for Industry. Control of Nitrosamine Impurities in Human Drugs, 2021. https://www.fda.gov/media/141720/download (accessed 2025-10-08).

(3) USP, USP General Chapter <1469>, “Nitrosamine Impurities” (Rockville, MD, 2021).

(4) You, C.; Ho, T.; Rucker, V.; Yeh, J.; Wang, L. A Simple and Universal Headspace GC-FID Method for Accurate Quantitation of Volatile Amines in Pharmaceuticals. Anal. Methods 2023, 15, 4427−4433. DOI: 10.1039/d3ay00956d

(5) Langford, V. S.; Perkins, M. J. SIFT-MS: From Method Concept to Routine Analysis; RSC Books, 2025.

(6) Perkins, M. J.; Hastie, C. J.; Langford, V. S. Quantitative Analysis of NDMA in Drug Products: A Proposed High-throughput Approach Using Headspace-SIFT-MS. AppliedChem 2024, 4, 107–121. DOI: 10.3390/appliedchem4010008

(7) Aitcheson, F.; Mathew, J.; Pelet, W. Nitrosamine Analysis from Aqueous Solutions Using Headspace-SIFT-MS. Syft Technologies Application Note, 2024. Available online: https://bit.ly/3YoTR5p (accessed 2025-10-08).

(8) Španěl, P.; Smith, D. Selected Ion Flow Tube Studies of the Reactions of H₃O⁺, NO⁺, and O₂⁺ with Several Amines and Some Other Nitrogen-Containing Molecules. Int. J. Mass Spectrom. 1998, 176, 203–211. DOI: 10.1016/S1387-3806(98)14031-9

(9) Smith, D.; Španěl, P.; Demarais, N.; Langford, V. S.; McEwan, M. J. Recent Developments and Applications of [SIFT-MS]. Mass Spec. Rev.2025, 44, 101–134. DOI: 10.1002/mas.21835

(10) Langford, V. S.; Perkins, M. J. Improved Volatiles Analysis Workflows Using Automated [SIFT-MS]. Anal. Methods 2024, 16, 8119–8138. DOI: 10.1039/d4ay01707b

(11) Perkins, M. J.; Silva, L. P.; Langford, V. S. Evaluation of Solvent Compatibilities for Headspace-SIFT-MS Analysis of Pharmaceutical Products. Analytica 2023, 4, 313–335. DOI: 10.3390/analytica4030024

(12) Perkins, M. J.; Langford, V. S. Leveraging Variable Sample Injection Speeds for Simplified Sensitivity Changes and Calibration. Syft Technologies Application Note, 2025. Available online: https://bit.ly/4aJwurd (accessed 2025-10-15).