NDMA in macrolides: GC-MS/MS method for its detection and study of its formation
European Journal of Pharmaceutical Sciences, Volume 211, 2025, 107135: Graphical abstract
The goal of this study is to investigate the potential of macrolide antibiotics, which contain dimethylamino groups, as precursors for the formation of N-nitrosodimethylamine (NDMA) in pharmaceutical products. Using a direct injection GC–MS/MS method, the researchers quantified NDMA levels in macrolide drug substances and tablets, identifying significant concentrations, particularly in azithromycin and spiramycin, which exceeded acceptable intake limits.
The study also explored the impact of solubility on analytical methods and demonstrated that a stable drug form, such as azithromycin dihydrate, can reduce NDMA formation. Additionally, findings showed that NDMA levels increase during storage, underlining the importance of careful formulation and monitoring.
The original article
NDMA in macrolides: GC-MS/MS method for its detection and study of its formation
Nejc Golob, Rok Grahek, Robert Roškar
European Journal of Pharmaceutical Sciences, Volume 211, 2025, 107135
https://doi.org/10.1016/j.ejps.2025.107135
licensed under CC-BY 4.0
Selected sections from the article follow. Formats and hyperlinks were adapted from the original.
In recent years, N-nitrosamines have been in the spotlight of the pharmaceutical industry and pharmaceutical regulatory authorities (EMA, 2021; FDA, 2021). Their presence in drug substances (DSs) and drug products (DPs) has been associated with the manufacturing process of DSs, the production of DPs and the storage of DPs including degradation processes that occur over time (Akkaraju et al., 2023; Horne et al., 2023; Wichitnithad et al., 2023).
Although nitrosamine drug substance-related impurities (NDSRIs) have recently gained the most interest (FDA, 2023; Schlingemann et al., 2023), the most recognized and prevalent nitrosamine to date is N-nitrosodimethylamine (NDMA) (Schmidtsdorff et al., 2022a). NDMA was the first nitrosamine detected in sartan medicines in 2018 (EMA, 2020a), with its presence linked to the use of specific reagents and solvents in the tetrazole ring formation step as a part of the DS synthesis. A similar case was found in the DS synthesis of pioglitazone HCl, where traces of sodium nitrite and solvent resulted in the formation of NDMA (EMA, 2020b). Other sources of NDMA have been detected in DPs such as metformin (MET) medicines, where its presence was related to dimethylamine (DMA) traces from the DS synthesis and nitrite present in the formulation (Schlingemann et al., 2022). In ranitidine medicines, the presence of nitrite in the DP facilitated an internal reaction of the DS, leading to the formation of NDMA (King et al. 2020). The degradation of the DS to DMA, along with the presence of nitrites from excipients, was the cause for the detection of NDMA in afatinib tablets (Golob et al., 2023). The potential risk of NDMA contamination has also been associated with the use of blister materials containing nitrocellulose, such as lidding foils and printing inks (Golob et al., 2022; Schlingemann et al., 2022). The severity of all the mentioned sources has led to multiple recalls of medicines containing NDMA worldwide (Bharate, 2021; Charoo et al., 2023; Schmidtsdorff et al., 2022b).
NDMA has been classified by The International Agency for Research on Cancer (IARC) (Li et al., 2021) as probably carcinogenic to humans (group 2A) and regulatory authorities have limited its acceptable intake (AI) to 96 ng per day due to its genotoxic properties (Paustenbach et al., 2024). In addition, new general chapters have been introduced in the European Pharmacopoeia (EDQM, 2020) and the United States Pharmacopeia (USP, 2022). These chapters encompass analytical procedures for the determination of NDMA content, including GC–MS or GC–MS/MS methods (due to the volatility properties of NDMA) and LC-MS/MS or LC–HRMS methods, focusing mainly on sartan DSs and DPs. Such methods with mass spectrometry detection are necessary to achieve sufficient specificity and sensitivity for the detection of NDMA below the AI limit (Parr and Joseph, 2019; Yang et al., 2023). Nevertheless, the proposed methods have been associated with several deficiencies, such as NDMA in-situ formation during headspace (HS) injection GC–MS analysis (Abe et al., 2020) or during sample storage prior to direct injection (DI) GC-MS/MS analysis (Fritzsche et al., 2022), and the co-elution of NDMA with dimethylformamide in LC-MS/MS analysis (Yang et al., 2020). It is also important to consider the sample preparation procedure prior to analysis, as inefficient extraction may lead to falsely lower results, while complete dissolution of the DS and/or excipients can cause matrix effects, interferences during analysis, or even contamination of the mass spectrometer (USP, 2019, 2021). Published methods, where multiple nitrosamines are simultaneously determined in numerous pharmaceuticals, require complex sample preparation prior to analysis (Planinšek et al., 2024; Planinšek and Roškar, 2025). Therefore, alternative analytical methods, such as GC–MS/MS analysis with solid phase microextraction (SPME) (Alshehri et al., 2020; Chang et al., 2022), have been proposed for NDMA testing in DSs and DPs.
In this paper, we aim to shed light on another class of DSs known as macrolide antibiotics, which were shown to be potential precursors of NDMA in wastewater (Farre et al., 2016; Leavey-Roback et al., 2016; Roccaro et al., 2020). Our first objective was to implement a GC–MS/MS based analytical method for the determination of NDMA in the group of macrolide antibiotics. The feasibility of a DI method for the analysis of macrolide DSs and DPs was confirmed and the method qualified, which has not yet been published. To avoid high column load and the formation of artifacts in the GC injector, the SPME method was tested as an alternative approach for the analysis of NDMA in macrolides. Secondly, the potential for the formation of NDMA from macrolides was investigated due to the presence of a dimethylamino group, a structural element that could yield NDMA (Fig. 1). Several DSs and DPs containing macrolides were analyzed and further subjected to an accelerated stability study to confirm that the NDMA content may increase during storage.
2. Materials, methods and sample preparation
2.2. Methods
2.2.1. GC–MS/MS method (DI and SPME)
A gas chromatograph 7890B GC coupled to a triple-quadrupole mass spectrometer 7000C GC/MS (Agilent, Santa Clara, CA, USA) was employed for the analysis of NDMA. An Agilent VF-WAXms column (30 m × 0.25 mm, 1.00 µm film thickness) (Santa Clara, CA, USA) was used for the analysis. Helium carrier gas was maintained at a constant flow of 1.0 mL/min. The injection port was maintained at 250 °C and used in pulsed splitless mode (30 psi to 0.5 min, 75 mL/min at 0.5 min). The injection volume was 2 µL in a case of DI. In a case of SPME injection, 85 μm CAR/PDMS SPME fiber (57295-U) from Supelco (St. Louis, MO, USA) was used and vials were incubated at 60 °C for 20 min, followed by SPME adsorption for 15 min. The GC oven temperature was programmed as follows: starting temperature of 40 °C (held for 0.5 min) and increasing to 250 °C at 20 °C/min, held for 3.0 min (SPME injection) or 10.0 min (DI). Ionization was carried out in the electron-impact mode (45 eV). The ion source and the transfer line temperatures were 230 °C and 250 °C, respectively. The temperatures of the quadrupoles MS1 and MS2 were kept at 150 °C. After SPME injection, a post bakeout of the SPME fiber was performed at 250 °C for 5 min. NDMA was monitored in the multiple reaction monitoring mode, where it was quantified with the MS/MS transition 74 m/z → 44 m/z (CE: 5 eV) and qualified with the MS/MS transition 74 m/z → 42 m/z (CE: 20 eV), whereas NDMA-d6 was quantified with the MS/MS transition 80 m/z → 50 m/z (CE: 5 eV).
2.2.2. LC–HRMS method
An ultra-high performance liquid chromatograph Thermo Scientific Vanquish Horizon coupled to a high-resolution mass spectrometer (LC–HRMS) Thermo Scientific Orbitrap Exploris 120 (Thermo Fisher Scientific, Waltham, MA, USA) was used for additional analysis of NDMA. The chromatographic separation was achieved using a Waters Acquity UPLC HSS T3 column (1.8 µm, 100 × 2.1 mm) (Waters Corporation, Milford, MA, USA) operated at 50 °C. 0.1 % formic acid as mobile phase (MP) A and methanol as MP B were used for the analysis. Constant flow rate of 0.40 mL/min and gradient as follows were employed: 2 % MP B was maintained for 1.0 min, then increased to 22 % MP B in 2.0 min, further increased to 80 % MP B in 3.0 min and maintained constant for 2.0 min. The total runtime, including a 3.0-min equilibration period, amounted to 11.0 min. The injection volume was 10 µL.
HRMS detection was performed in positive ionization mode with a selected ion monitoring scan of 75.0553 m/z and an isolation width of 0.4 m/z. The Orbitrap detector with a resolution of 120,000 was utilized for detection. Ionization was achieved using an atmospheric pressure chemical ionization probe with a vaporizer temperature of 350 °C and an ion transfer tube temperature of 275 °C. The sheath gas was set at 70 Arb and the auxiliary gas at 5 Arb. A divert valve prior to the mass spectrometer was set to MS between 1.25 min and 2.1 min.
3. Results
3.4. Determination of NDMA in macrolides
To assess the potential of macrolides to produce NDMA, several macrolide DSs and FCTs were examined. Aside from the initially analyzed samples (Fig. 2), additional samples were analyzed for traces of NDMA. The analysis revealed 496.3 ppb of NDMA in CLA 1 DS, followed by 52.9 ppb and 25.2 ppb in SPI and AZI 1HDR DSs, respectively. In FCTs, the highest NDMA content was found in SPI FCT, followed by AZI 1HDR 1 – 3 FCTs (Table 3). In contrast to the 1HDR samples, no NDMA traces or levels below the LOQ (5.0 ppb) were determined in the DS and FCT samples of the AZI 2 HDR. Similarly, NDMA below the LOQ (10.0 ppb) was detected in CLA 2 DS and no NDMA was found in CLA 250 mg and 500 mg FCTs. As a negative control, CLI Ph and CLI HCl DSs were analyzed and no traces of NDMA were detected.
European Journal of Pharmaceutical Sciences, Volume 211, 2025, 107135: Table 3. NDMA traces found in macrolide DSs and FCTs, and CLI DSs (negative control).
3.5. Formation of NDMA under accelerated conditions
Samples of macrolide DSs and FCTs were subjected to accelerated stability testing at 60 °C for 14 days, and analyzed for traces of NDMA (Fig. 4, Fig. 5). DS analysis showed significantly higher amounts of NDMA in all stressed samples in which the t = 0 analysis already revealed NDMA, except for CLA 1 DS, where NDMA remained at approximately the same level. Similarly, no notable increase was noticed in AZI 2HDR DS, where at the t = 0 no NDMA was found (Fig. 4). The highest increase in absolute NDMA content was noticed in cases of SPI and ROX DSs, where levels rose by 81.7 ppb and 58.2 ppb, respectively. The highest NDMA content increase relative to the t = 0 was detected with CLA 2 DS, where the level increased by approximately 14-fold. FCT analysis showed higher NDMA content in stressed samples of AZI 1HDR 2 and SPI FCTs, while NDMA levels in other FCTs remained within the range of t = 0 detection (Fig. 5).
European Journal of Pharmaceutical Sciences, Volume 211, 2025, 107135: Fig. 4. NDMA levels in macrolide and CLI DSs (negative control) at t = 0 and after accelerated stability testing at 60 °C for 14 days. In brackets are estimated levels below the LOQ.
European Journal of Pharmaceutical Sciences, Volume 211, 2025, 107135: Fig. 5. NDMA levels in macrolide FCTs at t = 0 and after accelerated stability testing at 60 °C for 14 days. In brackets are estimated levels below the LOQ.
As a negative control, stressed samples of CLI Ph and CLI HCl DSs were analyzed and no NDMA was detected.
5. Conclusion
The DI GC–MS/MS method has been demonstrated to be suitable for the analysis of NDMA in the group of macrolide antibiotics for the first time. The method was qualified for the analysis of NDMA in multiple macrolide matrices. It has been shown that the accurate determination of NDMA in macrolides is strongly influenced by sample preparation. The complete dissolution of macrolide DSs in an organic solvent and the additional use of pyrrolidine as a scavenger proved to be the appropriate approach for the accurate analysis of NDMA. The SPME GC-MS/MS method was found to be unsuitable as the extraction of NDMA from poorly water-soluble macrolide DSs was proven to be limited, but its feasibility was verified for a water-soluble MET. The important finding regarding DS solubility, extraction efficiency and accurate determination of NDMA can be extended beyond macrolides and applied to the whole range of methods, especially those analyzing multiple nitrosamines in several DSs and DPs with different physico-chemical properties. Failure to account for poor solubility can result in misleading analytical results, potentially underestimating the true NDMA content. Furthermore, the presence of a dimethylamino group in the structure of macrolides was confirmed to pose a risk for NDMA formation in pharmaceuticals, which was most pronounced in SPI, containing two such structural elements. CLI as a negative control, without dimethylamino group in the structure, was incapable of NDMA formation. The accelerated stability study showed that the NDMA content in macrolide DSs and FCTs can substantially increase during storage. In the case of AZI, it has been shown that certain forms of DS exhibit greater resistance to nitrosylation, even though they may contain vulnerable reaction sites. Significant amounts of NDMA in macrolide FCTs far exceeded the AI limit, therefore calling for increased attention.
