(T1430-10-58) Understanding Nitrite Loss in Pharmaceutical Drug Products and its Impact on N‑Nitrosamine Formation

Purpose: N-Nitrosamines (N-NA) are a class of potentially mutagenic impurities in pharmaceutical products with acceptable intakes typically ranging from 26.5 to 1500 ng/day. Nitrite—a reactant in nitrosation chemistry—is commonly detected at the ppb- or ppm-level in drug product (DP) excipients. Due to the nitrosation rate-dependency on nitrite concentration, higher levels of nitrite increase N-NA formation in DP. Therefore, minimizing nitrite in DP is a viable approach for N-NA control.¹ Understanding solid-state nitrite stability in DP may provide foundational knowledge to 1) appropriately design experiments to project N-NA formation risk over DP shelf-life and 2) contextualize predictive modelling vs. experimental results for DP N-NA growth. Furthermore, knowledge of the conditions that impact nitrite stability in the solid-state may inform strategies to purge nitrite from excipients prior to incorporation into the DP. To evaluate nitrite stability in DP, placebo tablets were formulated without an active pharmaceutical ingredient (API) to remove competing nitrosation pathways. The placebo tablets were prepared with and without fumaric acid to modulate the solid-state microenvironment pH. The nitrite content in the placebo tablets was measured over 30-days at a series of temperatures, humidities, and storage configurations.

Methods: Tablet preparation: Two lots of 100-ppm nitrite-spiked placebo tablets were compressed from dry granulated blends prepared with microcrystalline cellulose, croscarmellose sodium, mannitol, and magnesium stearate excipients. Fumaric acid (2 wt%) was added to one blend lot as a formulation pH modifier. Stability studies:i) Open storage conditions: Tablets were added to 20 mL glass vials (15 tablets/vial/timepoint) and placed, uncapped, in a Bahnson ES2000 or ThermoFisher Scientific Forma environmental chamber equilibrated to 40°C/75%RH, 25°C/75%RH, or 25°C/60%RH. ii) Packaged storage configuration: Tablets (15 tablets/bottle/timepoint) were packaged in induction-sealed 45 mL high-density polyethylene (HDPE) bottles with 3 g of desiccant and stored in a Bahnson ES2000 environmental chamber equilibrated to 40°C/75%RH. The relative humidity inside the induction-sealed 45 mL HDPE bottle was measured by placing a MadgeTech MicroRHTemp probe in the bottle. Ion chromatography: Nitrite and nitrate quantitation was conducted via ion chromatography using a Dionex ICS 6000 (ThermoFisher Scientific) with suppressed conductivity detection. The injection volume was 100 µL and a gradient elution using aqueous KOH concentrations ranging from 10 mM to 54 or 100 mM was used. Aqueous 10 mM NaOH was used as the sample preparation diluent.

Results: The slurry pH of tablets with and without fumaric acid was measured to be approximately 3.2 and 6.4, respectively. A pH-dependent loss of nitrite was observed in placebo tablets containing a 100-ppm nitrite spike when stored open in the 25°C/60%RH environmental chamber (Figure 1). Exposure of the placebo tablets to 40°C/75%RH, a common accelerated stability condition, further increased nitrite loss over 30-days and afforded ≥ 95% nitrite reduction irrespective of formulation pH. To isolate the impact of humidity on nitrite stability, the placebo tablets were exposed to 60%RH and 75%RH at a fixed 25°C temperature (Figure 1). After 30-days, the nitrite in tablets stored at 75%RH was 2% (2 wt% fumaric acid) or 4% (0 wt% fumaric acid) of that at 60%RH. This humidity trend was corroborated when placebo tablets with 0 wt% fumaric acid were packaged in an induction-sealed, desiccated HDPE bottle and stored in a 40°C/75%RH environmental chamber. The tablets in the desiccated HDPE bottle—with a measured %RH of ~2%—had no discernable nitrite loss after 20-days (Figure 2). In contrast, the tablets stored open at 40°C/75%RH had ≥ 95% nitrite loss over 20-days. At all temperatures, humidities, and tablet storage configurations evaluated, nitrate content did not appear to increase. This suggests that nitrite oxidation to nitrate is not a significant contributor to nitrite loss under these conditions.

Conclusion: The notable decrease in the measured nitrite in placebo tablets stored in open containers at 25°C/60%RH, 25°C/75%RH, and 40°C/75%RH raises the question of what pathways lead to nitrite consumption in the absence of a nitrosatable API. Possible nitrite loss routes stem from nitrous acid generation. Nitrous acid is a precursor to, and in equilibrium with, several volatile nitrosating agents (e.g., N₂O₄, N₂O₃,), which are gases at the conditions evaluated. The O‑nitrosation of hydroxyl-functionalized DP excipients is also feasible and could contribute to a reduction in the measured inorganic nitrite, if not hydrolyzed during the analytical sample preparation and analysis.^2,3 The conversion of nitrite to volatile nitrosating agents under sufficiently humid conditions suggests that tablets stored in open storage configurations may not accurately represent nitrosation kinetics in packaged DP. This highlights the importance of understanding the impact of headspace on nitrite loss and N-NA growth during DP stability studies. These results also motivate further studies to establish correlations between nitrite loss and N-NA formation in DP.

References: (1) Moser, J.; Ashworth, I. W.; Harris, L.; Hillier, M. C.; Nanda, K. K.; Scrivens, G. N-Nitrosamine Formation in Pharmaceutical Solid Drug Products: Experimental Observations. Journal of Pharmaceutical Sciences 2023, 112 (5), 1255–1267. https://doi.org/10.1016/j.xphs.2023.01.027.
(2) Aldred, S. E.; Williams, D. L. H. Direct Measurement of the Rate Constants in the Reaction of Nitrous Acid with Methanol. J. Chem. Soc., Chem. Commun. 1980, No. 3, 73. https://doi.org/10.1039/c39800000073.
(3) Aldred, S. E.; Williams, D. L. H.; Garley, M. Kinetics and Mechanism of the Nitrosation of Alcohols, Carbohydrates, and a Thiol. J. Chem. Soc., Perkin Trans. 2 1982, No. 7, 777. https://doi.org/10.1039/p29820000777.

Acknowledgements: The authors thank Chris Smith, Fiona Nkala, Richard Wadden, Brad Peori, and Deepika Parasuraman for IC method development. The authors also acknowledge nitrosamine working group member Dan Li for helpful discussions.

Figure 1. Measured nitrite and nitrate content over time in tablets with slurry pH values of 3.2 and 6.4 that were stored open in environmental chambers at 40°C/75%RH, 25°C/75%RH, and 25°C/60%RH.

Figure 2. Measured nitrite and nitrate content over time in tablets with a slurry pH value of 6.4 that were stored open in an environmental chamber (40°C/75%RH) or in a desiccated, induction-sealed HDPE bottle (40°C/2%RH).