AAPS PharmSci360 -Nitrosamines

[Naiffer_Host]

The American Association of Pharmaceutical Scientist conference will be on October 20-23 in Salt Lake City, UT.

Flagging few of the posters related to Nitrosamines

• (T1430-11-60) Determining Impact of Particle Size and Crystal Quality on Formation of Nitrosamine in API and Drug Formulations. Tuesday, October 22, 2024
• (T1330-11-62) Laboratory Study in Gastric Nitrosamine Formation. Tuesday, October 22, 2024
• (W1030-01-01) Nitrosamine Mitigation Case Study: The Importance of Utilizing a Multi-Pronged Approach to Reduce Impurities. Wednesday, October 23, 2024

Who is going to be there? If you see me in the hallways, I’ll be carrying the Nitrosamines Exchange badges!

[jason.brown]

I will be there with bells on.

We have 2 of these posters and are hoping for a third. Definitely need to catch up.

[MarkS]

I am trying to see if I can tag the trip into something else as well, so that I can be there.

[jason.brown]

Looking forward to seeing you at AAPS next week. Hope more are attending! If there is a big enough group, we should grab a meal together.

[Naiffer_Host]

Send me a message with you phone number so I can create a group and coordinate the meet up! -Naiffer

[Naiffer_Host]

USP and Community members were present at AAPS. It was so great to meet in person and connect with our community members!

[Naiffer_Host]

Very interesting posters at AAPS:

[Naiffer_Host]

(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. Redirecting.
(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. Direct measurement of the rate constants in the reaction of nitrous acid with methanol - Journal of the Chemical Society, Chemical Communications (RSC Publishing).
(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. Kinetics and mechanism of the nitrosation of alcohols, carbohydrates, and a thiol - Journal of the Chemical Society, Perkin Transactions 2 (RSC Publishing).

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).

[Naiffer_Host]

(T1530-11-62) The Necessity of Using LC-MS/MS Technology to Investigate the Presence of N-Nitrosamines Impurities in Pharmaceutical Drug Products

Purpose: Since 2018 there has been an increased attention from regulatory authorities towards the possible presence of nitrosamine impurities in human medicinal products containing chemically synthesized active pharmaceutical ingredients and biologically active substances. The focus on N-Nitrosamines is triggered by the fact that these chemical compounds are classified as probable human carcinogens and exhibit a greater correlation between mutagenicity and carcinogenicity compared to non-nitrosamine compounds. Nitrosamines are formed due to a specific chemical reaction whereby both nitrites, known to be oxidizing by nature, and amines (functional group) are present in specific conditions. Currently identified sources of nitrosamine impurities include the use or presence of nitrites in production processes, contaminated raw materials, cross contamination, degradation reactions, the use of certain packaging materials: Figure 1. Important to note that the potential source isn’t only the chemical formation during the DP manufacturing process. The European Medicines Agency (EMA) has defined a process with in a first step a thorough risk assessment and if needed, in step 2 confirmatory testing. The latter is a challenging analytical requirement as not only the appropriate mass spectrometry based equipment should be used, but also the detection limits (low) and the matrix (challenging) contribute to this analytical complexity. For the US market the FDA has mainly followed the EMA focus and attention on the N-Nitrosamines in finished DP with some small differences in its approach e.g. on the limits to be applied for the analytical testing. The identification and subsequent quantification (when needed) is an analytical challenge requiring state-of-the-art equipment, e.g. a Liquid Chromatography Triple Quad MS system, exhibiting enough analytical sensitivity for the most difficult N-Nitrosamines whereby the latter is often a combination of the intrinsic (low) sensitivity, the low thresholds (application and dose depending) and potential matrix interferences. An experimental case is described to illustrate the challenges in N-Nitrosamine testing in drug products as well as the need of LC-MS/MS equipment and appropriate, optimized methodologies. The importance of the establishment of the AET concentration is also demonstrated.

Methods: Standard solutions of nitrosamines are purchased commercially with a CoA. Solvents used in the chromatographic analysis and in the preparation of standards or spike solutions are of high quality grades to reduce the background presence of the target species. The equipment used in these case studies is an Agilent LC-MS/MS with Masshunter Acq software 10.1.

Results: The first challenge in each study on N-Nitrosamines concerns the establishment of the analytical evaluation threshold (AET) is deduced from the Acceptable Intake (AI) Limit specific for each N-Nitrosamine (Table 1) and the maximum daily dose (MDD) which is application dependent. The AET concentration calculated guides the development (and potential validation) of the method for the detection and confirmation of a specific N-Nitrosamine in the sample, e.g. the challenge in specificity/selectivity. Table 1 provides some AI limits for common N-Nitrosamines and a calculation of the AET concentration in various applications, e.g. Large Volume Parenterals (LVP), IV bags or syringes used for the administration of vaccines. It’s clear the key parameter, next to the AI limit, is the established daily dose leading to target analytical concentrations from as high as 26.5 µg/L to ultra trace concentrations of 0.5 ng/L. N-Nitrosodiproplyamine and N-Nitrosopiperidine (Fig 2) were at first the subject of a method development/feasibility study leading to the definition of the appropriate column to obtain sufficient separation, the ionization mode (APCI or ESI, positive or negative) and MRM transitions.Peak separation was obtained and subsequently a matrix spike was compared to the standard in solvent for the same species to assess specificity requirements. From the examples in Figure 2 it’s obvious that the selection of the appropriate column to separate the multiple N-Nitrosamines peaks is important, but more crucial is selecting the right MRM transitions to guarantee specificity and ultimately being able to distinguish between the actual presence of a target N-Nitrosamine and a false positive. Other challenges are a.o. the search for a suitable sample preparation procedure, establishing a realistic concentration factor, selection of a suited extraction solvent, the internal standard(s), evaluation on background presence of the target species. Potential sources are e.g. gloves, solvents, filters, polymeric materials used for the container closure systems.

Conclusion: Risk evaluation of all data available (e.g. E&L data) is the first step, a focus on drug products with high daily doses should be the next as these exhibit the lowest analytical evaluation threshold concentration. In view of the analytical challenges special attention should be given to the appropriate instrument (LC-MS/MS) and analytical column to obtain sufficient sensitivity and peak separation. Specificity is obvious crucial in this analysis to positively identify any present N-Nitrosamine and more challenges could be present in case of low AET levels, complex drug product matrix and respective mass spectral interferences with the selection of the appropriate MRM settings being key in the latter.

References: EMA updated Appendix 1 on “Acceptable Intakes (Ais) for N-nitrosamines” on 11th May 2024, EMA/15440/2024/rev.4 (NcWP; non-clinical Working Group)
FDA‘s Control of Nitrosamine Impurities in Human Drugs (Guidance for industry)

Table 1: Selected N-Nitrosamines and their Acceptable Intake limit (EMA limit) with examples of calculating the AET as a function of the application.

Figure 1: Visualization of the potential sources of N-Nitrosamines in pharmaceutical drug products.

Figure 2: LC-MS/MS analysis of a drug product (after appropriate sample prep) with APCI + mode (sample, spiked sample and standard in solvent) for N-Nitrosopiperidine (upper; MRM from 115.1 to 41.1) and N-Nitrosopropylamine (lower; MRM from 131.0 to 43.1).

[Naiffer_Host]

(M1230-09-48) Role of Low Nitrite (LN) Excipients in Mitigating N-Nitrosamine Drug Substance-Related Impurity (NDSRI) Formation in Drug Products

Purpose: N-Nitrosamines, a class of chemical impurities and degradants, are a significant concern in the pharmaceutical industry. They arise from the reaction of endemic nitrites with vulnerable secondary amines that may be present in drug substances or other impurities left behind from the manufacturing process (Figure 1). N-Nitrosamines are either proven or highly suspected carcinogens. N-Nitrosamines found in drug products have thus become one of the fastest-growing causes of drug product recalls in the last decade. While the full scope of the dangers of these impurities is not fully understood, modes of control of their formation have become a focus of effort for pharmaceutical scientists. The primary control mode has so far included better purification of drug substances to remove vulnerable secondary amines’ residual impurities. However, some drug substances contain the vulnerable secondary amine as part of the drug structure. In these cases, careful control of nitrite present in even a low-proportion excipient like crospovidone becomes a vital consideration.

Methods: Several formulations of Propranolol HCl 40 mg immediate-release tablets were manufactured to assess the efficacy of crospovidone type b ( < 15% on 63 µm screen) , Polyplasdone™ XL-10 LN (nitrite < 100 ppb), and Ultra 10 (nitrite < 100 ppb, peroxide < 50 ppm) in mitigating the formation of N-Nitroso-propranolol (NNP) from Propranolol HCl. These formulations were compared against standard copovidone type b (Table 1). All tablets were characterized for dimensions, hardness, friability, and dissolution release profile. Dissolution testing was conducted in 900 mL of 0.1N HCl maintained at 37°C with USP Apparatus 1 (baskets) at 100 RPM with samples taken every 5 minutes up to 30 minutes, quantitation by UV detection at 289 nm. Tablets were stored at 40°C and 75%RH for 2 weeks and 1, 3, and 6 months in HIS sealed 60 cc HDPE bottles (to simulate aging up to 1.5 years) and tested for the presence of NNP, ensuring a comprehensive assessment of the potential impurity. NNP determination: Samples and standards were prepared in 15:85 water: methanol. LC/MS/MS was performed on an Agilent 6420 QQQ LC/MS with 1260 Infinity Binary Modular HPLC and MassHunter software.

Results: All tablet formulations were comparable in hardness, friability, disintegration time, and dissolution release profile. All tablets initially contained 0.3-0.4 ng of NNP per mg of API. Without aging, some NNP was present in all formulations at about the same ng/mg of API level. During the aging study, however, it was evident that tight control of nitrites in even the low-weight ratio contributors to the formulation can significantly affect the amount of NDSRIs formed. In this instance, the only excipient variable was the type of crospovidone, which was only 5% of the total formulation. When the nitrite level is restricted to < 100 ppb in the starting excipient, as in the case of XL-10 LN and Ultra-10 grades, the NNP formation rate is significantly reduced (Figure 3).

Conclusion: Propranolol HCl Tablet formulations with LN, Ultra, or standard crospovidone grades show similar tablet properties and dissolution profiles. After 3 months at aggressive storage conditions, the LN grade contributed less than 1.0 ng of NNP formation to the total formulation content. This indicates that the LN grade is the best choice for the APIs containing secondary amines.

References: Alberto Berardi, M. J. (2023). Modeling the Impact of Excipients Selection on Nitrosamine Formation towards Risk Mitigation. Pharmaceutics, 1-15.
European Medicines Agency. (1 July 2024). Acceptable intakes (AIs) established for N-nitrosamines. Amsterdam: the agency of the European Union.
Food and Drug Administration. (3/5/2024). FDA Recommended AI Limits for Certain Hypothetical NDSRIs. Rockville, MD: FDA Dockets Management Staff (HFA-305).

Figure 1: Propranolol to N-nitroso-propranolol (NNP), Acceptable Intake (AI) limits: EMA1 and FDA2; 1500 ng/day

Table 1: Propranolol HCl Formulations

Figure 2. NNP Growth in Propranolol HCl 40 mg tablets (240mg as average maximum dosage) under accelerated aging conditions (6-month data pending)

[Naiffer_Host]

(W1030-01-01) Nitrosamine Mitigation Case Study: The Importance of Utilizing a Multi-Pronged Approach to Reduce Impurities

Purpose: Nitrosamine reduction is a top priority for the entire industry, with recent FDA guidance setting ambitious timelines for NDSRI compliance. Each API and drug product formulation is different, a one-size-fits-all additive strategy cannot be applied. In this presentation, we walk through Adare’s experience reducing the presence of nitrosamines in our own products over the last two years and detail our system of best practices in nitrosamine mitigation. This study specifically speaks to reducing the formation of NDSRIs formed from secondary and dimethyl tertiary amines, which the FDA has identified as most problematic. These functional groups can readily react with trace nitrite contamination present in the drug product to form NDSRI impurities. While the new NDSRI guidance specifically applies to marketed products, drugmakers with products in development containing these high-risk functional groups would be well advised to assess and address risks proactively. This minimizes the risk of late-stage reformulation, added costs, and product launch delays.

Methods: Nitrosamine mitigation was studied through the evaluation of nitrite scavengers, amino acids, and pH modification. Materials were chosen due to their chemical properties, inclusion in the FDA Inactive Ingredient Database, and compatibility with the API and formulation. The study was designed to characterize the solid state reactivity and the importance of the API microenvironment. This process utilized a screening process where chemicals were placed in binary mixtures with the API with added sodium nitrite to mimic real world situations. The most promising chemicals, concentrations, and mixtures were used in the subsequent step where they were mixed either with a lab blend or ground drug product at various concentrations. Multiple concentrations were used to gauge the response. The mixtures were dried then placed in a temperature and humidity-controlled environment for 2-4 weeks.

Results: 70-80% reduction in NDSRI formation vs. this control in the API Microenvironment for multiple samples. 84% reduction in NDSRI formation vs. control was achieved in the Solid State Stress Study, including the full formulation. All controls reached 50% to 200% of the regulatory limit, confirming a real-world environment. Traditional nitrite scavengers show the greatest reduction in nitrosamine formation in both the API microenvironment & drug product mixtures. Marked contrast in results from various amino acids.

Conclusion: Mitigation achieved is sufficient for practical use to meet regulatory requirements. An environment simulating real world time on the shelf was achieved. Previously unidentified reaction mechanisms observed in amino acids. Orthogonality was shown, increasing the design space as formulations and processes evolve.

Image 1: N-Nitrosopropranolol Mass Spectra

Image 2: Screening/API Microenvironment Results utilizing traditional nitrite scavengers and diazotization reaction scavengers vs. control (100%)

Image 3: Full formulation Solid State Stress Study Results of traditional nitrite scavengers and diazotization reaction scavengers vs. control (100%)

[Naiffer_Host]

(T1330-11-62) Laboratory Study in Gastric Nitrosamine Formation

Purpose: Nitrosamine reduction is a top priority for the entire industry, with recent FDA guidance setting ambitious timelines for nitrosamine drug substance-related impurities (NDSRI) compliance. Endogenous formation of nitrosamines from secondary amines has long been understood. However, there is no current in vitro test to approximate this phenomenon. This study specifically speaks to measuring the formation of NDSRIs from secondary and dimethyl tertiary amines, which the FDA has identified as most problematic. These functional groups can readily react with nitrite present in the gastric tract to form NDSRI impurities.

Methods: In vivo conditions were simulated utilizing in vitro USP Dissolution Apparatus 2 with a well characterized drug product known to form an NDSRI. Dissolution conditions were selected to mimic conditions at various states in the feeding cycle including biorelevant pH, salts, enzymes, and volume. The appropriate nitrite quantity was established based upon IARC Vol. 94, Ingested Nitrate and Nitrite, and Cyanobacterial Peptide Toxins and the extrapolated maximum nitrite quantity during a meal. This was coupled with LC/MS analysis to achieve the desired sensitivity and selectivity.

Results: Each set of biorelevant conditions performed showed NDSRI concentrations substantially higher than the regulatory limit of 2.3µg/g. Most experiments exceeded this threshold 50-fold. This was true whether a simple buffer was used or simulated gut fluids.

Conclusion: Nitrite concentrations and pH alone are sufficient to drive the reaction. Given the similarity in gastric conditions to the widely accepted NAP test, which is designed to show the possibility of nitrosamine formation, these results are not surprising. Future work should be performed based on product specific pharmacokinetics. Interspecies NOx conversion in the more complex in vivo environment may reduce the effect of nitrite concentration differences.

References: Raisfeld-Danse IH, Chen J. Drug interactions. III. Formation of nitrosamines from therapeutic drugs. Formation, mutagenic properties and safety assessment of propranolol hydrochloride with respect to the intragastric formation of N-nitrosopropranolol under conditions found in patients. J Pharmacol Exp Ther. 1983 Jun;225(3):713-9. PMID: 6345752.
IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. IARC monographs on the evaluation of carcinogenic risks to humans. Ingested nitrate and nitrite, and cyanobacterial peptide toxins.

Image 1: N-Nitrosopropranolol Mass Spectra

Image 2: NDSRI formation in reference to API mass, pH 3.0 Fasted State Simulated Gastric Fluid/pH 6.8 Fasted State Simulated Intestinal Fluid with Enzymes

Image 3: NDSRI formation in reference to API mass, pH 1.6 Fasted State Simulated Gastric Fluid/pH 6.8 Fasted State Simulated Intestinal Fluid

[Naiffer_Host]

(T1230-10-58) Evaluation of Mitigation Approaches for Nitrosamine Formation in Binary Blends

Purpose: N-Nitrosamines are a class of potentially mutagenic carcinogens, some of which have been observed in pharmaceutical products. Nitrosamines could possibly form from the nitrite impurities in the excipients reacting with vulnerable secondary amines on the active pharmaceutical ingredient. It is hypothesized that the application of nitrite scavenging film converts the nitrite impurities into non-nitrosating agents, leading to mitigating the nitrosamine formation.

Methods: To test this hypothesis, experiments were designed containing binary blend of lisinopril (LSN) model drug substance and excipients exposed to 75%RH at 60 °C. The influence of water on nitrosamine formation was examined at various (11 to 75%RH) humidity conditions. To minimize nitrosamine formation, various formulation approaches such as coating with SiO2 (R200 (hydrophilic) and R972 (hydrophobic) grades), addition of antioxidants (cysteine (CYS) and propyl gallate (PG) and use of nitrite scavenging films (N-Sorb 36 and N-Sorb 38) Aptar CPS technologies, Auburn, AL, have been explored. N-Nitroso lisinopril (N-NO LSN) was quantitated using Vanquish Tandem Liquid Chromatography (LC) system and a Orbitrap Exploris™ 240 mass spectrometer (MS) (Thermo Scientific™, Waltham, MA) with a retention time of 4.3 min.

Results: It was observed that there is a good correlation between the relative humidity and the presence of nitrite in the formation of N-NO LSN impurity. The proposed mechanism of nitrosamine formation entails the following sequence: sorption of moisture by excipients to form a saturated solution layer → followed by dissolution of lisinopril → thereby facilitating the nitrosation reaction. The first mitigation approach, coating of LSN with SiO2 R200 and R972 grades resulted in more than 1.5 folds higher N-NO LSN formation. The second strategy, i.e., addition of CYS and PG antioxidants, displayed slight reduction (~ 20%) in nitrosamine formation. Finally, the nitrosamine impurity formation was significantly reduced in the presence of N-Sorb 36 and N-Sorb 38 films. The results demonstrated approximately 80% potential reduction of nitrosamine formation on day 3 with N-Sorb 36 films.

Conclusion: The nitrite scavenging films indicated a significant reduction in the nitrosamine formation as compared to widely used coating agents and antioxidants. This strategy offers an innovative solution to address the growing demand for nitrosamine remediation in the pharmaceutical industry.

Figure 1 Effect of nitrite scavenging films (N-sorb 36) on N-nitroso lisinopril formation in binary LSN-excipients blends at 60 °C/75%RH

[Naiffer_Host]

(T1430-11-60) Determining Impact of Particle Size and Crystal Quality on Formation of Nitrosamine in API and Drug Formulations

Purpose: The purpose of this project is to develop an understanding of the role of solid-state reactive species (SSRS) in the formation of nitrosamines, and to describe methodologies that can be used to both determine their concentration and to mitigate their formation.

Methods: The SSRS within several model compounds including 4-phenylpiperdine, ranitidine hydrochloride, were created with different manufacturing methods. Samples were cryo-milled or ball-milled for different length. The amount of SSRS was quantified with Solid-state NMR 1H T1 relaxation time (T1). Both milled and bulk samples were stored in different accelerated conditions. Ranitidine hydrochloride would degrade to N-nitrosodimethylamine (NDMA) and 4-phenylpiperdine would degrade to N-nitroso-4-phenylpiperdine (NiPP) at presence of nitrite. The levels of degradation product were measured by LC-MS/MS or LCMS at multiple time points. The surface area of milled sample was measured by Brunauer-Emmett-Teller (BET) surface area analyzer. After understanding the impact of SSRS in drug substance degradation process, this research will then focus on stability of drug substance/drug formulations under the influence of SSRS. The same model compounds will be tested with different formulations, primarily differing in the total nitrite content in the formulations. Total nitrite content is the limiting factor in the nitrosamine formation. The effect of SSRS and nitrite content on the degradation rate will be both evaluated.

Results: The preliminary data shown that the SSRS, specifically crystal defect, could be generated in multiple model compounds through cryo-milling such as lactose monohydrate. The SEM image indicated the difference between unmilled and 60min cryo-milled lactose monohydrate crystal sample. Our group also proved the T1 for lactose monohydrate was decreased when the milling time increased. This result indicated that the T1 could be used to identify the presence of SSRS (Figure.1). To correlate the T1 with the solid-state degradation rate, our group used Aspirin and Gabapentin as the model compound. Aspirin will degrade to salicylic acid and gabapentin will degrade to Gabapentin lactam in solid-state. Our group measured the stability of milled Aspirin and Gabapentin samples in accelerate condition and T1 for those samples after milling. The data shown that the increasing the milling time could increase the amount of crystal defects in the powder sample, in the other words, the sample contained more SSRS. When the sample contained more SSRS, the sample crystal quality decreased. As a result, the T1 decreased, and the reactivity of the sample increased due to the low crystal quality. As depicted in Figure 2, unmilled Aspirin had a T1 of approximately 57 seconds with 83.7% remaining post-stability test. In contrast, 60 minutes cryo-milled Aspirin sample exhibited a reduced T1 of 14 seconds, with no detectable Aspirin remaining after the stability test. The same phenomena were observed in Aspirin formulation and Gabapentin system. This result indicated that the T1 can serve as a predictive indicator of the degradation rate of drug substances or formulations in the solid state, influenced by the presence of SSRS. Once we established the relationship between T1 and the solid-state degradation rate, our attention shifted to drug substances that degraded into nitrosamines. Ranitidine hydrochloride and 4-phenylpiperidine hydrochloride were chosen as model compounds. Stability testing of ranitidine hydrochloride indicated that NDMA might further degrade under conditions of high humidity. Additionally, NDMA levels in all ranitidine hydrochloride samples stored at various temperatures under sealed conditions was higher than as-received samples. The stability of cryo-milled ranitidine hydrochloride samples under sealed condition at 60 °C shown that the longer cryo-milled sample had worse stability comparing with the unmilled sample. However, ranitidine hydrochloride had a very short T 1 relaxation time due to the methyl groups attached on the structure. These methyl groups can spin and act as relaxation “sinks,” significantly reducing the T1. Our group is currently attempting to run the samples at lower temperatures to decrease the spinning of the methyl groups and thereby increase the T1. We are also exploring the possibility of increasing the T1 by deuteration of the methyl groups to mitigate the “sink” effect. The other model compound in our study, 4-phenylpiperidine and its salt form, 4-phenylpiperidine hydrochloride, is currently undergoing stability testing. We are evaluating multiple sample types, including milled samples, samples with low levels of sodium nitrite impurities, and drug formulations. Our results shown that T1 was decreased when the milling time was increased for both 4-phenylpiperdine and 4-phenylpiperdine hydrochloride samples. (Figure 3)

Conclusion: Based on our preliminary data and experimental results, we conclude the following: SSRS can be produced through the grinding or milling of materials. The quantity of SSRS correlates with both degradation rates and 1H T1 solid-state NMR relaxation times. Ranitidine hydrochloride samples degrade more rapidly the longer they are milled. The degradation product of ranitidine hydrochloride, NDMA, may further degrade under conditions of high humidity. 4-Phenylpiperidine is an effective model compound for correlating degradation due to SSRS with 1H T1 solid-state NMR relaxation times.

References: Wexler, P. and B. D. Anderson (2005). Encyclopedia of toxicology, Academic Press.
Akkaraju, H., Tatia, R., Mane, S. S., Khade, A. B., andDengale, S. J. (2023) A comprehensive review of sources of nitrosamine contamination of pharmaceutical substances and products Regulatory Toxicology and Pharmacology 105355,
López-Rodríguez, R., McManus, J. A., Murphy, N. S., Ott, M. A., & Burns, M. J. (2020). Pathways for N-nitroso compound formation: secondary amines and beyond. Organic Process Research & Development, 24(9), 1558-1585.
Harmon, P. (2023). “Ranitidine: a proposed mechanistic rationale for NDMA formation and a potential control strategy.” Journal of Pharmaceutical Sciences 112(5): 1220-1224.
King, F. J., et al. (2020). “Ranitidine—investigations into the root cause for the presence of N-nitroso-N, N-dimethylamine in ranitidine hydrochloride drug substances and associated drug products.” Organic process research & development 24(12): 2915-2926.
Yokoo, H., et al. (2023). “Advanced Solid-State NMR Analysis of Two Crystal Forms of Ranitidine Hydrochloride: Detection of 1H–14N Intra-/Intermolecular Correlations.” Chemical and Pharmaceutical Bulletin 71(1): 58-63.
United States Pharmacopeial Convention, “United States Pharmacopeia and National Formulary (USP 42-NF 37).” Rockville, MD, U.S.A., pp. 3811–3812, 2019.
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Lactose monohydrate cryomilled sample SEM image and Solid-state NMR 1H T1 relaxation time.

Gabapentin and Aspirin milled/unmilled sample stability data.

Ranitidine Hydrochloride and 4-phenylpiperdine stability and T1 data.