Analytical Strategies for the Detection and Quantification of Nitrosamine Impurities in Pharmaceuticals

Brief overview of N-nitrosamine impurities and their significance in pharmaceuticals

N-Nitrosamine impurities, organic compounds characterized by the nitroso (N−N=O) functional group, have emerged as a significant concern within the global pharmaceutical landscape since 2018. Their presence in active pharmaceutical ingredients (APIs), drug products, and various other matrices is problematic due to their potential toxicity, carcinogenicity, and mutagenicity, even at low levels. Exposure in humans is linked to severe health risks, including various cancers which is the leading cause of death worldwide, amounting to nearly 7.6 million deaths globally (liver, lungs, esophagus, stomach, kidneys etc.), metabolic disturbances, reproductive harm, DNA damage, and potentially neurodegenerative diseases like Alzheimer's and Parkinson's. Given these potential risks, strict control and accurate quantification of these impurities in medicines are paramount for patient safety.^1–4  

Historical context of nitrosamine contamination issues (e.g., sartans, ranitidine, metformin)

While known chemically for over 150 years and recognized as potential carcinogens since the 1950s, N-nitrosamines surged into pharmaceutical focus in July 2018.  

Sartans (ARBs): The issue began with the detection of N-nitrosodimethylamine (NDMA) in valsartan API manufactured in China. This discovery triggered widespread investigations and subsequent recalls. Soon after, NDMA and another impurity, N-nitrosodiethylamine (NDEA), were found in other angiotensin II receptor blockers (ARBs) like losartan and irbesartan, leading to further recalls involving numerous manufacturers. Initial investigations suggested specific synthetic manufacturing processes were responsible for their formation.  

Ranitidine: In September 2019, regulatory authorities announced unacceptable levels of NDMA contamination in ranitidine products (commonly known as Zantac). This led to market withdrawals and suspensions in various countries, including the USA, Canada, and Australia.  

Metformin: Following the ranitidine findings, NDMA contamination was noted in some metformin products used for type 2 diabetes in late 2019 and early 2020. Voluntary recalls were initiated by several companies.  

Other Medications: The issue has since expanded beyond these initial drug classes, with nitrosamine impurities being reported in varenicline (Champix), rifampicin, and others, prompting further recalls and heightened regulatory scrutiny across the pharmaceutical industry.⁵

Importance of sensitive and selective analytical methods for detection and quantification

The extremely low acceptable intake limits set by regulatory agencies (e.g., typically ranging from 26.5 to 96 ng/day for single impurities per ICH M7) necessitate the development and implementation of highly sensitive and selective analytical methodologies. Traditional analytical techniques like standard HPLC-UV or GC often lack the capability to reliably detect and quantify these impurities at the required trace levels. The initial failure to detect NDMA in valsartan highlighted the critical need for more advanced methods. Consequently, techniques such as Liquid Chromatography-Mass Spectrometry (LC-MS, LC-MS/MS, LC-HRMS) and Gas Chromatography-Mass Spectrometry (GC-MS, GC-MS/MS), sometimes coupled with enhanced sample preparation, have become essential tools. These methods must exhibit adequate precision and accuracy to ensure the safety and quality of pharmaceutical products. ^6–10 

Scope and objectives of the review article

This review aims to provide a comprehensive overview of the analytical strategies employed for the detection and quantification of N-nitrosamine impurities in pharmaceuticals. It will delve into the challenges associated with nitrosamine analysis, explore various sample preparation techniques, and critically evaluate the predominant instrumental methods, including HPLC, GC, SFC, and particularly hyphenated mass spectrometric techniques (LC-MS and GC-MS). The review will summarize methods applied to commonly affected drug classes (sartans, ranitidine, metformin) as well as recently implicated products. Furthermore, it will touch upon the regulatory context and validation requirements pertinent to these analytical approaches. The overall objective is to consolidate current knowledge on analytical methodologies, offering insights into best practices and potential future directions for ensuring the reliable control of N-nitrosamine impurities in pharmaceutical products.^11–21

Understanding N-Nitrosamine Impurities

Chemical structure and definition

N-Nitrosamines are organic compounds defined by the presence of a nitroso functional group (N−N=O) attached to an amine nitrogen. The general chemical structure involves a nitrogen atom bonded to another nitrogen atom, which is, in turn, double-bonded to an oxygen atom, with the first nitrogen also bonded to two organic substituents (R1 and R2). While nitroso groups can also attach to carbon (C-nitroso), sulfur (S-nitroso), or oxygen (O-nitroso), N-nitrosamines specifically refer to compounds with the R_1?R₂?N−N=O structure. They are often formed via the reaction of secondary or tertiary amines (or related structures like amides, carbamates) with nitrosating agents.

Table 1 Common N-Nitrosamine Impurities and Regulatory Limits

(MAI: Maximum Allowable Intake; NA: Not Available)

Sources of N-Nitrosamine Impurities in Pharmaceuticals

The presence of N-nitrosamine impurities in pharmaceutical products can arise from multiple sources throughout the product lifecycle. Identifying these sources is crucial for risk assessment and mitigation.  

• API Manufacturing Process:
  • Raw Materials/Starting Materials/Intermediates: Contamination can originate from impurities present in these initial materials. The use of certain reagents, solvents, and catalysts can also introduce risks.  
  • Synthesis Route: Specific reaction steps, particularly those involving sodium nitrite (NaNO2) or other nitrosating agents (often used to quench residual azides), can lead to nitrosamine formation. The use of solvents like DMF, NMP, DMA, or amine bases like TBA can degrade or contain amine precursors.  
  • Process Conditions: Reaction conditions such as temperature, pH (especially acidic conditions), and the order of reagent addition can influence nitrosamine formation. Inadequate process optimization or purification steps may fail to remove these impurities.  
  • Recovered Materials: Use of recovered solvents, reagents, or catalysts without adequate purification can reintroduce contaminants.  
• Drug Product Formulation:
  • Excipients: Many common pharmaceutical excipients (e.g., povidone, crospovidone, croscarmellose sodium, lactose, starches) can contain trace levels of nitrites or nitrates. Excipients themselves can also contain or degrade to form amine precursors. Formaldehyde, found as an impurity or degradation product in excipients like PEG and polysorbates, can catalyze nitrosation.  
  • API-Excipient Interactions: Interactions between the API and reactive impurities in excipients can lead to nitrosamine formation during formulation or storage.  
  • Manufacturing Process: Conditions during formulation, such as the presence of water, heat (e.g., during drying), and acidic pH, can facilitate nitrosamine formation.  
• Degradation and Storage:
  • N-nitrosamines can form over time in the final drug product due to the degradation of the API or excipients, especially under certain storage conditions (temperature, humidity). The presence of nitrites in packaging materials (e.g., nitrocellulose blister lidding foil) has also been identified as a potential source.  
• Other Sources: Contamination from external sources like water used in processing, packaging materials (e.g., printing inks, rubber), and even environmental factors is possible

Table 2 Sources of N-Nitrosamine Impurities

Mechanism of Formation

The formation of N-nitrosamines generally requires two key components: a nitrosating agent and a secondary or tertiary amine, typically reacting under acidic conditions.  

• Nitrosating Agents: These are species capable of donating a nitroso group (N=O). Common sources relevant to pharmaceuticals include:
  • Nitrous acid (HNO2), formed from nitrites (e.g., NaNO2) under acidic conditions. HNO2 can exist in equilibrium with dinitrogen trioxide (N2O3), another nitrosating species.  
  • Nitrogen oxides (NOx), present as contaminants or formed from degradation, can react to form nitrites or directly participate in nitrosation.  
  • Nitrosyl halides or other reactive nitrogen species formed under specific process conditions.
• Amine Precursors:
  • Secondary amines (R2NH) are most readily nitrosated.  
  • Tertiary amines (R3N) can also undergo nitrosative cleavage to form nitrosamines.  
  • Other nitrogen-containing functional groups like amides, carbamates, and urea derivatives can potentially form nitrosamines.  
  • Sources include the API itself, related substances, impurities in raw materials/reagents/solvents (e.g., dimethylamine impurity in DMF leading to NDMA), or degradation products.  
• Reaction Conditions:
  • pH: Nitrosation is typically favored under acidic conditions (which promote HNO2 formation), although the optimal pH depends on the specific amine (related to amine basicity and deprotonation).  
  • Temperature: Elevated temperatures can increase reaction rates.  
  • Catalysts: Certain species, like formaldehyde or other aldehydes, can catalyze the reaction. The presence of water is also often important.^22–27

Table 3 N-Nitrosamine Formation

Toxicological Significance

N-nitrosamines are classified as probable human carcinogens (IARC Group 2A) and are known mutagens.  

• Carcinogenicity & Mutagenicity: They are considered potent carcinogens, with evidence from numerous animal studies showing tumor formation in various organs. Their mutagenicity arises from their ability to chemically interact with DNA after metabolic activation, potentially leading to genetic mutations.  
• Regulatory Classification: Due to these properties, they are treated as "cohort of concern" impurities under guidelines like ICH M7(R1), which addresses DNA reactive (mutagenic) impurities.  
• Acceptable Limits: Extremely low limits are set to control exposure.
  • Threshold of Toxicological Concern (TTC): For mutagenic carcinogens like nitrosamines, a TTC-based limit of 1.5 µg/day is often referenced from ICH M7, representing a negligible lifetime cancer risk.  
  • Maximum Allowable Intake (MAI) / Acceptable Intake (AI): Compound-specific limits (often expressed in ng/day) are established based on carcinogenic potency data where available. Examples include NDEA (26.5 ng/day) and NDMA (96.0 ng/day). These limits apply to single nitrosamines; adjustments are needed if multiple nitrosamines are present. EMA has introduced approaches like the Carcinogenic Potency Categorization Approach (CPCA) and Enhanced Ames Test (EAT) to help establish AIs for new nitrosamines.  
• Risk Assessment: Predicting mutagenicity and carcinogenicity often involves in silico assessments using QSAR methodologies (expert rule-based and statistical) as per ICH M7.  ^28–31

Table 4 Toxicological Significance

Analytical Strategies for Detection and Quantification

The accurate detection and quantification of N-nitrosamine impurities represent a formidable challenge in pharmaceutical analysis. This stems primarily from the stringent regulatory limits imposed due to their potential carcinogenicity and the inherent complexity of pharmaceutical formulations. Traditional analytical methods often fall short of the required sensitivity and selectivity, necessitating the adoption of advanced, highly sophisticated analytical strategies.

• Ultra-Trace Level Quantification: N-nitrosamines must typically be quantified at the nanogram (ng) or parts-per-billion (ppb) level to comply with regulatory acceptable intake (AI) limits, which can range from <20 ng/day to ~100 ng/day depending on the specific compound and regulatory guidance. Achieving adequate sensitivity (LOQ) is therefore paramount.  
• Matrix Complexity and Interference: Pharmaceutical products consist of the active pharmaceutical ingredient (API) and various excipients. These matrix components can significantly interfere with nitrosamine analysis, particularly in mass spectrometry (MS), causing ion suppression or enhancement, and potentially masking the target analyte signal. Effective sample clean-up is critical to mitigate these effects.  
• Analyte Properties: N-nitrosamines exhibit diverse physicochemical properties. Some are volatile (e.g., NDMA, NDEA), making them amenable to Gas Chromatography (GC), while others are less volatile or thermally sensitive, necessitating Liquid Chromatography (LC) approaches.  
• Artifactual Formation: There is a risk of artifactual formation of nitrosamines during the analytical process itself, for example, in hot GC injectors or under harsh sample preparation conditions if precursors (amines and nitrosating agents) are present. Analytical conditions must be carefully optimized to prevent in situ formation.  
• Selectivity: Ensuring the method can distinguish the target nitrosamine(s) from structurally similar compounds, isomers, and isobaric interferences within the complex matrix requires highly selective techniques, typically involving tandem mass spectrometry (MS/MS) or high-resolution mass spectrometry (HRMS).^32–35

Sample Preparation Techniques

Robust sample preparation is indispensable for isolating nitrosamines from the sample matrix, concentrating them to detectable levels, and removing interfering substances prior to instrumental analysis. Techniques referenced in the provided article and commonly employed include:

• Solid-Phase Extraction (SPE): A cornerstone technique for sample clean-up and concentration, utilizing cartridges packed with specific sorbents to selectively retain nitrosamines while washing away matrix components. Online SPE configurations enhance automation and throughput.  
• Liquid-Liquid Extraction (LLE): A conventional technique partitioning analytes based on differential solubility between two immiscible liquid phases.
• Dispersive Liquid-Liquid Microextraction (DLLME): A miniaturized LLE method using small volumes of extraction and disperser solvents, offering high enrichment factors and reduced solvent consumption.  
• Solid-Phase Microextraction (SPME): Employs a coated fiber to extract analytes, particularly useful for volatile/semi-volatile compounds from the sample headspace (HS-SPME) prior to GC analysis. It can minimize thermal degradation associated with direct injection.  
• Other Techniques: Methods like precipitation (to remove proteins or macromolecules), simple dilution ("dilute-and-shoot" for less complex samples or highly sensitive instruments), and specific chemical derivatizations (e.g., for fluorescence detection) are also utilized depending on the specific application and analytical technique.

Chromatographic Techniques

Chromatography separates the nitrosamines from each other and from remaining matrix components before detection.

• High-Performance Liquid Chromatography (HPLC):
  • The workhorse for separating non-volatile or thermally labile compounds. Ultra-High-Performance Liquid Chromatography (UHPLC/UPLC) is preferred for its speed, resolution, and efficiency.
  • Detection: While UV detection is common in HPLC, it generally lacks the sensitivity for trace nitrosamine analysis. Fluorescence detection (FLD) can be used after specific derivatization procedures. Primarily, HPLC/UHPLC serves as the inlet for mass spectrometers.  
• Gas Chromatography (GC):
  • Well-suited for volatile nitrosamines (e.g., NDMA, NDEA, NEIPA, NDIPA).  
  • Sample Introduction: Headspace (HS) injection is common, minimizing matrix contamination and potential thermal degradation in the injector. SPME is also frequently coupled.  
  • Detection: While detectors like Nitrogen Phosphorus Detector (NPD) exist, mass spectrometry (GC-MS/MS) is the standard for achieving the required sensitivity and selectivity.  
• Supercritical Fluid Chromatography (SFC):
  • Uses a supercritical fluid (typically CO2) as the mobile phase, offering orthogonal selectivity compared to LC and GC.
  • It has been applied successfully for the rapid analysis of nitrosamines in various drug substances and formulations, sometimes simultaneously with other related impurities. It can be coupled with MS/MS detection.^36,37

Hyphenated Techniques (Mass Spectrometry Based)

Coupling chromatography with mass spectrometry provides the necessary sensitivity and selectivity for definitive trace-level nitrosamine analysis.

• Liquid Chromatography-Mass Spectrometry (LC-MS):
  • The most versatile and widely reported technique, suitable for a broad range of nitrosamines, including less volatile and thermally unstable ones.  
  • Ionization: Electrospray Ionization (ESI) and Atmospheric Pressure Chemical Ionization (APCI) are common interfaces enabling analyte ionization prior to MS analysis.  
  • Analyzers:
    • Tandem MS (MS/MS): Typically using Triple Quadrupole (QqQ) instruments operating in Multiple Reaction Monitoring (MRM) mode provides excellent sensitivity and selectivity for targeted quantification.  
    • High-Resolution MS (HRMS): Instruments like Time-of-Flight (TOF) or Orbitrap offer high mass accuracy and resolution, facilitating confident identification, structural elucidation, and discrimination from isobaric interferences, often used for screening and confirmation.  
• Gas Chromatography-Mass Spectrometry (GC-MS):
  • Preferred for volatile nitrosamines.  
  • Analyzers: Tandem MS (GC-MS/MS) is crucial for achieving low detection limits and selectivity in complex matrices. HRMS (e.g., GC-Orbitrap MS) is also employed for accurate mass measurements.²³

Other Techniques

Capillary Electrophoresis (CE)-MS: While less common for routine pharmaceutical analysis, CE offers high separation efficiency based on charge-to-size ratio. CE-MS has demonstrated utility for specific applications, such as analyzing tobacco-specific nitrosamines in biological fluids.  

Comparison of Analytical Techniques

The choice of technique depends on the specific nitrosamine(s), the matrix, required sensitivity, available instrumentation, and the analytical goal (screening vs. quantification).

• Sensitivity & Selectivity: Hyphenated MS techniques (LC-MS/MS, GC-MS/MS, LC-HRMS, GC-HRMS) offer superior sensitivity and selectivity compared to conventional HPLC-UV/FLD or GC-NPD, essential for meeting regulatory limits. HRMS provides higher certainty in identification than MRM-based QqQ methods.  
• Applicability: LC-MS is more broadly applicable across different nitrosamine polarities and volatilities. GC-MS is excellent for volatile species. SFC offers an alternative selectivity profile.  
• Throughput & Automation: Modern UHPLC and automated sample preparation systems (e.g., online SPE) enhance throughput. Headspace GC methods can also be readily automated. SFC can offer speed advantages.  
• Limitations: Potential for artifact formation (especially in GC), matrix effects (MS), cost and complexity of advanced instrumentation.

Method Validation Considerations

Validation according to ICH Q2(R1) and relevant regulatory guidance is mandatory. Specific considerations for nitrosamine methods include:

• Achieving Low LOQ/LOD: Demonstrating quantification limits sufficiently below the regulatory AI limit is critical.  
• Specificity/Selectivity: Proving the method can unequivocally quantify the target nitrosamine(s) in the presence of matrix components, isomers, and potential interferences. Use of MS/MS or HRMS greatly aids in demonstrating specificity.
• Accuracy & Precision: Assessing recovery and repeatability at low concentration levels relevant to the specification limits.
• Matrix Effect Evaluation: Quantifying and mitigating ion suppression/enhancement in MS-based methods, often using matrix-matched standards or stable isotope-labeled internal standards.
• Control of Artifact Formation: Demonstrating that the analytical procedure itself does not generate the target nitrosamine.^38–41

Table 5 Analytical Strategies

Review of Analytical Methods for Specific Pharmaceuticals

Following the initial discovery of N-nitrosamine impurities in valsartan, extensive analytical efforts have been directed towards developing and validating methods for various pharmaceutical products. The choice of methodology often depends on the specific nitrosamine(s) of concern, the drug substance properties, and the matrix complexity

Sartans (Valsartan, Losartan, etc.)

A significant number of methods have been published for angiotensin II receptor blockers (ARBs) due to the initial recalls. Both LC-MS/MS and GC-MS/MS techniques are prevalent. LC-MS/MS methods, often employing APCI or ESI sources, target a range of nitrosamines including NDMA, NDEA, NMBA, NEIPA, NDIPA, and NDBA. GC-MS/MS methods, frequently using headspace sampling, are also widely applied, particularly for the more volatile nitrosamines like NDMA and NDEA. SFC methods have also been developed for sartan analysis, offering alternative selectivity. Regulatory bodies like the US FDA, EMA, and national laboratories (Swissmedic, TFDA, German OMCL) have published official methods utilizing these techniques.

Ranitidine and Other H2 Blockers (Famotidine, Nizatidine)

NDMA was the primary concern in ranitidine. LC-MS/MS is the dominant technique reported for its quantification in both drug substances and products. Methods often incorporate strategies to avoid in situ formation of NDMA during analysis, such as using specific sample preparation or avoiding high temperatures. GC-MS methods, including HS-SPME-GC-MS, have also been explored as alternatives. Fewer methods are specifically cited in the source article for famotidine and nizatidine, though GC-MS has been used for nizatidine

Metformin

NDMA has been the main impurity investigated in metformin products. Numerous LC-MS/MS and LC-HRMS methods have been developed by regulatory agencies (US FDA) and researchers for NDMA quantification, often targeting low ppb levels. GC-MS/MS methods, including full evaporation static headspace techniques, have also been reported for analyzing NDMA and other potential nitrosamines in metformin. The potential role of excipients and formulation processes in NDMA formation in metformin has been a focus

Recently Highlighted Drugs

Analytical methods are being developed accordingly:

• Rifampicin: LC-MS/MS methods targeting 1-methyl-4-nitrosopiperazine (MNP) have been reported.  
• Atorvastatin & Itraconazole: An LC-UV method using formic acid-aided sample preparation was reported for eight nitrosamines in these poorly water-soluble drugs.  
• Propranolol: LC-MS methods for N-nitrosopropranolol impurity quantification are available.  
• Duloxetine: An LC-MS/MS (APCI) method for NDMA, NDIPA, and N-nitroso duloxetine was developed.  
• Rivaroxaban: An LC-MS/MS method quantified a specific N-nitrosamine derivative (N-(2-hydroxyethyl)-N-phenylnitrous amide).  
• Pioglitazone, Cilostazol, Sunitinib: SFC-MS/MS and GC-MS methods have been reported for screening or quantifying nitrosamines in these products.
• Methods using HPLC or LC-MS have also been cited for enalapril, lisinopril, and others.²⁵

Analysis in Different Matrices

While the primary focus is on APIs and finished drug products, analytical methods have been adapted for various matrices:

APIs & Drug Products: The majority of methods focus on these, employing techniques suited to the specific drug and impurity.  

Excipients: Analysis of excipients for nitrosating agents (nitrites) or amine precursors is crucial for risk assessment, though specific methods are less detailed in the reviewed article's tables. Databases tracking nitrite levels in excipients exist.  

Water: GC-MS and LC-MS methods are used to monitor nitrosamines in drinking water and wastewater, relevant as both a potential contamination source and an environmental concern.  

Biological Samples: LC-MS/MS and CE-MS methods have been developed for quantifying nitrosamines (e.g., NDMA, tobacco-specific nitrosamines) in plasma, urine, or serum for exposure or pharmacokinetic studies.⁴²

Table 6 Predominant Techniques for Major Drug Classes

Future Perspectives & Recommendations

The ongoing challenge of controlling N-nitrosamine impurities in pharmaceuticals necessitates continuous innovation in analytical methodologies, risk assessment strategies, and collaborative efforts. While significant progress has been made, particularly in deploying sensitive hyphenated techniques, several areas warrant future focus.

Emerging Analytical Technologies and Potential Improvements

The demand for ever-increasing sensitivity, selectivity, and throughput drives the exploration of new analytical frontiers.

• Advanced MS Techniques: Further development and application of high-resolution mass spectrometry (HRMS), such as Orbitrap and advanced TOF technologies coupled with both LC and GC, can enhance confident identification of novel or unexpected nitrosamines and improve selectivity in complex matrices. Techniques like ion mobility spectrometry (IMS)-MS may offer additional separation dimensions to resolve challenging isomers.
• Miniaturization and Automation: Integrating microfluidics, developing more automated sample preparation techniques (e.g., enhanced online SPE, robotic systems), and faster chromatographic methods (e.g., advancements in UHPLC, multi-dimensional chromatography) can increase sample throughput and reduce turnaround times, crucial for routine quality control.
• Novel Detection Principles: Research into alternative detection methods or sensors that offer high sensitivity and selectivity specifically for the nitroso functional group could potentially lead to faster screening tools, although MS remains the gold standard for confirmation and quantification. Instrument manufacturers are encouraged to develop more advanced, sensitive, and selective tools specifically targeting nitrosamine detectability.  
• Data Analysis: Utilizing advanced data processing algorithms, including machine learning (ML) and artificial intelligence (AI), could improve signal processing, deconvolution in complex chromatograms, and automated identification of potential nitrosamines in large datasets generated by HRMS screening.

Need for Standardized Methods

While regulatory agencies have published some official methods (e.g., by US FDA, Ph. Eur.), the literature contains a vast array of customized methods developed by different laboratories. This highlights a potential need for greater harmonization and standardization.  

• Reference Methods & Materials: Development of universally accepted reference methods and certified reference materials for a wider range of nitrosamines, especially drug substance-related nitrosamine impurities (NDSRIs), would facilitate inter-laboratory comparisons, ensure consistency in testing, and support method validation across the industry.
• Method Lifecycle Management: Adopting analytical lifecycle management principles can ensure methods remain robust and fit-for-purpose throughout their use.

Advances in Predictive Tools for Risk Assessment

Predictive tools play a vital role in proactively assessing the risk of nitrosamine formation.

• In Silico Models: Continued refinement of in silico tools, including Quantitative Structure-Activity Relationship (QSAR) models, is essential for predicting mutagenicity and carcinogenic potency, particularly for novel NDSRIs. Approaches mentioned include expert rule-based and statistical methods, quantum mechanical calculations (e.g., for DNA reactivity prediction), and structural similarity analyses to identify appropriate surrogates for toxicological assessment. Web-based automated tools are also emerging for rapid risk categorization. Integration of larger datasets and advanced modeling techniques (e.g., AI/ML) could improve the accuracy and scope of these predictions.  
• Formation Pathway Modeling: Developing kinetic models to predict the likelihood and extent of nitrosamine formation under specific manufacturing process conditions or during storage could further enhance risk assessment and mitigation efforts.

Recommendations for Future Research Directions

Based on current challenges and opportunities, future research should focus on several key areas:  

• Fundamental Research: Continued investigation into the fundamental mechanisms of nitrosamine formation and degradation under various pharmaceutical-relevant conditions (including API synthesis, formulation processes, and storage) is needed.
• Analytical Methodology: Development of more advanced, sensitive, and selective analytical methods using cutting-edge techniques (LC-MS, GC-MS, CE-MS, SFC-MS) remains a priority, especially for novel NDSRIs and challenging matrices. Research into innovative sample preparation techniques that are both efficient and minimize artifactual formation is also crucial.  
• Mitigation Strategies: Exploring and validating effective strategies to prevent nitrosamine formation during manufacturing (e.g., alternative reagents, process controls, use of inhibitors) requires further research.
• Toxicological Understanding: Expanding the toxicological database, particularly for NDSRIs, through methods like the Enhanced Ames Test (EAT) and establishing reliable carcinogenic potency data is critical for setting scientifically justified acceptable intake limits.  
• Collaboration: Enhanced collaboration among regulatory authorities, research institutions (including government and academic labs), and pharmaceutical companies is strongly recommended to foster innovation, share knowledge, and develop harmonized approaches to manage nitrosamine risks effectively.⁴³

Table 7 Future Perspectives & Recommendations