Introduction

Since July 2018, global regulatory agencies have flagged angiotensin II receptor blockers (ARBs)due to suspected contamination with nitrosamines, including the carcinogen N-nitroso dimethylamine (NDMA) found in valsartan¹. As a result, the Food and drug administration (FDA) in the United states (US) issued voluntary recalls, and recalls were implemented widely throughout Europe. Next, it was discovered that losartan and valsartan contained N-nitrosoN-methyl-4-aminobutyric acid (NMBA) and N-nitrosodiethylamine (NDEA), respectively. Efforts are underway to investigate nitrosamine contamination in ARB formulations, with concerns centered around tetrazole formation during Active Pharmaceutical Ingredient (API) production. The contamination issue has extended to other drugs like ranitidine and metformin, where NDMA contamination led to market withdrawal of ranitidine in 2020  ^2,3. Ranitidine’s NDMA levels    can increase over time, especially at higher temperatures. Metformin’s NDMA contamination is likely due to finished  product degradation under specific conditions ⁴.

Brief Evolution of the Generation of N-Nitrosamines in Pharmaceutical Products

Concerns about N-nitrosamines (NAs) began in the late 1800s, but their dangers became evident in 1954, leading to their classification as carcinogens in 1970. They are now found in tobacco, food, medicine, and possibly the gut ⁵ . International council for harmonization (ICH) guideline M7R1confirmed this classification, classifying N-nitrosamines and their precursors as members of a “cohort of concern” because of their high mutagenic potency. Significant drug recalls involving N-nitrosamine have occurred in recent years ⁶. The recalls included valsartan, angiotensin II receptor antagonists, anti-diabetic medications, and ranitidine due to harmful impurities⁷. Regulations to prevent such contamination were incorporated in replies from regulatory agencies like the European medicine agency (EMA) and FDA ^8,9. Additionally, research on the use of nitrite scavenger’s dates back to the 1960s, when it was discovered that ascorbic acid might decrease the formation of N-nitrosamines by converting nitrous acid more precisely, the anhydride Dinitrogen trioxide (N₂O₃) to Nitric oxide (NO) ¹⁰. Studies carried out in the 1970s focused on inhibiting agents, namely the interaction between ascorbic acid and nitrite, in order to reduce nitrosation ¹¹. This preventive measure was tested in various pharmaceutical systems, including piperazine and tetracycline drugs, to reduce nitrosamine synthesis¹².

Potential origins of Nitrosamine Impurities in Pharmaceuticals

Angiotensin II Receptor Blockers (ARBs)

Nitrosamine impurities in ARBs, particularly tetrazole-based ones like losartan and valsartan, result from nitrosation during API production, often linked to recycled catalysts or solvents. Non-tetrazole ARBs, like telmisartan and eprosartan, are less affected ¹³. To avoid using hazardous hydrazoic acid, azides and nitriles can be converted into a tetrazole ring through a [1 + 3] cycloaddition process¹⁴. The formation of tetrazole using azide reagents is typically carried out in the concluding stage of sartan synthesis ¹⁵ . Despite their human health hazards, organometallic azide derivatives like trimethyltin azide (Me₃SnN₃) and tributyltin azide (BuSnN₃) and sodium azide (NaN₃) are preferred as azide reagents because they are easy to handle and dispose of. Sodium nitrite is usually added in an acidic environment to neutralize any unreacted azides that remain after the tetrazole production process¹⁶. The disposal of alkyl amine residues, unreacted azides, or contaminated solvents can unintentionally form nitrosamines, posing a contamination risk. The recall of valsartan, the first ARB with NDMA presence, was linked to NDMA forming from dimethylamine (DMA), a residual impurity in N,N-dimethylformamide (DMF) used in API synthesis¹⁷. During ARB production, DMF can degrade into dimethylamine (DMA), while triethylamine (TEA) used in losartan synthesis may be contaminated with diethylamine (DEA), which is linked to the presence of NDEA ¹⁸  which is a known precursor of NDEA as reported previously. Additionally, TEA can undergo direct dealkylation to form DEA when exposed to nitrous acid or nitrite, as shown in Figure 1. Additional nitrosamine compounds include N, Nitrosodiisopropylamine (NDIPA), NMBA and N- nitrosoethylisopropylamine (NEIPA) have also been detected in ARBs in addition to NDEA. Initially, NMBA contamination was detected in both irbesartan and losartan. The precursor of NMBA is N-Methyl amino N-butyric acid (MBA), that might develop as a result of N-methylpyrrolidinone (NMP) deterioration, an organic solvent utilized during tetrazole synthesis. N-ethyl N-isopropylamine  (EIPA) and N,N  -diisopropylamine (DIPA), which act as the corresponding precursors of NEIPA and NDIPA, are examples of dealkylative compounds that can be produced from NDEA, N,N -diisopropylethylamine (DIPEA), a frequent base in API synthesis. Tetrazole-containing ARBs may be contaminated with various nitrosamines due to different amine reagents used in their synthesis. Risk assessments and manufacturer recalls have identified azido impurities as novel by-products, although the nitrosamine issue has not been fully resolved ¹⁹. Azido impurities, specifically 4- (azidomethyl)-[1,10-biphenyl]-2-carbonitrile and 5-(4′-(azidomethyl)-[1,10-biphenyl]-2-yl)-1H-tetrazole (AZTT), arise from a secondary reaction between sodium azide and the left-over intermediates from the step before it. ARB medications with azido impurities, like losartan forming AZTT, have led to recalls by the Canadian FDA. To reduce nitrosamine contamination, avoiding nitrosating agents is advised. China uses a risky hydrogen peroxide quenching method for losartan, while triaryl phosphine can alternatively remove residual azides via the Staudinger reaction ²⁰. In a European patent, the utilization of triphenylphosphine for azide quenching has been described as a method for in-process remediation ²¹. Triphenylphosphine reacts with an azide to form a phosphazide intermediate, which releases nitrogen (N₂) to generate iminophorane. An aqueous workup then yields amine derivatives and phosphine oxide. To prevent nitrosamine contamination during ARBs synthesis, use minimal triphenylphosphine instead of sodium nitrite for azide removal.

Histamine -2 (H2) Receptor Antagonists

NDMA, associated with the ozonation and chlorination processes, raises environmental issues ²². Efforts to remove NDMA revealed a link between ranitidine and NDMA during wastewater treatment. The dimethylamine side chain and nitro group in nizatidine and ranitidine are associated with increased NDMA formation ²³. Ranitidine can produce NDMA directly, while its impurities release NDMA at a notably faster rate. The proposed mechanism involves impurity degradation, inherent instability of the API, and contamination during the manufacturing process ²⁴ . Shown in figure 2.

Figure 1: Possible root causes of nitrosamines and azido impurities in angiotensin II receptor blockers. Click here to view Figure

Ranitidine’s NDMA contamination increases with storage time and temperature, with a strong link between ranitidine, DMA, and NDMA formation, raising potential cancer risks ²⁵. Controlling and keeping an eye on stability-related parameters including temperature, light, and humidity during drug production and storage is advised as a solution to this problem ²⁶. Ranitidine’s temperature-dependent NDMA production requires controlled storage: 2-8°C for parenteral forms and below 25°C for solid forms. Regular NDMA level checks during storage are advised, and a comprehensive strategy to assess and mitigate NDMA risks in ranitidine is essential for safety.

Anti-diabetic Drugs

Metformin, a guanidine derivative like dimethyl guanidine, lacks a nitro group unlike ranitidine. Thus, nitrosamine synthesis requires an external nitro-sating agent ²⁷.

Figure 2: Possible root causes of N-nitroso dimethylamine (NDMA) contamination in ranitidine and nizatidine and their pharmaceutical products. Click here to view Figure

Health officials are concerned about NDMA contamination in metformin, linked to environmental factors and insufficient breakdown mechanisms (Figure 3). A 2019 study identified degradation by-products, and the FDA found excess NDMA in 2020  ²⁸  Top of FormBottom of Form.  Nitrosation during wet granulation and drying can cause NDMA contamination in tablets. Avoiding nitrate-containing excipients and implementing strict quality control for metformin and pioglitazone is essential to protect patient safety.

Antimicrobial Agents

In August 2020, the FDA detected nitrosamine impurities 1-cyclopentyl-4-nitrosopiperazine (CPNP) and 1-methyl-4-nitrosopiperazine (MNP) in rifapentine and rifampin (Figure 4), used for tuberculosis. CPNP likely forms during rifapentine synthesis, while MNP’s source remains unclear, possibly linked to rifampicin. Many US products exceed acceptable nitrosamine limits, with CPNP in rifapentine at 0.1 ppm and MNP in rifampin at 0.16 ppm. The FDA set higher limits (20 ppm for CPNP and 5 ppm for MNP) to ensure drug availability, while Singapore’s Health Sciences Authority (HAS) approved rifampin with trace MNP.

Figure 3: Possible root causes of N-nitroso dimethylamine (NDMA) contamination in metformin and its pharmaceutical products. Click here to view Figure

Nitrosamine toxophores generally imply lower carcinogenicity than NDMA²⁹. NDMA’s interim Acceptable Intake (AI) replaces strict limits, requiring manufacturers to prevent contamination and regulators to enforce oversight. Rifapentine and Rifampicin may be contaminated with CPNP and MNP via nitrosating chemicals reacting with piperazine ³⁰.  This  emphasizes Strict quality control is vital for detecting and removing contaminants that endanger patient safety during  pharmaceutical production.

Other Medicine

Nitrosamine detection in ARBs has highlighted tetrazole-containing drugs, like sartans, as prone to contamination, affecting medications like ceftezole and letrozole, leading to shortages, treatment changes, and wastewater concerns ³¹.Chloramine disinfection can lead to nitrosamine formation,³² in APIs like nizatidine, ranitidine, clarithromycin, metformin, and others. Lidocaine may be contaminated with NDEA. Ozonation in sewage treatment helps minimize NDMA formation by facilitating N-oxide production through electron donation ³³. Nitrosamine formation in wastewater, linked to amine-based medicines, challenges drug manufacturers to ensure medication safety through rigorous risk assessment and quality control.

Figure 4: Potential root causes of contamination with MNP in rifampin and CPNP in rifapentine. Click here to view Figure

Amines as N-Nitrosamine Precursors

Nitrosamines in pharmaceuticals are a critical concern due to their carcinogenic potential. They can originate from APIs, solvents, reagents, and degradation during storage. Nitrosamines form via nitrosation of amines by nitrosating agents, with formation influenced by pH, strong acids reduce reactivity and nitrosamine levels, while very low pH decreases nitrosation. Common nitro-sating agents include Nitrous acid (HNO₂), nitrite (NO₂), N₂O₃, Dinitrogen tetroxide (N₂O₄₎, Nitrosyl Chloride (NOCl), nitrosonium tetrafluoroborate (NOBF₄), and nitro thiocyanate (CN₂OS). Controlling these conditions can significantly reduce nitrosamine formation, ensuring product safety and regulatory compliance³⁴.

Nitrosation Mechanisms for Different Amines (Figure 5)

Primary Amines

The compound is susceptible to undergoing nitrosation, a chemical process where it reacts to form unstable N-nitroso compounds. These compounds can subsequently decompose into diazonium salts which are reactive intermediates in various chemical reactions.

Secondary Amines

Convert to nitrosamines more promptly, making them the most reactive in forming nitrosamine contaminants.

Tertiary Amines

This compound can react with nitrites to form N-nitrosamines, following dealkylation to produce secondary amines ³⁵.

Geno toxicity and carcinogenicity in nitrosamines

Nitrosamine contaminants, such as NDEA, present genotoxic risks in pharmaceuticals and are bioactivated by CYP2A6 and cytochrome P450 enzymes ³⁶. CYP2E1 converts nitrosamines into α-hydroxy nitrosamine, forming alkylating electrophiles that bind Deoxyribonucleic acid (DNA) and proteins, creating adducts^37,38. Alkylating agents target DNA’s N⁷ guanine, causing SN² alkylation. Alkyl diazonium ions can also target O⁴ of thymine and O⁶ of guanine, leading to stable DNA modifications that may result in mutations and potentially cause cancer. For example, Translation changes from G: A can occur when O⁶ methylguanine and O⁶ ethylguanine adducts are misinterpreted as adenine during DNA replication. This misinterpretation can lead to point mutations, altering the genetic code. O⁴-alkylthymine adducts are mistakenly recognized as cytosine during DNA replication, which result in translation alterations from T: A to C: G. In vivo experiments on rat and mouse livers exposed to NDEA or NDMA have shown the separation of Ethyl and Methyl DNA adducts, mainly at N⁷G, however  alkylation at other sites such as O²T, O⁶G, N³A and O⁴T was also noted, ³⁹ Compared to NDMA, NDEA generated a greater ratio of O⁶G to N⁷G adducts, which suggests that different alkylation sites on guanines have different distributions shown in Figure 6. Aldehydes produced during the bioactivation of N-nitroso dialkylamines, such as formaldehyde a carcinogen classified by World health organization/International agency For Research on Cancer (WHO/IARC), form DNA adducts and contribute to the genotoxic effects of alkyl diazonium ions ⁴⁰. NDMA and other N-nitroso dialkylamines are strong carcinogens as they alkylate DNA via aldehydes and alkyl-diazonium ions, resulting in increased DNA damage. This is demonstrated by the detection of DNA adducts such as N6-hydroxymethyl-2′-deoxyadenosine and di-(N6-deoxyadenosyl)-methane in the lungs and liver of rats following NDMA injections, which can cause mutations and cancer ^41,42 .NDMA and NDEA generate reactive oxygen species (ROS) in Caco-2 cells, leading to disrupted gene expression ⁴³. Since 1978, the IARC has confirmed that animals exposed to NDMA and NDEA are suitable models for studying their combined carcinogenic effects ⁴⁴. 

Figure 5: Pathways of nitrosamine formation between nitrosating agents and primary, secondary, tertiary amines and quaternary amines. Click here to view Figure

According to the ICH M7R1 guideline, consuming N-nitrosamines at interim-limit levels over a lifetime would result in fewer than one additional cancer case per 100,000 people⁴⁶. The EMA flagged potential cancer risks from prolonged NDMA exposure in valsartan, but a Danish study found no significant risk, with an adjusted hazard ratio of 1.09 (95% Confidence interval: 0.85–1.41). These findings highlight the need for strict regulatory oversight to ensure drug safety.

Figure 6: Bioactivation of nitrosamines to reactive species and the major nucleobase adducts subsequently formed. Click here to view Figure

Compounds capable of inhibiting or preventing the development of N-nitrosamines of in pharmaceutical product

Substances can reduce N-nitrosamine synthesis via three main mechanisms: nitration of phenol, redox pathways, and diazotization of primary amines as shown in figure 7 ⁴⁷. Effective blocking agents convert nitro-sating agents into non-nitrosating NO, competing with amines based on their quantities ⁴⁸. Lipid and water-soluble nitrite scavengers, like ascorbic acid, bisulfite, and cysteine, block nitrosation, though phenolics may facilitate trans-nitrosation ⁴⁹. Fat-soluble antioxidants like α-tocopherol, butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) are also used, but their efficacy may be limited. Solid oral dosage forms can benefit from FDA approved “inactive ingredients” ⁵⁰. Table 1 lists nitrite scavengers suitable for these forms.

Figure 7: Proposed Mechanisms of the Main scavenging Pathways of Nitrosating Agents. Click here to view Figure

Ascorbic Acid

Ascorbic acid effectively counteracts various nitro-sating agents and is safe for pharmaceutical use. It excels in weakly acidic and aqueous environments, producing NO from N₂O₃, Nitryl ion (H₂NO₂), and NOX ⁴⁸. Despite NO’s potential oxidation to nitro-sating agents in aerobic conditions, ascorbic acid remains a potent scavenger, especially in anaerobic settings ⁵¹. A stoichiometric model in drug formulations calculates the necessary ascorbic acid based on anticipated nitrosating agent levels during storage. In solid forms, particle size and distribution affect scavenging efficacy, with wet granulation providing uniform distribution. Research confirms that ascorbic acid effectively prevents nitrosation, significantly reducing N-nitrosamine formation in tablets and other pharmaceuticals  ⁵² .

Table 1: Nitrite scavengers that may be used in solid oral

α-Tocopherol

Preventing N-nitrosamine production depends significantly on the antioxidant ability of α-tocopherol, which reduces nitro-sating agents to non-nitro-sating molecules ⁵³. The fully substituted aromatic ring of α-tocopherol minimizes C-nitrosation,  ⁵⁴   with its effectiveness relying on the non-esterified form due to radical production at the hydroxyl group during oxidation. In lipophilic media, α-tocopherol works well, also performing in aqueous environments where the molar ratio of tocopherol to nitrite affects NA suppression ⁵⁵. Optimal nitrite reduction occurs at pH 2-3, where less than 25% remains after an hour, versus over 95% at pH 5. α-Tocopherol, especially when combined with ascorbic acid, reduces nitrite faster than γ-tocopherol, significantly lowering nitrosamine production in lipid-rich matrices like bacon and tablets ⁵⁶.

Amino Acid

Amino acids with primary amines or thiol groups, except proline, efficiently scavenge nitrites via diazotization reactions, ^57,58  producing unstable diazo intermediates that decompose into alcohols and nitrogen through the Van Slyke reaction ⁵⁹. L-cysteine effectively inhibits nitrosamine and N-nitrosonornicotine synthesis, outperforming other amino acids like serine, alanine, and proline. Recent screenings indicate variable efficacy with temperature, but L-cysteine’s rapid nitrite reduction and scavenging capability, attributed to its thiol group, make it ideal for solid-state tests ⁶⁰.

Other Substances

Substances like resveratrol, BHT, BHA, maltol, and propyl gallate show potential as excipients to reduce nitrosamine formation. Nitrosamine conversion decreased by 40-50% with lysine and glycine, and by up to 90% with histidine at pH 3.0 and 60°C. However, at ambient temperature and pH 3.0, scavenging effectiveness of histidine, arginine, glycine, and lysine was modest. Poorly water-soluble scavengers, such as resveratrol, BHA, and propyl gallate, are unsuitable for liquid applications ⁵⁰.

Regulatory considerations regarding the use of tocopherol and ascorbic acid in pharmaceutical    products.

Global regulatory bodies like FDA, EMA, Pharmaceuticals and Medical Devices agency (PMDA), Health Canada, and others collaborate to minimize nitrosamine contamination in pharmaceuticals due to widespread manufacturing networks and distribution.

Europe

The executive director of the EMA began a review of nitrosamine impurities in chemically synthesized API in September 2019 following the discovery of N-nitrosamine contamination in pharmaceutical goods in Europe. This evaluation was expanded to cover all human pharmaceuticals in 2020. It is up to the producers or Marketing Authorization Holders (MAHs) to determine the risk of N-nitrosamine contamination ⁶¹. MAHs must evaluate risk, test for nitrosamine presence, and implement risk mitigation strategies, like adding nitrite scavengers, demonstrating effectiveness through finished product testing.

USA

In response to the finding of N-nitrosamine contamination in pharmaceuticals, the US FDA released Industry Guidance in September 2020. The steps for evaluating and controlling the risk of N-nitrosamine contamination in medications were described in this guidance. This document outlines mitigation techniques and sets timelines for drug makers to complete phases of pending and approved applications, aligning with EMA’s three-step procedures ⁶². Mitigation techniques for N-nitrosamines include antioxidants like α-tocopherol and ascorbic acid, which effectively inhibit their formation in pharmaceuticals, highlighting their importance in reducing contamination risks ⁶³.

Acceptable intake (AI) of nitrosamine in drugs

The acceptable intake (AI) of nitrosamines in drugs is based on toxicity data to minimize cancer risk, with regulatory agencies setting AI limits using toxicological assessments like TD50 values. AI values determine specific nitrosamine limits for medications, usually in parts per million (ppm). Accurate analytical techniques with appropriate limits of quantification (LOQ) are crucial. The daily limit for nitrosamine pollutants is set at 26.5 nanograms, ensuring overall exposure remains below harmful levels ⁶⁴.Temporary threshold standards for nitrosamines in medications is given in table 2.

Analytical Technique

Various analytical techniques like Ultra performance liquid chromatography (UPLC), Gas chromatography (GC), Liquid chromatography- tandem mass spectroscopy (LC-MS/MS), and High-pressure liquid chromatography (HPLC) are used to detect and quantify nitrosamine contaminants in ranitidine and sartans, ensuring pharmaceutical safety.

HPLC

In the past decade, HPLC has revolutionized chemistry and pharmaceuticals by effectively separating complex biological mixtures ⁶⁵. Numerous HPLC techniques feature in United states Pharmacopoeia (USP) and European Pharmacopoeia (EU). Stationary phases like C18/Phenylhexyl are popular for nitrosamine analysis, with common detectors being Diode array detector (DAD) (230-233 nm) and Ultraviolet detection (UV) (228 nm). Innovations like Chemiluminescence detector (LC-PR-CLD) enhance nitrosamine detection. Valsartan’s NDMA detection limits are 0.00085 µg/mL limit of detection (LOD) and 0.00285 µg/mL (LOQ) ⁶⁶. Table 3 provides specifics on a number of documented LC-MS techniques for nitrosamine impurity detection.

Table 2: Temporary threshold standards for nitrosamines in medications

LC-MS/MS

LC-MS integrates liquid chromatography with mass spectrometry to separate, quantify, and precisely identify complex mixtures, including polar and unstable molecules ⁶⁷ . Ionization methods include Electrospray ionization (ESI) and  Atmosperic pressure chemical ionization (APCI) , crucial for detecting nitrosamine contamination in food, beverages, and pharmaceuticals. LC-MS/MS optimizes parameters like cone voltage and collision energy for precise Quality assurance and control (QA/QC) of nitrosamine impurities. Studies have shown nitrosamine contaminants in medications like valsartan and ranitidine using LC-MS/MS. Researchers identified eight nitrosamine impurities, including NDEA and NDMA, emphasizing the technique’s critical role in ensuring pharmaceutical safety and compliance ⁶⁸. Some reported LC-MS/MS methods are given in table 4.

GC-QTOF, GC-MS, GC-MS/MS, and GC-MS-Head Space

An essential tool for examining volatile substances and medicinal components is GC-MS. For nitrosamine detection, nitrogen chemiluminescence and nitrogen-phosphorus detectors are preferred  ⁶⁹ . NDMA and other nitrosamines are commonly analyzed in medications like valsartan, using Deutero N-Nitrosodimethylamine (d6-NDMA) as an internal standard. Methods like Quadrupole time-of-flight mass spectrometer (GC-QTOF), Gas chromatography (GC), GC-MS, and GC-MS-Head Space methods detect nitrosamines in ranitidine and sartans. The US FDA used GC-Head Space to identify four nitrosamines in valsartan, with detection limits of 0.01-0.025 ppm. Different sartans were tested for the presence of NDEA and NDMA using the official medicine control laboratory (OMCL’s) LGL Method. The permitted limits for these nitrosamine impurities per 320 mg of API are 0.080 ppm for NDEA and 0.10 ppm for NDMA, according to this procedure ⁷⁰. Few reported GC-MS methodologies for detecting nitrosamine impurities are given in table 5.

Non-Chromatographic Method

Various techniques for analyzing NAs include molecularly imprinted polymers with impedimetric sensors (LOD: 0.85 µg/L), chemiluminescence, and UV-photolysis ⁷¹. In the food industry, spectrophotometry, following the Griess reaction, is used for high concentrations of NAs but has limitations due to variability in Nitrate (NO₃₎ and Nitrogen dioxide (NO₂) ion generation during NDEA analysis ⁷² Reaction-based colorimetric assays, like the Eisenbrand-Preussman reaction, detect nitrite from cleaved nitrosamines but have high detection limits and require large nitrosamine concentrations. Walsh et al. (2005) developed a yeast-based biosensor using genetically modified yeast with a DNA damage reporter system (RAD54-GFP) and cytochrome P450 enzymes, detecting NDMA at 1.6 mg/L. Bui et al. developed a similar system with detection at 3 mg/L. While these methods are useful, they may face challenges in pharmaceutical testing due to high active pharmaceutical ingredient concentrations ⁷³.

Table 3: lists the specific HPLC techniques that have been published for nitrosamine impurity detection.

Different methods of sample preparation are available for separating nitrosamine impurities

Several methods are used for preparing samples to separate nitrosamine impurities in pharmaceutical products.

Solid-Phase Extraction (SPE)

Utilizes a solid sorbent to selectively retain nitrosamines from the sample matrix, followed by elution for analysis.

Liquid-Liquid Extraction (LLE)

Involves partitioning nitrosamines between two immiscible liquid phases, typically using organic solvents, followed by separation and concentration.

Derivatization followed by Extraction

Converts nitrosamines into more detectable or extractable forms prior to analysis, enhancing sensitivity.

Precipitation Techniques

Involves adding reagents to selectively precipitate nitrosamines for isolation.

Solid-Phase Microextraction (SPME)

Uses a coated fiber to extract and concentrate nitrosamines from the sample matrix ⁷⁴.

Each method offers unique advantages based on sample complexity and sensitivity. DCM is a common solvent for nitrosamine extraction, selected per study needs.

Table 4: Disclosed limited techniques for nitrosamine impurity detection using LC-MS.

Table 5: Few reported GC-MS methodologies for detecting nitrosamine impurities.

The effects of nitrosamine contamination and measures for its mitigation

Global ARB recalls due to nitrosamine contamination highlight the need for improved safety measures by manufacturers, regulators, and healthcare professionals.

Risk Evaluation

Preemptive risk assessment for nitrosamine contamination is essential throughout a medicine’s shelf life, starting from production. This is particularly important in API production, especially during tetrazole formation in ARB synthesis. Detailed synthetic route information is vital for identifying impurities, though often omitted in ASEAN registrations. Additional precautions include setting impurity limits and managing trace amines in solvents. In September 2019, the EMA requested nitrosamine evaluations for concerning drugs within six months, prioritizing risk management ⁷⁵. Tetrazole-containing drugs are high-priority for nitrosamine identification due to patient exposure risks. Risk assessment may prompt process modifications based on lab tests, with a strong understanding of organic chemistry being essential ^76,77. Mass spectrometry is vital for nitrosamine detection, but screening for unknown pollutants can be challenging due to equipment accessibility and affordability issues in GC-MS and LC-MS analysis

Impurity control for Nitrosamines

Controlling contaminants in drug ingredients, excipients, and final products complies with ICH quality principles and pharmacopeial standards. Pharmacopeial monographs establish limits for nitrosamine traces, but addressing mutagenic contaminants like nitrosamines remains challenging despite ICH M7 guidelines. Precise analytical techniques are crucial to avoid contamination during nitrosamine analysis in APIs and medicinal products. Toxicity data inform acceptable limits considering future synergistic effects. N-nitroso dialkylamines analysis may integrate into standard quality control due to carcinogenic concerns, possibly revising monographs. High-risk medications like tetrazole-containing ARBs ⁷⁸, warrant nitrosamine analysis, though pharmacopeial monographs may not cover all types. Regulatory agencies collaborate with producers to identify nitrosamine sources. Balancing patient carcinogen exposure risks with medication availability is challenging, mitigated by short-term measures to prevent shortages. Healthcare professionals play a critical role in patient communication, emphasizing adherence and exploring alternative therapies during recalls amidst the nitrosamine crisis. Various have implemented specific risk mitigation efforts to tackle the emerging crisis of nitrosamine contamination in pharmaceuticals, (Shown in table 6).

Table 6: Country Specific Risk Mitigation Efforts to Address the Emerging Nitrosamine contamination crises.

Conclusion

N-nitrosamines, known carcinogens found in medications like antidiabetic drugs and Histamine-2 receptor blockers, have prompted stringent testing and mitigation efforts by regulatory bodies. This review highlighted the need for robust detection methods, used of nitrite scavengers, and ongoing challenges in accurately assessing and managing risks. Tetrazole-based drugs and related amine analogs are particularly affected, necessitating adherence to ICH M7 (R1) guidelines and US Pharmacopeia Chapter <1469> for risk assessment and control. Regulatory bodies, including the US FDA, CDSCO, and EMA, are setting limits and requiring precise quantification to address these issues. Present review concluded that to improve safety, proactive risk management, enhanced industry collaboration, and updated regulatory guidelines are essential, along with incorporating country-specific risk mitigation strategies to manage emerging threats and ensure global drug quality.

Acknowledgement  

The authors would like to thank Dr. Sanjay Kshirsagar sir, Suman Kahandal and Dr. M.R.N Shaikh sir for their continuous guidance and support.

Conflict of Interest

The authors do not have any conflict of interest.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article. 

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

Author contribution

Snehal ukhade: The primary author who wrote the article, conducted the literature review, and synthesized the information

Sandeep S. Sonawane: Provided supervision, guidance, critical revisions, and overall support for the project.

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