An update on latest regulatory guidelines and analytical methodologies for N-nitrosamine impurities in pharmaceutical products – 2024

Abstract

Controlling or eliminating N-nitrosamine impurities in pharmaceutical products has become a significant challenge for both drug manufacturers and regulatory authorities. This difficulty is particularly pronounced in light of the recent increase in nitrosamine drug substance-related impurities, which have raised concerns about the safety and efficacy of various medications. Additionally, the pharmaceutical industry faces the challenge of developing analytical methods that are not only sensitive and selective but also precise and accurate. These methods are crucial for the reliable quantification of low levels of N-nitrosamine impurities, ensuring compliance with stringent current regulatory guidelines. The intricate nature of detecting these impurities at such low levels necessitates the use of cutting-edge analytical techniques, such as liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. In light of these challenges, this review article explores the latest regulatory guidelines and analytical methodologies concerning N-nitrosamine impurities in various pharmaceutical products for 2024. Findings from this review article help provide valuable insights for researchers and industry professionals to enhance the safety and quality of pharmaceutical products.

Introduction

N-Nitrosamine impurities are a class of organic compounds characterized by the presence of the nitroso functional group (N–N=O). In the domain of organic chemistry, “nitroso” denotes a functional group where the nitric oxide (N−N=O) moiety is bonded to an organic structure. These nitroso groups can be classified into several categories as C-nitroso compounds (such as nitroso alkanes, represented as R−N=O), S-nitroso compounds (such as nitroso thiols, denoted as RS−N=O), N-nitroso compounds (which include nitrosamines (NAs), structured as RN(−R′)−N=O) and O-nitroso compounds (such as alkyl nitrites, indicated as RO−N=O). N-Nitrosamines typically exhibit a general structure shown in Figure 1.1 These impurities can form quite readily, often resulting from reactions involving secondary and tertiary amines, amides, carbamates, and urea derivatives in combination with nitrites or other nitrogen-containing groups.2

Figure 1.

Chemical structure of N-nitrosamine.

It illustrates the chemical structure of N-nitrosamine, a class of compounds characterized by the presence of a nitroso group (-NO) attached to an amine. N-nitrosamines are known for their potential carcinogenic properties and are commonly found as impurities in various pharmaceutical products. The structure shown highlights the key functional groups and bonding patterns that define N-nitrosamines.

This review article delves into the latest regulatory guidelines and analytical methodologies concerning N-nitrosamine impurities, developed and published in 2024. It covers a range of pharmaceutical products, including sitagliptin, sartans, rifampicin, aceclofenac, levosulpiride, metformin, ranitidine, valsartan, varenicline tartrate, duloxetine, ciprofloxacin, nizatidine, losartan, mirabegron, rifapentine and rasagiline. Figure 2 shows structures of drugs containing N-nitrosoamine impurities. Table 1 shows product names, therapeutic areas, and treatment details of drugs containing N-nitrosoamine impurities. These hazardous N-nitrosamine impurities are known to pose significant risks to human health, including the potential to cause cancer. The information presented in this article is particularly valuable for drug manufacturers and researchers, as it helps them understand and align with the latest updates and changes in regulatory standards.

Figure 2.

Structures of drugs containing N-nitrosoamine impurities.

Table 1.

Product names, therapeutic areas, and treatment details of drugs containing N-nitrosoamine impurities

No.	Product name	Therapeutic area/class	Drug used for treatment
1	Sitagliptin	Type 2 diabetes/dipeptidyl peptidase-4 inhibitor	Sitagliptin is used to treat type 2 diabetes by increasing insulin levels in the body and reducing blood sugar. It is often used in combination with diet and exercise.
2	Sartans	Angiotensin II receptor blockers	Sartans are a family of medicines known as angiotensin II receptor blockers that are used for treating high blood pressure and heart failure.
3	Rifampicin/Rifampin	Antimicrobial	Rifampin is an antibiotic that is used to treat or prevent tuberculosis. Rifampin may also be used to reduce certain bacteria in your nose and throat that could cause meningitis or other infections.
4	Aceclofenac	Non-steroidal anti-inflammatory drug	Aceclofenac is a nonsteroidal anti-inflammatory drug used for the treatment of pain and inflammation in conditions such as arthritis, musculoskeletal disorders, ankylosing spondylitis, osteoarthritis, and rheumatoid arthritis.
5	Levosulpiride	Antipsychotic medicine	Levosulpiride is a dopamine antagonist medication used for the treatment of psychotic disorders like schizophrenia, major depressive disorder, nausea and vomiting, and gastroparesis. It is primarily used for its gut motility properties to improve food movement and treat disorders of the stomach and intestines. It is also used to manage mental health conditions like schizophrenia. Additionally, it is used to treat gastric problems such as nausea, vomiting, heartburn, and indigestion.
6	Metformin	Type 2 diabetes	Metformin is used to treat type 2 diabetes and is the preferred therapy for people with type 2 diabetes who do not have any contraindications to using metformin (such as kidney disease, metabolic acidosis, or poor liver function). It may be used in combination with other medications for type 2 diabetes.
7	Ranitidine	Histamine H2 antagonist	Ranitidine is used for treating and preventing ulcers in the stomach and intestines. It is also used to treat conditions where the stomach produces too much acid, such as Zollinger-Ellison syndrome. Additionally, Ranitidine is prescribed for treating pulmonary arterial hypertension and secondary Raynaud’s phenomenon.
8	Varenicline tartrate	Smoking cessation agents	Varenicline tartrate is used for treatment of smoking cessation. It helps people quit smoking by blocking the effect of nicotine on the brain and preventing cravings to smoke cigarettes. Quitting smoking lowers the risk of heart and lung disease, as well as cancer.
9	Duloxetine	Selective serotonin and norepinephrine reuptake inhibitors	Duloxetine is used for the treatment of major depressive disorder, generalized anxiety disorder, diabetic peripheral neuropathic pain, fibromyalgia, and chronic musculoskeletal pain. It is also used for pulmonary arterial hypertension.
10	Ciprofloxacin	Fluoroquinolone	Ciprofloxacin is used to treat bacterial infections in many different parts of the body. Ciprofloxacin oral liquid and tablets are also used to treat anthrax infection after inhalational exposure. This medicine is also used to treat and prevent plague (including pneumonic and septicemic plague).
11	Nizatidine	Esophagitis/histamine H2 antagonists	Nizatidine is used for treating and preventing ulcers, as well as for treating heartburn and erosive esophagitis caused by gastroesophageal reflux disease.
12	Mirabegron	Overactive bladder	Mirabegron is used in adults to treat overactive bladder with symptoms of frequent or urgent urination and urinary incontinence. Mirabegron is sometimes used together with another medicine called solifenacin (Vesicare). Mirabegron is used in children to treat neurogenic detrusor overactivity.
13	Rifapentine	Endothelin receptor antagonists	Rifapentine is an antibiotic used to treat tuberculosis. It is used together with other medications to treat active tuberculosis in adults and children who are at least 12 yr old. It is also used to keep inactive (latent) tuberculosis from becoming active in adults and children who are at least 2 yr old.
14	Rasagiline	Monoamine oxidase type B inhibitors	Rasagiline is used to treat symptoms of Parkinson’s disease (stiffness, tremors, spasms, poor muscle control). Rasagiline is sometimes used with another medicine called levodopa.

Search Strategy

The relevant literature was retrieved through an electronic search of Google Scholar databases from January 1 to December 31, 2024. The search strategy and selection criteria included mostly the following keywords: N-nitrosamine impurities, pharmaceutical products, latest analytical methodologies, cutting-edge analytical techniques, latest regulatory guidelines, liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry, method development, method validation, identification, quantification, and year 2024. Various combinations of these search terms were used to comprehensively access the literature. Initially, the relevance of the title and abstract to our target content related to N-nitrosamine impurities was reviewed. If deemed relevant, the entire paper was accessed to ensure it contained suitable descriptions of N-nitrosamine impurities. Articles published in English and focusing on the latest regulatory guidelines and analytical methodologies for N-nitrosamine impurities in pharmaceutical products were included in this article.

Mechanism of Formation of N-Nitrosamine Impurities

To generate N-nitrosamine impurities in a compound, two essential elements are required: a nitrosating agent and a secondary or tertiary amine. These components must be mixed in an acidic environment.3 Additionally, a tetrazole ring can be synthesized using azide-containing reagents such as sodium azide, tributyl azide, and trimethyltin azide. In the chemical synthesis of specific active pharmaceutical ingredients (APIs), solvents such as dimethylformamide, N-methyl-2-pyrrolidone, and triethylamine are commonly utilized. Even with thorough purification processes, these solvents can sometimes leave behind trace residues in the final products, which may contribute to the development of N-nitrosamine impurities. Figure 3 shows the mechanism of formation of N-nitrosodiethylamine. In addition, nitrogen oxides, including nitric oxide and nitrogen dioxide, play a significant role in the formation of nitrites. These nitrites can subsequently react with amines to produce N-nitrosamines, which are known to be harmful due to their carcinogenic properties. Figure 4 shows the formation of NAs from nitrite and amines.

Figure 3.

Mechanism of formation of N-nitrosodiethylamine.

It illustrates the chemical mechanism involved in the formation of NDEA. The process begins with the reaction of dimethylamine with nitrosating agents, such as nitrite ions (NO₂^–), under acidic conditions. The nitrosation reaction leads to the formation of NDEA, a potent carcinogen. The figure highlights the key intermediates and reaction steps, including the formation of the nitrosamine group (-N=O) and the subsequent stabilization of the NDEA impurity.

Figure 4.

Formation of nitrosoamine from nitrite and amines.

In the chemical structure of N-nitrosamines, R1 and R2 represent organic groups or substituents attached to the nitrogen atom. These groups can vary, leading to different N-nitrosamine compounds. For example, in N-nitrosodimethylamine (NDMA), both R1 and R2 are methyl groups (CH₃).

Latest Regulatory Guidelines for N-Nitrosamine Impurities

Considering the importance of human health, many regulatory agencies have recently revised and published updated guidelines concerning N-nitrosamine impurities. These impurities can pose significant risks, as they are associated with potential carcinogenic effects. Regulatory bodies, such as the United States Food and Drug Administration (FDA) and European Medicines Agency (EMA), play a vital role in overseeing pharmaceutical manufacturers to ensure the safety and quality of medications. By establishing stringent regulations, these agencies aim to minimize the presence of harmful impurities in drug products. The updated guidelines not only provide a framework for identifying and quantifying N-nitrosamines but also emphasize the need for robust manufacturing practices and thorough purification processes. This proactive approach is essential for safeguarding public health and maintaining trust in the pharmaceutical industry.

In September 2024, the FDA updated the guidelines on “Control of Nitrosamine Impurities in Human Drugs” Revision 2 wherein it introduced several significant changes aimed at enhancing drug safety. Key updates include the classification of NAs into common types and nitrosoamine drug substance-related impurities (NDSRIs), allowing for more targeted risk assessments. The acceptable intake (AI) limits have been revised based on the carcinogenic potential of various NAs, with new interim limits established for specific impurities. Additionally, the guidelines emphasize enhanced risk assessment protocols, providing detailed recommendations for testing methods and analytical approaches to detect these impurities effectively. Clear implementation timelines have also been set, ensuring that manufacturers understand their compliance obligations. These changes reflect the FDA’s commitment to addressing the risks associated with nitrosamine (NA) impurities in pharmaceutica.4

In January 2024 (at its 177^th session in November 2023), the European Pharmacopoeia implemented a new strategy for managing N-nitrosamine impurities in its monographs, focusing on streamlining regulations. Key changes include the removal of specific production sections from individual active substance monographs, as general requirements in revised monograph 2034 will now apply universally. Specifications for N-nitrosamine impurities will only be included in the tests section if the impurity is deemed process-related or a degradation impurity. Additionally, for medicinal product monographs, specifications will be introduced only when justified, recognizing that the risk of N-nitrosamine formation varies significantly based on product composition and manufacturing conditions. This approach aims to enhance safety while ensuring consistency across monographs, reflecting a proactive stance in addressing potential impurities in pharmaceuticals.5

In June 2024, the International Council for Harmonization (ICH) announced significant updates regarding NA impurities and real-world data. A key change is the forthcoming addendum to the M7 guideline, which will establish daily AI limits for NA impurities, enhancing safety assessments for pharmaceuticals. Additionally, a reflection paper will be released to harmonize the use of real-world data in evaluating drug efficacy, focusing on generating real-world evidence. These documents aim to provide clearer guidance and improve regulatory consistency across regions. The ICH proactive approach reflects its commitment to addressing emerging safety concerns and leveraging real-world data for better health outcomes.6

In May 2024, the European Directorate for the Quality of Medicines & HealthCare implemented significant changes to address N-nitrosamine contamination. A key update is the approval of a new strategy for managing N-nitrosamine impurities in individual monographs, which includes the removal of specific production sections related to these impurities. This aligns with the general requirements outlined in revised monographs. Additionally, the European Directorate for the Quality of Medicines & HealthCare published the first official analytical procedures for determining NDSRIs, enhancing detection capabilities. The focus remains on ensuring that active substances and medicines meet stringent safety standards, reflecting ongoing collaboration with regulatory authorities and manufacturers to mitigate contamination risks effectively. These initiatives underscore the European Directorate for the Quality of Medicines & HealthCare commitment to protecting public health in Europe.7

In March 2024, Health Canada updated its guidance on NA impurities, incorporating several significant changes. The revised document clarifies when confirmatory testing results are not required, particularly for non-mutagenic NAs. It also outlines procedures for managing Step 3 changes related to risk mitigation measures, allowing for certain changes to be submitted as Level III - Annual Notifications. Notably, the established AI limits have been renumbered to Appendix 1 and are now accessible via a dedicated tab on Health Canada’s website, facilitating easier updates and searches. The update includes additional AI limits for several NAs, enhancing the framework for assessing contamination risks in medications. These changes reflect Health Canada’s commitment to ensuring drug safety and improving regulatory clarity.8

In October 2024, the Therapeutic Goods Administration updated its guidance on AI limits for NA impurities in medicines. Key changes include increased AI limits for certain NAs, aligning with recent determinations from the EMA. The update provides additional clarification for sponsors and manufacturers regarding Therapeutic Goods Administration expectations, ensuring a better understanding of compliance requirements. Minor editorial amendments were made to enhance clarity and usability of the guidance documents. Furthermore, the Therapeutic Goods Administration included internationally determined AI limits for various NAs, reinforcing its commitment to drug safety. These updates aim to mitigate contamination risks and protect public health effectively.9

In April 2024, the World Health Organization updated its good manufacturing practices guidelines to enhance the prevention and control of NA contamination in pharmaceutical products. The new guidelines apply to all manufacturers of excipients, APIs, and finished products. Key changes include a comprehensive definition of NAs and identification of impurities of concern. The guidelines emphasize the importance of conducting thorough root cause analyses and risk assessments to identify potential sources of contamination. Additionally, manufacturers are encouraged to implement robust control measures and monitoring systems to mitigate risks effectively. These updates reflect World Health Organization’s commitment to ensuring the safety and quality of pharmaceutical products globally, providing a clearer framework for manufacturers to follow in addressing NA contamination.10

Latest Analytical Methodologies for N-Nitrosamine Impurities

Analytical methodologies for detecting and accurately quantifying N-nitrosamine impurities in pharmaceutical products are crucial for ensuring drug safety and efficacy. These methodologies enable manufacturers to identify the presence of potentially harmful NAs, which are classified as probable human carcinogens. Accurate detection is essential not only for compliance with regulatory standards set by agencies like the FDA and EMA but also for maintaining public trust in pharmaceutical products. Advanced techniques such as liquid chromatography coupled with mass spectrometry (LC-MS) and gas chromatography (GC) are commonly employed due to their sensitivity and specificity, allowing for the detection of trace levels of impurities. Furthermore, robust analytical methods facilitate thorough risk assessments and help manufacturers implement effective control measures during production. By ensuring that NA levels remain within acceptable limits, these methodologies play a vital role in safeguarding public health, preventing contamination, and ultimately contributing to the overall quality of pharmaceutical products. As the industry evolves, continuous advancements in analytical techniques will be essential to address emerging challenges related to NA contamination.

A thorough review of the literature reveals that a variety of sensitive and selective analytical techniques have been developed, validated, and published by researchers, regulatory bodies, and pharmaceutical manufacturers. These methodologies are specifically designed to detect N-nitrosamine impurities in pharmaceutical products, ensuring compliance with safety standards and enhancing product quality. The advancements in these analytical methods reflect a concerted effort to address the potential risks associated with N-nitrosamine impurities.

In 2024, Zheng et al.11 reported a practical high-performance liquid chromatography-mass spectrometry (HPLC-MS) method for the analysis of NA drug substance related impurities using an inexpensive single quadrupole mass spectrometer. This method provides the desired specificity and sensitivity for the analysis of 7-nitroso-3-(trifluoromethyl)-5,6,7,8 tetrahydro-[1,2,4] triazolo [4,3-a] pyrazine and can be easily implemented in an analytical lab. The limit of quantitation is 0.5 ng/mL, corresponding to 0.1 ppm with respect to 5 mg/mL sitagliptin. The method has been successfully validated as per ICH guidelines.

Kartop et al.12 has been developed and validated a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) based analytical method for the risk assessment of NAs in pharmaceutical products and packaging materials. This method is useful for N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitroso-N-methyl-4-aminobutyric acid (NMBA), N-nitrosodiisopropylamine (NDIPA), N-nitroso-ethyl-isopropylamine (NEIPA), N-nitrosodibutylamine (NDBA) and 1-methyl-4-nitrosopiperazine (MeNP) N-nitrosamine compounds in API and drug products (DPs) as well as in primary packaging materials, one of the risk sources. This validated method was applied to check the NA content may occur from container, blister, printed aluminum foil, nasal spray, and eye drop packaging materials as part of the extractable and leachable studies arising from interactions between the product and the primary packaging.

Planinšek Parfant and Roškar13 developed and validated a method on a comprehensive approach for N-nitrosamine determination in pharmaceuticals using a novel hydrophilic interaction liquid chromatography based solid phase extraction and liquid chromatography high-resolution mass spectrometry (LC-HRMS). This analytical approach was utilized to examine 26 commercially available and expired DPs. Three NAs (NDMA, NDEA, and NDBA) were detected, only NDMA exceeded the limits in expired DPs by up to 32-fold. It was found that special care should be taken when handling samples as NDMA content can be decreased by almost 50% if samples are not prepared immediately. The approach was tested on 59 different APIs and was confirmed as reliable tool for routine monitoring of 15 NAs in various DPs.

Jiang et al.14 developed and validated a method for identification and quantitative analysis of genotoxic impurities in rifampicin: Development and validation of a targeted LC-MS/MS method for 1-amino-4-methylpiperazine. This method was established and validated for detecting the genotoxic impurity, 1-amino-4-methylpiperazine, adhering to the ICH guidelines, which include specificity, linearity, detection and quantification limits, accuracy, precision, and robustness. These developments improve the quality control strategy for genotoxic impurities in rifampicin, ensuring product safety.

Nakka et al.15 published a novel and eco-friendly ultra performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS) method for the quantification of ACF-NDSRI (aceclofenac nitroso drug substance related impurity) from aceclofenac drug substance and combination formulations. The authors developed a simple, accurate, and highly sensitive UPLC-MS/MS method for the quantification of ACF-NDSRI from ACF drug substances and combination formulations. This current method could be used to quantify ACF-NDSRI in drug substance, as well as combination formulations during commercial release and stability testing. Authors also used enhanced tools, such as green analytical procedure index, analytical GREEnness metric (AGREE), and analytical eco scale to establish the greenness and eco-friendliness of the current method.

Vikram et al.16 reported a trace-level quantification of NDMA in levosulpuride API and tablet formulation using ultra-fast liquid chromatography-mass spectrometry (UFLC-MS/MS). This method is sensitive and selective for NDMA in levosulpuride drug substance and tablet formulations and can be employed for routine quality control analysis in pharmaceutical industry. In addition, the authors evaluated the greenness of the developed method using the green analytical procedure index, AGREE, and assessment evaluation and standardization metrics. These assessments confirm that the method adheres to green chemistry principles, minimizing the use of hazardous substances and implementing effective waste management practices and this report concluded the method as not only scientifically robust but also environmentally responsible.

Perkins et al.17 reported a method on quantitative analysis of NDMA in drug products: A proposed high-throughput approach using headspace–selected ion flow tube mass spectrometry. Through the novel application of the multiple headspace extraction technique, NDMA was quantified directly and rapidly from the drug product without dissolution, at levels well below the regulatory AI of 96 ng/day. A comparative analysis of recalled metformin using multiple headspace extraction-selected ion flow tube mass spectrometry and a conventional LC-MS/MS method showed good agreement.

Batista Junior et al.18 reported an agile and accurate approach for N-nitrosamines detection and quantification in medicines by direct analysis in real time-mass spectrometry (DART-MS). DART-MS has emerged as a prominent ambient ionization technique for pharmaceutical analysis due to its high-throughput capability, simplicity, and minimal sample preparation requirements. Thus, in this study DART-MS was evaluated for the screening and quantification of NAs in medicines. The DART-MS technique demonstrated to be an alternative method to determine NAs in medicines, aligning with the principles of green chemistry.

Zhang et al.19 development and validation of an LC-MS/MS method for screening and quantification of trace N-nitrosamines in a pharmaceutical formulation. This method highlighted the possibility of using quantitative structure-retention relationship modeling to facilitate liquid chromatography development. This in silico approach could be extended to the other NAs under the oversight of EMA. This method is developed and validated for 4-nitrosamines as a limit test and as an assay for N-nitrosomethylphenylamine and N-nitrosodiphenylamine.

Batista Junior et al.20 reported a method on development of a reliable method for determination of N-nitrosamines in medicines using disposable pipette extraction and HPLC-MS analysis. The dynamic pseudo-extraction high-performance liquid chromatography-mass spectrometry (DPX/HPLC-MS) technique offers a faster and cost-effective method for analyzing NAs in medicines compared to traditional approaches. Besides, this method reduces solvent consumption and residue generation, enhancing environmental sustainability according to green chemistry principles.

Hao et al.21 reported a method on exploration and detection of NA impurity nitroso-STG-19 in sitagliptin tablets and API as well as nitrites in excipients by LC-MS/MS. This is a derivatization method for the detection of nitrite salts at lower concentration to select applicable excipients to decelerate the generation of 7-nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyrazine (NTTP). It is a sensitive and accurate method, suitable for screening appropriate pharmaceutical excipients.

Vidyamani et al.22 development and validation of a rapid and sensitive liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS/MS) method for the determination of three N-nitrosamine impurities in varenicline tartrate drug substance and drug products. The method can be routinely applied for the detection of N-nitrosamine impurities (NDMA, NDEA and N-nitroso varenicline) in varenicline tartrate drug substance and drug products.

Fukuda et al.23 reported a simple and practical method for the quantitative high-sensitivity analysis of N-nitroso duloxetine in duloxetine drug products utilizing LC-MS/MS. The applicability of the method for the content determination of N-nitroso duloxetine in a variety of duloxetine drug products was demonstrated. The method aids the risk assessment process of N-nitroso duloxetine in duloxetine drug products through providing a fast and reliable quantitation.

Öncü et al.24 reported a LC-MS/MS investigation method for NA impurities in certain sartan group medicinal products available in Istanbul, Türkiye. The goal of this method was to assess the amounts of six different NAs in sartan group medicines purchased from formal pharmacies in Istanbul, Türkiye, using a validated LC-MS/MS method.

Nakka et al.25 reported a method on synthesis and trace-level quantification of mutagenic and cohort-of-concern ciprofloxacin NDSRIs and other nitroso impurities using ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method optimization using I-optimal mixture design. Also, quantitative structure-activity relationship methodology was employed to assess and categorize the genotoxicity of all ciprofloxacin N-nitroso impurities. Analytical quality by design driven I-optimal mixture design was used to optimize the mixture of solvents in the method. The current method is capable of determining the trace levels of three N-nitroso ciprofloxacin impurities simultaneously from the marketed tablet dosage forms for commercial release and stability testing.

Dhorajiya et al.26 reported a novel method for monitoring of carcinogenic impurity of N-nitrosamine in nizatidine pharmaceutical products using ultra high-pressure liquid chromatography triple quadrupole mass spectrometry (UHPLC–MS/MS). The developed UHPLC–MS/MS method offers a simple, precise, accurate, and selective approach for monitoring NDMA levels in nizatidine formulations available in Australia, promising enhanced sensitivity and specificity with limits of quantification in the ppb and sub-ppb ranges.

Pereira dos Santos et al.27 reported a method on water as a green solvent for sustainable sample preparation: single drop microextraction of N-nitrosamines from losartan tablets. This method pioneers using water as the extraction solvent in headspace single-drop microextraction for N-nitrosamines from losartan tablets. Additionally, the environmental sustainability of the method is assessed using the AGREE prep methodology, positioning it as an outstanding green alternative for determining hazardous contaminants in pharmaceutical products.

Dharani et al.28 reported a method on patient in-use stability testing of FDA-approved metformin combination products for N-nitrosamine impurity. The objective of the present study was to evaluate in-use stability of commercial metformin combination products for NDMA. The NDMA impurity level in some of the assessed products was above acceptable daily intake limit.

Panusa et al.29 reported a new ultra high-performance liquid chromatography tandem mass spectrometry method (UHPLC-MS/MS) to monitor 1-amino-4-methyl-piperazine and 1-methyl-4-nitroso piperazine in rifampicin hydrolysis: A standard addition approach to quantify NA in rifampicin. In this study, a suitable UHPLC-MS/MS method was developed to quantify both 1-amino-4-methyl-piperazine and 1-methyl-4-nitroso-piperazine during rifampicin hydrolysis in buffered aqueous solutions and in methanol.

Uppala et al.30 reported a method on development of an UPLC-MS/MS approach to detect and quantify N-nitroso mirabegron in mirabegron with enhanced sensitivity using ultra performance liquid chromatography tandem mass spectrometry. This method has been developed and validated in accordance with ICH Q2A guidelines. Thus, it is highly accurate, linear, sensitive, specific, effective, and precise for the concurrent quantitation of N-nitroso mirabegron impurity in the mirabegron drug, achieving reduced LOD and LOQ values.

Selaya et al.31 reported a headspace gas chromatography-mass spectrometry (GC-MS) method to quantify NA impurities and precursors in drug products: method validation and product testing. The method described for NMDA, NDEA, NDIPA, NEIPA, and dimethylformamide has been validated and applied to sartan and metformin drug products. This validated GC-MS method enable regulators and manufacturers to assess the safety of products before and after their release into the public market.

Tummala et al.32 reported a head space gas chromatography-tandem mass spectrometry method for the quantification of five nitrosoamine-genotoxic impurities (NDEA, NEIPA, NDIPA, N-nitrosodipropylamine (NDPA), and NDBA) in metformin hydrochloride. The proposed method demonstrated rapid analysis capability, high accuracy, sensitivity, and precision, making it a reliable approach for monitoring N-nitrosamines in metformin hydrochloride. This method was developed and validated according to ICH guidelines. The results obtained were within the sensitivity limits set by the U.S. Food and Drug Administration.

Carlos et al.33 developed and reported a sensitive LC-MS/MS method for simultaneous determination of three NAs (NDEA, NDIPA and NDBA) in losartan API as well as assessment of NAs formation. In this study, the authors developed and validated a sensitive, selective, and high-throughput LC-MS/MS method for the simultaneous determination of three NAs in losartan using an atmospheric-pressure chemical ionization source with multiple reaction monitoring acquisition method. Additionally, eight batches of commercial losartan products were analyzed, and none of them showed the presence of NAs.

Aishwarya et al.34 reported a method on comparative evaluation of antioxidants as potent inhibitors of N-nitrosodimethylamine formation in metformin hydrochloride drug products. In this method, the authors investigated the efficacy of three antioxidants (alpha-tocopherol, ascorbic acid, and trolox) as inhibitors of NDMA formation in metformin hydrochloride products under vulnerable storage conditions. The developed liquid chromatography-electrospray ionization-high resolution mass spectrometry method showed better sensitivity and reliability in quantifying NDMA levels in metformin hydrochloride drug products.

Li et al.35 reported a method for mitigating matrix effects for LC-MS/MS quantification of nitrosamine impurities in rifampin and rifapentine. In this study, the authors optimized and validated two LC-MS/MS methods for quantifying the nitrosamine impurities, 1-methyl-4-nitrosopiperazine and 1-cyclopentyl-4-nitrosopiperazine at levels as low as 0.05 ppm in the tuberculosis medications rifampin and rifapentine. The successful development and validation of these methods necessitated a comprehensive understanding of various factors, including the chemical and physical properties of NAs, their interactions with sample matrices, and the benefits and challenges of different sample preparation and quantification techniques. These factors collectively influence the overall performance of the methods.

Patel et al.36 successfully developed, validated and reported a liquid chromatography-triple quadrupole-tandem mass spectrometry method for the quantification of N-nitrosorasagiline in rasagiline drug substance and products/tablets. This method exhibited outstanding specificity, precision, accuracy, and robustness, making it ideal for quantifying N-nitrosorasagiline. The low limit of detection and limit of quantification values (ranging from 1 to 2 μg/g) highlight the methods high sensitivity, enabling quantification below the permitted limits. The method was validated according to ICH guidelines and is useful for quality control testing.

Manchuri et al.37 compiled and published an article on analytical methodologies to detect N-nitrosamine impurities in APIs, drug products and other matrices. This review article aims to concentrate on the products that are recently reported to contain N-nitrosamine impurities. These products include rifampicin, champix, famotidine, nizatidine, atorvastatin, bumetanide, itraconazole, diovan, enalapril, propranolol, lisinopril, duloxetine, rivaroxaban, pioglitazones, glifizones, cilostazol, and sunitinib.

In addition to the above N-nitrosamine impurities, there are few research articles published on genotoxic impurities as well (N-nitrosamine impurities are class of genotoxic impurities). Shaik et al.38 developed and published a novel liquid chromatography quadrupole time-of-flight mass spectrometry method for trace level identification and quantification of potential genotoxic impurity, 5-nitro-2-(propylthio)pyrimidine-4,6-diol in ticagrelor drug substance. This method is novel, fast, sensitive, accurate, precise, specific, simple, cost-effective, and linear over the established ranges. It is also fit for its intended purpose. Given the advanced technology of the liquid chromatography quadrupole time-of-flight mass spectrometry instrument, it excels in identifying and confirming precise m/z values with minimal error, especially when analyzing low concentration genotoxic impurities. Additionally, this method was developed and validated in accordance with the latest ICH M7 (R1) guidelines for detecting genotoxic impurities. Therefore, it is suitable for use in process control, quality control, impurity profiling, and stability investigations within the pharmaceutical industry. Shaik et al.39 also developed and published another novel UHPLC-MS/MS method for trace level identification and quantification of genotoxic impurity 2-(2-chloroethoxy) ethanol in quetiapine fumarate. The newly developed UHPLC-MS/MS “transition” method with a short run time is simple, sensitive, selective, accurate, precise, specific and cost-effective. This method is also useful for process control, quality control, impurity profiling, and stability studies within the pharmaceutical industry. Ali et al.40 also developed and published another novel liquid chromatography with quadrupole time-of-flight tandem mass spectroscopy method for ultra-trace level identification and quantification of the genotoxic impurity 2,6-diamino-5-nitropyrimidin-4(3H)-one in valganciclovir hydrochloride. This newly developed and validated LC–QTOF–MS/MS reverse phase method with a very short run time is time saving, cost effective, rapid, simple, specific, sensitive, precise, accurate, selective and linear from various stated ranges for identification and ultra-trace level quantification of genotoxic impurity in valganciclovir hydrochloride API. Moreover, this method is more consistent and convenient for routine analysis in the pharmaceutical industry.

An overview of current analytical methods that were developed and reported for the determination of N-nitrosamine impurities in various pharmaceutical products are presented in Table 2.

Table 2.

An overview of current analytical methods for the determination of N-nitrosamine impurities in pharmaceutical products

Product	Technique	Method	N-nitrosamine impurity	Limit of detection	Limit of quantification	Advantage	Reference
Sitagliptin	HPLC-MS	A practical HPLC-MS method for the analysis of nitrosamine drug substance related impurities using an inexpensive single quadrupole mass spectrometer	7-Nitroso-3-(trifluoromethyl)-5,6,7,8 tetrahydro-[1,2,4] triazolo [4,3-a] pyrazine (NTTP)	NR	0.1 ppm	Specific and sensitive method for the analysis of NTTP and can be easily implemented in an analytical lab	11
Sartans and packaging materials	LC-MS/MS	Evaluation of a novel LC-MS/MS based analytical method for the risk assessment of nitrosamines in pharmaceutical products and packaging materials	NDMA, NDEA, NMBA, NDIPA, NEIPA, NDBA, MeNP and primary packaging materials	NR	1 & 0.5 ng/mL	Highly reliable, fast, accurate, sensitive method for simultaneous detection of NAs even at low concentrations	12
59 different APIs	LC-HRMS	A comprehensive approach for N-nitrosamine determination in pharmaceuticals using a novel HILIC-based solid phase extraction and LC-HRMS	MeNP, NDELA, NDMA, NMOR, NMBA, NPYR, NMEA, NDEA, NPIP, NEIPA, NDIPA, NMPA, NDPA, NDBA, NDPhA	NR	0.8–2.9 μg/L	A versatile and innovative method using a unique sample clean-up procedure by solid phase extraction. Highly sensitive, selective, and robust method, can be further adapted to the specific API of interest or extended to the newly emerging NDSRIs. This method was applied for 59 different APIs and 15 NAs	13
Rifampicin	LC-MS/MS	Identification and quantitative analysis of genotoxic impurities in rifampicin: Development and validation of a targeted LC-MS/MS method for 1-amino-4-methylpiperazine	1-Amino-4-methyl piperazine	0.028 μg/mL	0.091 μg/mL	Useful for detecting genotoxic impurity as per ICH guidelines. Also, useful to define quality control strategy for genotoxic impurities in rifampicin	14
Aceclofenac	UPLC-ESI-MS	A novel and eco-friendly UPLC-ESI-MS method for the quantification of aceclofenac-NDSRI from aceclofenac drug substance and combination formulations	ACF-NDSRI	0.10 ng/mL	0.30 ng/mL	A simple, accurate, and highly sensitive method. Can be used to quantify ACF-NDSRI in DS as well as combination formulations during commercial release and stability testing	15
Levo sulpuride	UFLC-MS/MS	Trace-level quantification of NDMA in levosulpuride active pharmaceutical ingredient and tablet formulation using UFLC-MS/MS	NDMA	0.195 ppb	0.390 ppb	Highly sensitive and selective method. Adheres to green chemistry principles, minimizing the use of hazardous substances and implementing effective waste management practices	16
Metformin, ranitidine, & valsartan	Headspace–SIFT-MS	Quantitative analysis of NDMA in drug products: a proposed high-throughput approach using headspace–SIFT-MS	NDMA	NR	2 ng/g	Sensitive and selective method. Useful as a fast-screening tool in routine testing laboratories (QCs)	17
Sartan medicines	DART-MS	An agile and accurate approach for N-nitrosamines detection and quantification in medicines by DART-MS	NMEA NPYR NDEA NPIP NMOR NDBA	0.10 ng/mL 0.10 ng/mL 0.25 ng/mL 0.10 ng/mL 0.10 ng/mL 0.10 ng/mL	1.00 ng/mL 1.00 ng/mL 1.00 ng/mL 1.00 ng/mL 1.00 ng/mL 1.00 ng/mL	Efficient, sensitive and high-throughput method with the principles of green chemistry	18
Pharmaceutical formulation	LC-MS/MS	Development and validation of an LC-MS/MS method for screening and quantification of trace N-nitrosamines in a pharmaceutical formulation	NMBA NNK NMPA NDPhA	0.02 ng/mL 3.91 pg/mL	NR	QSRR modeling was used to facilitate the liquid chromatography development. This method is useful for analysis of 4-NAs	19
Sartan medicines	HPLC-MS	Development of a reliable method for determination of N-nitrosamines in medicines using disposable pipette extraction and HPLC-MS analysis	NMEA NPYR NDEA NPIP NMOR NDBA	0.5 ng/mL 0.5 ng/mL 0.5 ng/mL 0.5 ng/mL 0.5 ng/mL 0.5 ng/mL	1.0 ng/mL 1.0 ng/mL 1.0 ng/mL 1.0 ng/mL 1.0 ng/mL 1.0 ng/mL	Offer a faster and cost-effective method for analyzing NAs in medicines compared to traditional approaches. Reduce the solvent consumption and residue generation, enhancing the environmental sustainability according to green chemistry principles	20
Sitagliptin	LC-MS/MS	Exploration and detection of nitrosamine impurity nitroso-STG-19 in sitagliptin tablets and API as well as nitrites in excipients by LC-MS/MS methods	Nitroso-STG-19	NR	56 ppb (0.056 ng/mL)	Sensitive and accurate method, suitable for screening of excipients. Derivatization method for the detection of nitrite salts at lower concentration	21
Varenicline tartrate	LC-APCI-MS/MS	Development and validation of a rapid and sensitive LC-APCI-MS/MS method for the determination of three N-nitrosamine impurities in varenicline tartrate drug substance and drug products	NDMA, NDEA, N-nitroso varenicline	0.22 ppm	0.66 ppm	Novel, rapid, sensitive method with very low LOD and LOQ values. Method can be routinely applied for the detection of three NAs in both DS and DPs	22
Duloxetine	LC-MS/MS	Simple and practical method for the quantitative high-sensitivity analysis of N-nitroso duloxetine (NDXT) in duloxetine drug products utilizing LC-MS/MS	NDXT	0.02 ng/mL	0.075 ng/mL	Highly sensitive method for the determination of NDXT. The method aids the risk assessment process of NDXT in duloxetine DPs through providing a fast and reliable quantitation	23
Sartan products	LC-MS/MS	LC-MS/MS investigation of nitrosamine impurities in certain sartan group medicinal products available in Istanbul, Türkiye	NDBA NDIPA NEIPA NDEA NMBA NDMA	NR NR NR NR NR NR	1.0 μg/L 1.0 μg/L 1.0 μg/L 1.0 μg/L 1.0 μg/L 1.0 μg/L	A multi-analyte method for screening and identifying of six NAs in selected sartan products	24
Ciprofloxacin	UPLC-ESI-MS	Synthesis and trace-level quantification of mutagenic and cohort-of-concern ciprofloxacin NDSRIs and other nitroso impurities using UPLC-ESI-MS/MS method optimization using I-optimal mixture design	COX-N-nitroso impurity-1 COX-NDSRI COX-N-nitroso impurity-2	0.03 ng/mL 0.03 ng/mL 0.03 ng/mL	0.10 ng/mL 0.10 ng/mL 0.10 ng/mL	Rapid method for capable of determining the trace levels of three N-nitroso ciprofloxacin impurities from the marketed tablet dosage forms for commercial release and stability testing	25
Nizatidine	UHPLC–MS/MS	Novel method for monitoring of carcinogenic impurity of N-nitrosamine in nizatidine pharmaceutical products using ultra high-pressure liquid chromatography triple quadrupole mass spectrometry	NDMA	0.25 ng/mL	0.5 ng/mL	A simple, precise, accurate, and selective method for monitoring NDMA levels in nizatidine formulations available in Australia with ppb and sub-ppb limits of quantification	26
Losartan tablets	HS-SDME	Water as a green solvent for sustainable sample preparation: single drop microextraction of N-nitrosamines from losartan tablets	NDMA NDEA EIPNA NDIPA	50 ng/g 80 ng/g 50 ng/g 80 ng/g	NR NR NR NR	Method exhibits linear responses as well as demonstrating appropriate detectability, precision, and accuracy. Additionally, the environmental sustainability of the method is assessed using the AGREE prep methodology	27
Metformin & Metformin combination products	LC-MS	Patient in-use stability testing of FDA-approved metformin combination products for N-nitrosamine impurity	NDMA	NR	NR	Useful method for in-use stability testing of commercial metformin combination products for NDMA	28
Rifampicin	UHPLC-MS/MS	A new ultra high-performance liquid chromatography tandem mass spectrometry method to monitor 1-amino-4-methyl-piperazine and 1-methyl-4-nitroso piperazine in rifampicin hydrolysis: A standard addition approach to quantify nitrosamine in rifampicin	AMP, MNP	NR	Ammonium format: 50, 0.5 ng/mL Methanol: 2.5, 5 ng/mL	Sensitive, selective and suitable method to quantify AMP and MNP during RIF hydrolysis in buffered aqueous solutions and methanol	29
Mirabegron	UPLC MS/MS	Development of an UPLC-MS/MS approach to detect and quantify N-nitroso mirabegron in mirabegron	N-nitroso mirabegron	0.006 ppm	0.02 ppm	Accurate, linear, sensitive, specific, effective and precise method for the concurrent quantitation of N-nitroso mirabegron impurity with reduced LOD and LOQ values	30
Sartan & metformin products	Headspace GC-MS	A headspace GC-MS method to quantify nitrosamine impurities and precursors in drug products: method validation and product testing	NMDA, NDEA, NDIPA, NEIPA, DMF	NR	NR	Useful method for NMDA, NDEA, NDIPA, NEIPA, and DMF in sartan and metformin drug products. Method enables regulators and manufacturers to assess the safety of products	31
Metformin	GC-MS/MS	Head space GC-MS/MS method for quantification of five nitrosoamine-genotoxic impurities in metformin HCl	NDEA, NEIPA, NDIPA, NDPA, NDBA	0.001 ppm	0.004 ppm	The method demonstrated rapid analysis capability, high accuracy, sensitivity, and precision, making it a reliable approach for monitoring five N-nitrosamines in metformin HCl. Method was validated as per ICH guidelines	32
Losartan	LC-MS/MS	Development of a sensitive LC-MS/MS method for simultaneous determination of three nitrosamines in losartan API and assessment of nitrosamines formation	NDEA, NDIPA, NDBA	1.96, 1.7, 0.42 ng/mL	5.95, 3.2, 1.27 ng/mL	A sensitive, selective, and high-throughput LC-MS/MS method for the simultaneous determination of three NAs in losartan using an APCI source with multiple reaction monitoring acquisition method	33
Metformin	LC-HRMS	Comparative evaluation of antioxidants as potent inhibitors of N-nitrosodimethylamine formation in metformin hydrochloride drug products	NDMA	0.1 ng/mL	0.3 ng/mL	A better sensitive and reliable method for investigating the efficacy of three antioxidants as inhibitors of NDMA formation in metformin under vulnerable storage conditions	34
Rifampin & rifapentine	LC-MS/MS	Mitigating matrix effects for LC-MS/MS quantification of nitrosamine impurities in rifampin and rifapentine	1-Methyl-4-nitrosopiperazine, 1-cyclopentyl-4-nitrosopiperazine	NR	0.05 ppm	The methods are capable for quantifying NAs (MNP & CPNP) at levels as low as 0.05 ppm in the rifampin and rifapentine. Additionally, the matrix effects were effectively assessed and mitigated.	35
Rasagiline	LC-MS/MS	Trace level quantification of N-nitrosorasagiline in rasagiline tablets by LC-TQ-MS/MS	N-nitrosorasagiline	1 μg/g	2 μg/g	A sensitive, specific, precise, accurate, and robust method, validated according to ICH Q2 guidelines, is useful for quality control testing	36

-N=O: Nitrosamine group; ACF-NDSRI: Aceclofenac nitroso drug substance related impurity; AGREE: Analytical GREEnness metric approach; AMP: 1-amino-4-methyl-piperazine; APCI: atmospheric pressure chemical ionization; APIs: active pharmaceutical ingredients; COX: ciprofloxacin; COX-NDSRI: ciprofloxacin nitrosoamine drug substance related impurity; CPNP: 1-cyclopentyl-4-nitrosopiperazine; DART-MS: direct analysis in real time mass spectrometry; DMF: dimethylformamide; DPs: drug products; DS: drug substance; EIPNA: N-nitrosoethylisopropylamine; FDA: U.S. Food and Drug Administration; GC-MS/MS: gas chromatography tandem mass spectrometry; GC-MS: gas chromatography mass spectrometry; HCl: hydrochloric acid; HILIC: hydrophilic interaction liquid chromatography; HPLC-MS: high performance liquid chromatography mass spectrometry; HS-SDME: headspace single drop microextraction; ICH: International Council for Harmonization; LC-APCI-MS/MS: liquid chromatography atmospheric pressure chemical ionization mass spectrometry; LC-HRMS: liquid chromatography high resolution mass spectrometry; LC-MS/MS: liquid chromatography tandem mass spectrometry; LC-MS: liquid chromatography mass spectrometry; LC-TQ-MS/MS: liquid chromatography triple quadrupole tandem mass spectrometry; MeNP: 1-methyl-4-nitrosopiperazine; MNP: 1-methyl-4-nitroso piperazine; NaNO2: sodium nitrite; NAs: nitrosamines; NDBA: N-nitrosodibutylamine; NDEA: N-nitrosodiethylamine; NDELA: N-nitrosodiethanolamine; NDIPA: N-nitrosodiisopropylamine; NDMA: N-nitrosodimethylamine; NDPA: N-nitrosodipropylamine; NDPhA: N-nitrosodiphenylamine; NDSRIs: nitrosoamine drug substance related impurities; NDXT: N nitroso duloxetine; NEIPA: N-nitroso-ethyl-isopropylamine; NMBA: N-nitroso-N-methyl-4-aminobutyric acid; NMEA: N-nitrosomethylethylamine; NMOR: N-nitrosomorpholine; NMPA: N-nitroso-N-methylphenylamine; NNK: nicotine-derived nitrosamine ketone; -NO: nitroso group; NO2–: nitrite ions; NPIP: N-nitrosopiperidine; NPYR: N-nitrosopyrrolidine; NR: not reported; NTTP: 7-Nitroso-3-(trifluoromethyl)-5,6,7,8 tetrahydro-[1,2,4] triazolo [4,3-a] pyrazine; QCs: quality controls; QSRR: quantitative structure retention relationship; RIF: rifampicin; SIFT-MS: selected ion flow tube mass spectrometry; STG: sitagliptin; UFLC-MS/MS: ultra-fast liquid chromatography mass spectrometry; UHPLC–MS/MS: ultra high-pressure liquid chromatography triple quadrupole mass spectrometry; UPLC MS/MS: ultra performance liquid chromatography tandem mass; UPLC-ESI-MS: ultra performance liquid chromatography electrospray ionization mass spectrometry.

Future Recommendations

After reviewing the literature, research advancements, regulatory guidelines, industry status, and patient needs regarding N-nitrosamine impurities, the following future recommendations can be proposed.

• Manufacturers should conduct thorough risk assessments to identify potential sources of NA impurities during the entire lifecycle of drug products, from raw materials to final formulations.

• Implement robust analytical methods for confirmatory testing of N-nitrosamine impurities, ensuring that any detected impurities are reported and addressed promptly.

• Develop and document control strategies to mitigate the presence of N-nitrosamine impurities, especially focusing on the quality of excipients and raw materials.

• Stay updated with evolving regulatory guidelines, such as those from the FDA, which provide specific limits and testing recommendations for N-nitrosamine impurities.

• Provide ongoing training for staff involved in drug formulation and manufacturing to ensure awareness of NA risks and mitigation strategies.

Conclusions

This review article primarily focuses on the latest regulatory guidelines and analytical methodologies related to N-nitrosamine impurities that are developed and published in 2024 for various pharmaceutical products, including sitagliptin, sartans, rifampicin, aceclofenac, levosulpiride, metformin, ranitidine, valsartan, varenicline tartrate, duloxetine, ciprofloxacin, nizatidine, losartan, mirabegron, rifapentine and rasagiline. Essentially, these hazardous N-nitrosamine impurities pose significant risks to human health with the associated diseases (cancer). Most importantly, this information will be particularly useful and helpful for drug makers and researchers to understand and align with current updates and changes. This review article does not cover the information prior to 2024 related to N-nitrosamine impurities. It is limited to the latest regulatory guidelines and analytical methodologies published in 2024, including a few future recommendations.

Data availability statement:

No additional data are available.