Various Analytical Techniques used for Nitrosamine impurities in Pharmaceutical Dosage Forms: A Comprehensive Review

Kalpesh Upadhyay*, Jaswandi Mehetre, Vimal Kumar, Tushar Mehta,

Anirban Roy Chowdhury, Keyur Ahir

School of Pharmacy, ITM SLS Baroda University, Halol highway, Vadodara 391510, Gujarat, India.

*Corresponding Author E-mail: Kalpesh.aol@gmail.com, kalpeshu@amneal.com

ABSTRACT:

Nitrosamine impurities are a class of substances formed from the reaction of a nitroso compound and secondary or tertiary amines under acidic conditions. The detection of nitrosamine impurities in  Active Pharmaceutical Ingredients (APIs) and Finished Pharmaceuticals (FP) has raised significant health concerns due to their potent genotoxic carcinogenic properties. The presence of  low concentration levels of nitrosamine impurities has led to drug withdrawals and extensive investigations into their formation. For the estimation of such low level of impurities led to development of highly sensitive and selective analytical methods for the simultaneous determination and quantification of various nitrosamine impurities in active pharmaceutical ingredients (APIs) and Finished Pharmaceuticals (FP). This review evaluates common analytical techniques for nitrosamine impurities detection, including Gas Chromatography-Mass Spectrometry (GC-MS), Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). It compares their sensitivity, specificity, and practical applications, and discusses sample preparation methods such as Solid-Phase Extraction (SPE), Liquid-Liquid Extraction (LLE) etc. The choice of method depends on sample type, required sensitivity and as per regulatory expectation.

KEYWORDS: Nitrosamine Impurities, Analytical techniques, Gas Chromatography-Mass Spectrometry (GC-MS), Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS), Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS), Capillary Electrophoresis-Mass Spectrometry (CE-MS), Sample preparation, Solid-Phase Extraction (SPE), Liquid-Liquid Extraction (LLE).

INTRODUCTION OF NITROSAMINE IMPURITIES:

Nitrosamines have long been a concern in various foods,^1-3 beverages, water,⁴ tobacco products^5,6 cosmetic products⁷ and this issue has recently extended to pharmaceuticals^8,9. The formation of nitrosamines in sartan medicines has been associated with the presence of nitrosating agents, which in most cases are sodium nitrite as a reagent and starting materials, intermediates, reagents, solvents or catalysts as sources of secondary amines or secondary amine precursors. For example, the presence of NDMA in sartans class blood pressure medications (angiotensin-II-receptor antagonists) has been associated with the use of N, N-dimethylformamide as a solvent and sodium nitrite as a reagent in the formation of the tetrazole ring.⁸ This discovery led to numerous product recalls, resulting in sales losses and potential reputational damage. Currently, nitrosamines in at least four categories of drugs are under scrutiny. Many professionals anticipate that nitrosamine impurities testing could expand beyond active pharmaceutical ingredients (APIs) and finished drug products in the future. It has been discovered that N-nitroso drug substances can be formed by the reaction of the amine functionality of drug substance with a nitrosating agent, which in most cases is probably nitrite present as an impurity in excipients.^10,11

Classification of Impurities:

Pharmaceutical impurities are unwanted chemicals that form during the synthesis of APIs or through degradation, storage conditions, contamination, or excipient interactions. According to ICH M7 guidance, these impurities must be quantified at trace levels to eliminate the risk of carcinogenic effects for human consumption. In July 2018, the US FDA globally recalled and recommended that drug manufacturers quantify nitrosamine levels in their drugs. If these impurities exceed acceptable limits, they should be reduced or removed as per regulatory requirements. Nitrosamines pose a high potential risk of cancer, making even small quantities in APIs and drug products a significant health and safety concern. These impurities can form through various pathways, including degradation during formulation, drug storage conditions, or environmental contaminants.

List of Nitrosamine Impurities and Their Limits:

Nitrosamine impurities are part of the “cohort of concern” compounds, meaning the usual toxicological value threshold approach for determining acceptable concentrations does not apply. The FDA has set interim acceptable concentrations for major nitrosamine impurities based on animal toxicity studies, with safety levels shown in Table 1.

Table 1 Potential Possible Nitrosamine Impurities in APIs and FP.

Name of impurity	Structure	Allowable daily intake (AI Limit) (ng/day)
N-Nitroso dimethylamine (NDMA)		96
N-Nitrosodiethylamine (NDEA)		26.5
N-Nitrosodibutylamine (NDBA)		26.5
N-Nitroso-N-methyl-4-aminobutyric acid (NMBA)		96
N-Nitrosomethylphenylamine (NMPA)		26.5
N-Nitrosodisopropylamine (NDIPA)		26.5
N-Nitrosoisopropylethylamine (NIPEA)		26.5
N-Nitrosopyrrolidine (NPYR)		26.5
N-Nitrosopiperidine (NPIP)		26.5

Screening Nitrosamine Impurities:

Traditional spectral analysis methods struggle to detect nitrosamines due to their structural formula, except for NMBA, which shows high signal intensity because of its carboxyl group. Consequently, routine drug analysis methods lack the sensitivity and selectivity needed to determine nitrosamine impurities. Most existing methods use chromatographic separation (mainly HPLC) with MS detection. Single quadrupole detection methods can only determine nitrosamine concentrations at the detection limit.

Over the past seven years, various methods have been developed for individual impurities and drugs, but the issue of universal control methods remains unresolved due to equipment availability, product lines, and regulatory requirements. Currently, only four nitrosamine impurities (NDMA, NDEA, NMBA, NEIPA) are included in regulatory screening and monitoring requirements for sartan medicines, although over 12 compounds from this group have been studied.

Regulatory authorities like the FDA and EMA have developed methods for determining nitrosamines in Valsartan, Losartan, and Irbesartan. The FDA’s method, which uses rare and expensive equipment like LC-HRMS, can simultaneously determine six nitrosamine impurities, while the EMA’s method is used for two (NDMA, NDEA). However, these methods make routine quality control longer and more time-consuming.

A variety of analytical methods have been developed to suffice the needs of detecting and quantifying small molecule nitrosamines in pharmaceutical products. Among them majority of these procedures used Mass Spectrometer (MS) as the detector followed by either gas or liquid chromatographic separation (GC-MS, LC-MS/MS or LC-HRMS) to achieve the necessary sensitivity and selectivity.

Here we try to compile various analytical techniques published for determination of nitrosamine impurities in Pharmaceuticals are enlisted in Table 2. Mainly focused on GC-MS, LC-MS/MS and LC-HRMS.

Table 2 Comparison of the developed method with FDA and earlier methods.

Pharmaceutical product	Analyte	Instrument	LOQ	Linearity Range	Accuracy	Reference
Valsartan	NDMA	GC-MS	0.5 μg/g	0.005–0.2 μg/mL	—	12
Sartans, Metformin, anitidine	NDMA, NDEA	GC-MS	0.9 and 0.3 μg/kg	0.9-1000 μg/kg for NDMA 0.3-1000 for NDEA	95.0-105 % for NDMA and 93.6-104 % for NDEA	13
Losartan	NDMA, NDEA, DIPNA, EIPNA	GC-MS	25 ng/mL for NDMA and NDEA, 50 ng/mL for DIPNA, EIPNA	25 to 5000 ng/mL	− 7.04% to 7.25% (92.96% - 92.75%)	14
Valsartan	NDMA, NDEA, NEIPA, NDIPA, and NDBA	GC-MS	0.008,0.005,0.005, 0.0050.005,0.025 ppm	2.5 to 100 ng/mL	—	15
Valsartan	NDMA, NDEA	LC-MS/MS	0.00026 ppm (NDMA) and 0.00013 ppm (NDEA)	NDMA 0.000260-0.0500 ppm, NDEA 0.000130-0.0500 ppm, VLA 0.000562-0.108 ppm, and VLA-DIM 0.0000237-.0.00455 ppm	100.8% for NDMA, 98.4% for NDEA, 100.0% for VLA, 100.1% for VLA-DIM	16
Metformin	NDMA	LC-HRMS	3.0 ng/mL	3-10 ng/mL	—	17
Ranitidine	NDMA	LC-MS/MS	1.0 ng/mL	1-100 ng/mL	—	18
Rivaroxaban		LC-MS/MS	0.15 ng/mL	-	Acceptable range	19
Valsartan, Losartan, Irbesartan	12 NA’s	LC-MS/MS	50 ng/mL	2.5-50 ng/mL	81.4-117.0% (Intra-day) 81.7-117.5% (Inter-day)	20
Sartans	NDMA, NMEA, NDEA, NPyr, Nmor, NDPA, NPIP, NDBA	LC-MS/MS	1.0-4.74 ng/mL	2-100 ng/mL	82.1-114.1%	21
Olmesartan	NDMA	LC-MS/MS	0.12 µg/mL	0.34-50.6 µg/L	80.23-103.87 %	22
Valsartan	NDMA, NDEA, NEIPA, NDIPA, NMPhA, NDBA	LC-MS/MS	1.5 ppb	1.5-9 ppb	101.88-102.92 % for NDMA 99.11-102.78 % for NDEA 104.69-100.75 % for NEIPA 101.6-104.17 % for NDIPA 96.64-99.77 % For NMPhA 100.72-101.15 % for NDBA	23
Valsartan, Losartan, Irbesartan	NDMA, NMBA, NDEA, NEIPA	LC-MS/MS	0.4 ng/mL	0.4-1.1 ng/mL	96.1-98.3 % for NDMA 90.3-101.1 % for NDEA 98.3-100.7 % for NMBA 90.7-97.2% for NEIPA	24
Telmisartan	NDMA, NDEA, NEIPA, NMBA, NDIPA, NDBA	LC-MS/MS	0.004 ppm	0.002-2 ppm	97-98.5 % for NDMA 90.8-103.7 % for NDEA 97.7-101.4 5 for NEIPA 100.4-101.2 % for NDIPA 93.9-101.8% for NDBA 101.7-103.2% for NMBA	25
Sartan	NDMA, NDEA, NDBA, NDIPA	GC-MS/MS	0.008-0.500 ppm	3-60 ng/mL for NDMA and NDIPA 6-60 ng/mL for NDBA 0.8-16 ng/mL for NDEA	87.68-123.76%	26
Losartan	NDMA, NDEA, EIPNA, DIPNA	HS-GC-MS	25 ppb for NDMA and NDEA 50 ppb for DIPNA and EIPNA	25-5000 ng/mL	—	27

CONCLUSION:

Development of Analytical Methods:

The development of analytical methods to determine Nitrosamines impurities is the challenging task due to very low levels of impurities present in the complex matrices. The developed methods also need to be validated to conform to GMP requirements.

Several methods have been published by the FDA to cover NDMA and NDEA in different 'sartans'. The EMA has indicated the extension of measures to include more Nitrosamines. Other regulatory authorities (Canada, Switzerland and Singapore) have adopted their own measures and published analytical methods. Most of the methods used for testing Nitrosamines in drug substance and drug product utilize the chromatographic techniques such as reversed-phase liquid chromatography (LC) or gas chromatography (GC) combined with various detectors such as mass spectrometry (MS), Ultraviolet spectrophotometry (UV) or nitrogen chemiluminescence (NCD) etc. European network of Official Medicines Control Laboratories (OMCLs) has developed methods for the testing of specific Nitrosamines in sartans on the basis of different analytical principles. The Irish OMCL in the Public Analyst’s Laboratory in Galway (PALG), the French OMCL at the ANSM site in Montpellier, the Chemisches and Veterinär- Untersuchungsamt (CVUA) Karlsruhe as well as the LGL Bayern established analytical methods for quantification of Nitrosamine on behalf of the network. Additionally, the U.S. FDA, Health Canada and Swissmedic have published methods for the simultaneous determination of Nitrosamine.

These testing methods serve as a starting point for the development and validation of analytical methods, appropriate for other drug substance and drug products. The FDA says that these methods should be validated by the user if the resulting data are used to support a required quality assessment of the API or drug product or if the results are used in a regulatory submission.

There are analytical techniques for the quantification of Nitrosamines impurities in pharmaceutical drug substances and drug products.

· LC-MS/MS (Liquid chromatography with mass spectrometry)

· GC-MS/MS (Gas chromatography with mass spectrometry)

Liquid Chromatography with Mass Spectroscopy:

Liquid chromatography along with mass spectroscopy technique offers a faster alternative to the traditional GC-MS methods. The use of high-resolution accurate mass spectrometry helps to obtain good selectivity for the detection of both GC- detectable and GC-undetectable compounds along with thermally stable and unstable Nitrosamines. The FDA has observed that the GC-MS method for testing ARBs of Nitrosamine impurities is not suitable for testing ranitidine because heating the sample generates NDMA. An LC-HRMS method was subsequently developed by the FDA to measure the levels of NDMA in ranitidine drug substance and drug product following ICH Q2 (R1). The limit of detection (LOD) is 0.011ppm, limit of quantitation (LOQ) is 0.033 and the range of the method is 0.033 – 3.33 ppm. In addition, several methods using liquid chromatography-mass spectrometry (LC-MS) or LC- MS/MS have been reported in scientific literature. However, only a few studies have reported for NDMA analysis using conventional high-performance liquid chromatography (HPLC), especially in the drugs. HPLC is the most popular technique for quality control of APIs and products in routine analysis and it is preferable if NDMA impurity is simultaneously detected with drug substances by a single HPLC analysis. Thus, it is important to develop a fast and simple analytical method for NDMA in drugs by using HPLC.

Gas Chromatography with Mass Spectroscopy:

Gas chromatography along with mass spectroscopy (GC-MS) is the most frequently employed technique for the determination of Nitrosamines with lower molecular weight. The majority of recent publications employ GC-MS, GC–MS/MS or GC-HS-MS technique due to its high selectivity and low detection limit. The FDA has developed and validated a combined GC-HS- MS method for the simultaneous evaluation of four Nitrosamine impurities in Valsartan drug substance and drug products, these impurities are N-nitrosodimethylamine (NDMA), N- nitrosodiethylamine (NDEA), N-nitrosodiisopropylamine (NDIPA), and N- nitrosoethylisopropylamine (NEIPA). This method meets all the requirements of current regulations with sensitivity and repeatability and exceeds the expected requirements of the control limits. GC-TEA (Thermal energy analyzer) also provides high selectivity for Nitrosamines. The high molecular weight Nitrosamines are rather labile molecules, and it is impossible to determine by GC.

SUMMARY:

Liquid chromatography with mass spectrometry (LC-MS/MS) and Gas chromatography with mass spectrometry (GC-MS/MS) are analytical techniques which have been used to monitor the Nitrosamines impurities in pharmaceutical drug substances and pharmaceutical products. Looking at various validation criterions, an analytical method needs to meet, in today’s stringent quality requirement. High pressure liquid chromatography with mass spectroscopy (LC- MS/MS) is considered as the most versatile because of availability of different types of stationary phases, unlimited choice of mobile phases, varieties of detectors mass spectroscopy to be chosen from a range and as a whole applicability to a wide range of impurities which are not possible to analyze using other classical analytical techniques.

Gas chromatography with mass spectroscopy (GC-MS/MS) is also as the most varsities to quantification of trace level of Nitrosamines impurities with the help of Head Space and autosampler. There are a number of books and reviews available on this subject discussing various aspects of the techniques in detail. Many international symposia and conferences are held on this subject with highlights the importance of this technique. It is therefore thought worthwhile to develop new methods of analyzing Nitrosamine’s impurities in drugs using the technique of LC-MS/MS and GC-MS/MS at trace level of quantification.

The work will be done by analytical instrument for quantification of Nitrosamines impurities in pharmaceutical drug substances and drug products at Trace Level (ppm or ppb level).

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Received on 07.03.2025      Revised on 29.03.2025

Accepted on 15.04.2025 Published on 06.05.2025

Available online from May 10, 2025

Asian Journal of Pharmaceutical Analysis. 2025; 15(2):131-136.

DOI: 10.52711/2231-5675.2025.00021

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