24/07/2025, 11:23 Structure of Nitrosamine Drug Impurities and Implications for Chemical Analysis

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Structure of Nitrosamine Drug Impurities and

Implications for Chemical Analysis
August 29, 2021

Why is control of N-nitrosamine impurities in drugs

important?
Many nitrosamines (N-Nitrosamines) are potent genotoxins; some have been classified as possible
human carcinogens. Recently several toxic nitrosamines have been found as impurities in human
drugs. As a result of this discovery, 2020 FDA guidance (https://www.fda.gov/regulatory-
information/search-fda-guidance-documents/control-nitrosamine-impurities-human-drugs)
mandates control of N-nitrosamine impurities in pharmaceuticals. See our post ‘Control of
Nitrosamines in Human Drugs (https://acanthusresearch.com/uncategorized/control-of-
nitrosamine-impurities-in-human-drugs/)’ for more background information.
Let’s now focus on the structure of N-nitrosamines and implications for their chemical analysis.

Can N-Nitrosamines exist in two different stable

forms?
N-nitrosamines substituted with two different alkyl groups can exist in two different stable forms
distinguishable by several analytical methods.
Usually, N-nitrosamines are drawn as the structure shown on the left in (Fig.1). However, the
zwitterionic resonance structures shown on the right are stable enough to be observed by several
analytical methods (Ref. 1).

Figure 1: Traditional representation of N-nitrosamine structure (left) and rotameric structures

(right)
In the two zwitterionic resonance forms the rotation around the nitrogen-nitrogen bond is
restricted, leading to the two identifiable rotamers when R and R’ are not the same. The two
rotamers may differ in their overall dipole moment and therefore may posses different chemical

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24/07/2025, 11:23 Structure of Nitrosamine Drug Impurities and Implications for Chemical Analysis

and physical properties. The relative ratio of the two rotamers of N-nitrosamines depends on their
relative stability. Based on analogy to cis/trans (Z/E) isomerism in asymetric alkenes the rotamers
are sometimes referred to as cis (Z) or trans (E) forms.

Why do I see two species in NMR of some N-

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nitrosamines?

N-nitrosamine rotamers are observed by Nuclear Magnetic Resonance (NMR) spectroscopy. For
example, the two rotamers of N-nitroso methylethylamine show different chemical shifts (Ref 2):
chemical shifts of the methylene protons are 6.4 ppm and 5.8 ppm, for cis and trans forms,
respectively, in a ratio of 8 to 2 cis/trans.

Why do I see two peaks when analyzing N-

Nitrosamines by HPLC or GC?
High Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC) analysis of N-
nitrosamines may show two rotamers of the N-nitrosamine as two separate peaks. Whether two
peaks are observed depends on the structure of the N-nitrosamine (i.e., symmetric vs. asymmetric)
as well as the separation power of the analytical method.
Let’s consider HPLC analysis of seven FDA-identified N-nitrosamine impurities: N-
nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitroso-N-methyl-4-
aminobutanoic acid (NMBA), N-nitrosoisopropylethyl amine (NIPEA), N-nitrosomethyl-
phenylamine (NMPA), N-nitrosodiisopropylamine (NDIPA) and N-nitrosodibutylamine (NDBA).
Four of these compounds, namely NDMA, NDEA, NDIPA and NDBA, are symmetric dialkylamines.
These symmetric nitrosamines are expected to give a single peak since the zwitterionic forms are
equivalent. However, the chromatograms of NMBA, NIPEA and NMPA may show two peaks
resulting from the two stable rotamers. Note that during the chromatographic analysis of these
compounds, resolution of the two configurations may not have been achieved. In addition, the
minor rotamer may be missed during analysis, and only the major form reported.
At Acanthus Research, we have observed a set of two peaks in the analysis of several N-nitroso
derivatives of drug molecules. For example, the HPLC analysis of metopropol N-nitroso showed
two peaks at 8.9 min and 9.4 min; the HPLC trace of ramipril N-nitroso gave two peaks at 15.7 min
and 17.0 min. The proposed stable rotamers of metopropol N-nitroso and ramipril N-nitroso are
shown in Figure 2.

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24/07/2025, 11:23 Structure of Nitrosamine Drug Impurities and Implications for Chemical Analysis

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Figure 2: Stable rotamers of Metoprolol N-nitroso and Ramipril N-nitroso

In the case of metoprolol N-nitroso the rotamers ratio according to HPLC analysis is 13.5 to 86.2. The
two stable rotamers of ramipril N-nitroso appear to be nearly equivalent with a ratio of 47.4 to 50.5.
The HPLC analyses are consistent with NMR characterization.
Other N-nitroso derivatives of API’s that we observed as two separate speaks in HPLC
chromatograms are:
N-Desmethyl Clarithromycin N-Nitroso (https://acanthusresearch.com/products/uncategorized/n-
desmethyl-clarithromycin-n-nitroso/)
Rilpivirine N-Nitroso (https://acanthusresearch.com/products/drug-impurities-reference-
standards/rilpivirine-n-nitroso/)
Enalapril N-Nitroso (https://acanthusresearch.com/products/drug-impurities-reference-
standards/enalapril-n-nitroso/)
Lisinopril N-Nitroso (https://acanthusresearch.com/products/drug-impurities-reference-
standards/lisinopril-n-nitroso/)
Perindopril N-Nitroso (https://acanthusresearch.com/products/drug-impurities-reference-
standards/perindopril-n-nitroso/)

What is the take home message on the structure

of N-nitrosamines?
In short, it is important to consider the possibility of existence of stable of N-nitrosamine rotamers.
Most often the structure has been assumed to consist of a nitrogen-oxygen double bond (N-N=O).
However, in asymmetric secondary amines, restricted rotation around the N-N bond, which
assumes a double bond character, leads to two different stable rotamers, sometimes called trans
and cis isomers. These two forms may be observed by NMR and various chromatographic
techniques.
References:
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24/07/2025, 11:23 Structure of Nitrosamine Drug Impurities and Implications for Chemical Analysis

1. Jessica C. Beard and Timothy M. Swager, An Organic Chemist’s Guide to N‑Nitrosamines:

Their Structure, Reactivity, and Role as Contaminants, J. Org. Chem. 2021, 86, 2037−2057.
2. G. J. Karabatsos, R. A. Taller, Structural Studies by Nuclear Magnetic Resonance. IX.
Configurations and Conformations of N-Nitrosamines, J. Am. Chem. Soc. 1964, 20, 4373-4378.
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