Journal of Pharmaceutical Sciences 110 (2021) 3118−3128

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Journal of Pharmaceutical Sciences

jo urn a l h om ep ag e : w ww.jp harms ci.o rg

Review

Nitrosamine Contamination in Pharmaceuticals: Threat, Impact,

and Control
Bodin Tuesuwan, Vorasit Vongsutilers*
Department of Food and Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Chulalongkorn University, 254 Phayathai Rd., Bangkok
10330, Thailand

A R T I C L E I N F O A B S T R A C T

Article history: Nitrosamine-contaminated medicinal products have raised safety concerns towards the use of various drugs,
Received 15 February 2021 not only valsartan and all tetrazole-containing angiotensin II receptor blockers, but also ranitidine, metfor-
Revised 30 April 2021 min, and other medicines, many of which have been recalled and prone to shortage. At any stages, from drug
Accepted 30 April 2021
substance synthesis throughout each product’s lifetime, these impurities may evolve if an amine reacts with
Available online 11 May 2021
a nitrosating agent coexisting under appropriate conditions. Consequently, drug regulatory authorities
worldwide have established stringent guidelines on nitrosamine contamination for all drug products in the
Keywords:
market. This review encompasses various critical elements contributing to successful control measures
Global health
Regulatory science
against current and upcoming nitrosamine issues, ranging from accumulated knowledge of their toxicity
Mutation concerns and potential root causes, precise risk evaluation, as well as suitable analytical techniques with suf-
Toxicity ficient sensitivity for impurity determination. With all these tools equipped, the impact of nitrosamine con-
Mass spectrometry tamination in pharmaceuticals should be mitigated. An evaluation aid to tackle challenges in risk
Nitrosamine identification, as well as suitable industry-friendly analytical techniques to determine nitrosamines
NDMA and other mutagenic impurities, are among unmet needs that will significantly simplify the risk assessment
Angiotensin II receptor blockers process.
© 2021 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.

Introduction known mutagenic impurities and potential human carcinogens. The

Since July 2018, angiotensin II receptor blockers (ARBs) have been
flagged by regulatory authorities worldwide due to suspected con-
tamination with nitrosamines (or nitrosoamines), which are well
Abbreviations: A, adenine; Asp, aspartic acid; APCI, atmospheric pressure chemical
ionization; API, active pharmaceutical ingredient; ARBs, angiotensin II receptor block-
ers; BP, British Pharmacopoeia; C, cytosine; CPNP, 1-cyclopentyl-4-nitrosopiperazine;
DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; DNAs, deoxyribo-
nucleic acids; EDQM, European Directorate for the Quality of Medicines & HealthCare;
EMA, European Medicine Agency; ESI, electrospray ionization; G, guanine; GC−MS/
MS, gas chromatographs-tandem mass spectrometers; HRMS, high-resolution mass
spectrometer; IARC, International Agency for Research on Cancer; ICH, International
Council for harmonization of Technical Requirements for Pharmaceuticals for Human
Use; LC-MS/MS, liquid chromatographs-tandem mass spectrometers; LOQ, limit of
quantification; MNP, 1-methyl-4-nitrosopiperazine; NDEA, N-nitrosodiethylamine;
NDIPA, N-nitrosodiisopropylamine; NDMA, N-nitrosodimethylamine; NEIPA, N-nitro-
soethylisopropylamine; NMBA, N-nitroso-N-methyl-4-aminobutyric acid; NMP, N-
methyl pyrrolidinone; Ph. Eur., European Pharmacopoeia; Ph. Int., International Phar-
macopoeia; QQQ, triple quadrupole; QTRAP, quadrupole-ion trap; SPE, solid phase
extraction; SPME, solid phase microextraction; T, thymine; Tyr, tyrosine; U.S. FDA, U.S.
Food and Drug Administration; USP, United States Pharmacopeia; WHO, World Health
Organization.
* Corresponding author.
E-mail address: vorasit.v@chula.ac.th (V. Vongsutilers).
first incidence was reported after a concerning amount of N-nitroso-
dimethylamine (NDMA) was detected in valsartan. NDMA has been
shown to be a mutagenic carcinogen in various animal species.1 Cur-
rently, this agent is not only classified as “probably carcinogenic to
humans”2 by the International Agency for Research on Cancer (IARC),
World Health Organization (WHO), but also listed as “reasonably
anticipated to be human carcinogens”3 by the National Toxicology
Program, U.S. Department of Health and Human Services. As a result,
a lot of investigations were extensively undertaken, and the Euro-
pean Medicine Agency (EMA) was the first to announce a recall of
valsartan products in all European countries.4 Within one week, the
U.S. Food and Drug Administration (U.S. FDA) issued a voluntary
recall of several valsartan-containing preparations.5 A few months
later, the U.S. FDA also identified N-nitrosodiethylamine (NDEA) as
the second potentially mutagenic impurity in valsartan products.6 In
contrast, nitrosamine contamination in other sartans had not been
evidenced until March 2019, when the U.S. FDA revealed the exis-
tence of another nitrosamine, N-nitroso-N-methyl-4-aminobutyric
acid (NMBA), in losartan.7 In response to this challenging crisis, both
regulatory agencies and pharmaceutical industries worldwide have
been putting a lot of efforts into the investigations of suspected nitro-
samine contamination in ARB-containing formulations. Based on

https://doi.org/10.1016/j.xphs.2021.04.021
0022-3549/© 2021 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.
 B. Tuesuwan, V. Vongsutilers / Journal of Pharmaceutical Sciences 110 (2021) 3118−3128
investigation findings, N-nitrosodialkylamines, i.e., NDMA and NDEA,
as well as NMBA, are possibly generated during active pharmaceuti-
cal ingredient (API) production processes. For ARB raw materials, the
risks of nitrosamine contamination have been associated with the
tetrazole formation step.8
Recently, the global concern over nitrosamine contamination has
grown beyond ARBs to include the NDMA-contaminated ranitidine,
nizatidine, and metformin as revealed by the EMA and U.S. FDA.9−13
Several ranitidine products contain NDMA above the acceptable daily
intake limit of 0.32 ppm13. Unlike in sartans, the NDMA level in ranit-
idine can be elevated over its shelf life, especially at higher tempera-
tures. On April 1st, 2020, the U.S. FDA and EMA decided to withdraw
ranitidine from the market14,15 after more than three decades of clin-
ical uses.16 In case of metformin, NDMA has been detected in only
some finished products, not the API, suggesting that this contaminant
likely results from the degradation of finished products under certain
conditions.11 In addition, the most recently announced nitrosamine
impurities include 1-methyl-4-nitrosopiperazine (MNP) and 1-cyclo-
pentyl-4-nitrosopiperazine (CPNP) in rifampin and rifapentine,
respectively.17 Structures of medicines with nitrosamine issues and
nitrosamines found in pharmaceuticals are shown in Fig. 1.
While nitrosamines in ARBs could be described as the tip of the
iceberg, their contamination in other medications has also been
continuously reported as mentioned above and raised significant con-
cern about mutagenic impurities in pharmaceuticals. In this article, we
will discuss the toxicity of nitrosamines, as well as the root cause,
impact, and control of nitrosamine contamination in pharmaceuticals.
These lessons learned from the past could serve as a stepping stone to
effective management of emerging contamination issues.
Genotoxicity and Carcinogenicity of Nitrosamines
The nitrosamine contaminants in pharmaceuticals that have been
identified to date are mainly N-nitrosodialkylamines. Their genotox-
icity requires bioactivation through hydroxylation by cytochrome
P450s, particularly CYP2E1 (except for NDEA, which can also be bio-
activated by CYP2A6) .18 As shown in Fig. 2, the resulting a‑hydroxy
nitrosamine spontaneously decomposes into an aldehyde and a cor-
responding alkyldiazohydroxide, which successively gives rise to an
alkyldiazonium ion, followed by an alkyl carbonium ion. Such a
potent alkylating electrophile can then react and form adducts with
intracellular nucleophilic macromolecules, including proteins and
DNA.19−21 For example, methyldiazohydroxide and ethyldiazohydr-
oxide are the metabolically activated intermediates of NDMA and
NDEA, and ultimately get converted to methyldiazonium (Fig. 2) and
ethyldiazonium ions, respectively. These highly electrophilic alkyla-
tors trigger SN2 alkylation at DNA nucleophilic sites.22 The strong
nucleophilicity of the N7 atom in guanine (N7G) makes it a good
accessible and preferred site for any small and diffusible DNA alkylat-
ing agents like alkyldiazonium ions. However, these ions can also
preferentially attack O6 of guanine (O6G) and O4 of thymine (O4T) ,23
the alkylation of which yields chemically stable adducts with poten-
tial mutagenicity and carcinogenicity.24−28 O6-methyl- and O6-ethyl-
guanine adducts can be misread as adenines, thereby inducing G:
C ! A:T translation mutations. On the other hand, O4-alkylthymine
adducts in DNA are recognized as cytosines, thus mispairing with
guanines in DNA replication and causing T:A ! C:G translation
mutations.27,28 In vivo studies have demonstrated that methyl and
ethyl DNA adducts, mainly at N7G as expected, were isolated from
mouse and rat livers upon exposure to either NDMA or NDEA,
although alkylation at N3A, O6G, O2T, and O4T were also observed.
Interestingly, NDEA produced a higher ratio of O6G to N7G adducts
than NDMA did,23 indicating different distributions of these nitros-
amines among various alkylation sites on guanines.
3119
Besides the genotoxic effects of alkyldiazonium ions, the alde-
hydes simultaneously generated during of N-nitrosodialkylamines
bioactivation have also been shown to form adducts with DNAs.29
For example, a metabolite of NDMA is formaldehyde, which is classi-
fied by WHO/IARC as “carcinogenic to humans”30 and listed as
“known to be human carcinogens” by the National Toxicology Pro-
gram, U.S. Department of Health and Human Services.3 After a subcu-
taneous injection of NDMA, a formaldehyde adduct and a methylene
cross-link were identified as N6-hydroxymethyl-20 -deoxyadenosine
and di-(N6-deoxyadenosyl)-methane (Fig. 2), respectively, in rat liver
and lungs.31 Hence, NDMA and other N-nitrosodialkylamines could
be considered as bident carcinogens due to their ability to alkylate
DNA through both alkyldiazonium ions and aldehydes.31,32 In addi-
tion to DNA alkylation, NDMA and NDEA were also able to form reac-
tive oxygen species in Caco-2 cells, leading to deregulated gene
expression.33
Since 1978, the IARC has collectively published the carcinogenic
effects of NDMA and NDEA that were observed in animals.1 NDEA-
exposed animals have been established as a suitable experimental
model of hepatocellular carcinoma.34 Moreover, some experimental
data in rat models have shown that ethanol ingestion could induce
metabolic proteins like CYP2E1, leading to an increase in NDMA- and
NDEA-mediated DNA alkylation.35,36 Thus, the genotoxicity of N-
nitrosodialkylamines can be augmented by alcohol-induced CYP2E1.
Based on such evidence and the similarity of N-nitrosodialkylamine
biotransformation in human and rodent tissues, the IARC has classi-
fied NDMA and NDEA as probable human carcinogens (Group 2A) .1
According to the ICH Harmonised Guideline M7(R1), a lifetime intake
of N-nitrosamines at the interim-limit levels would cause less than
one additional case of cancer for every 100,000 population.37 After
extensive data review, the EMA has deduced that if 100,000 patients
had taken the maximum dose of valsartan contaminated with the
highest level of NDMA (based on the NDMA contamination data
reported in 2012 and estimated until 2018) daily for full six years,
this could result in 22 additional cases of cancer over their lifetime. In
the same manner, the EMA has concluded that in 100,000 patients
who had taken the largest dose of NDEA-containing valsartan (based
on the NDEA contamination data reported in 2014 and estimated till
2018) for the whole four years, there could be 8 extra cases of can-
cer.38 In contrast, a cohort study in Danish patients who had poten-
tially taken NDMA-contaminated valsartan suggested that there was
no correlation with the increased overall risk of cancer (the adjusted
hazard ratio was 1.09 with a 95% confidence interval of 0.85 to 1.41)
in these users.39 However, the duration of this study was too short to
evaluate lifetime events, and thus, a continuous monitoring program
would be beneficial.
Source of Nitrosamine Contamination in Pharmaceuticals
Nitrosamine impurities in food and water have been extensively
studied in terms of their formation, detection, and control. Secondary
amines, tertiary amines, and other amine derivatives, such as cyana-
mides, guanidines, amidines, hydroxylamines, hydrazines, hydra-
zones, and hydrazides have been reported to be nitrosamine
precursors.40 Nitrosation of these compounds with nitrous acid
(HNO2) or nitrite (NO2 ) under acidic conditions represents a common
pathway of nitrosamine formation as illustrated in Fig. 3A, whereas
nitrosation of primary amines yields unstable monosubstituted
nitrosamines which spontaneously decompose. In the presence of O2,
other nitrosating agents, e.g., nitrosyl chloride (NOCl), nitrogen
oxides (N2O3, N2O4), nitrosonium tetrafluoroborate (NOBF4) ,40 and
nitric oxide (NO) ,41 have also been reported to produce nitrosamines.
Contamination of nitrosamines in pharmaceuticals is suspected to
occur when a precursor amine coexists with a nitrosating agent
under nitrosation conditions, such as acidic conditions for nitrite.
3120 B. Tuesuwan, V. Vongsutilers / Journal of Pharmaceutical Sciences 110 (2021) 3118−3128

Figure 1. Chemical structures of the medicines with nitrosamine issues and various nitrosamine impurities reportedly identified in pharmaceuticals to date.
 B. Tuesuwan, V. Vongsutilers / Journal of Pharmaceutical Sciences 110 (2021) 3118−3128
Figure 2. Bioactivation of nitrosamines to reactive species and the major nucleobase adducts subsequently formed.
Therefore, investigations have been conducted to identify the root
cause(s) of nitrosamine contamination in pharmaceuticals.
Angiotensin II Receptor Blockers
The synthetic routes for ARB drug substances, whose structures
are provided in Fig. 1, generally include multistep reactions to fur-
nish biphenyl analogs with an acidic center, such as the tetrazole
ring present in five sartans, including valsartan, losartan, irbesar-
tan, olmesartan, and candesartan. This acidic functional group and
also a carboxyl moiety in ARB structures have been suggested to
mimic the Tyr4 phenol or the Asp1 carboxyl group of angioten-
sin,42 and hence, appear to be crucial for their pharmacological
activity. Between the two, however, a biphenyl tetrazole offers
superior metabolic stability and bioavailability. It is therefore
included in a majority of ARB molecules currently available in the
market, with the exception of eprosartan, telmisartan, and azilsar-
tan that contain a carboxyl substituent instead (Fig. 1). Unfortu-
nately, installation of a tetrazole itself has been proposed to be the
root of nitrosamine contamination.8 In one step, a biphenyl nitrile
can be easily transformed into a biphenyl tetrazole via an addition
reaction with an azide,43 e.g., sodium azide (NaN3), trimethyltin
azide (Me3SnN3), or tributyltin azide (Bu3SnN3) (Fig. 4). Due to
their explosive nature and human toxicity potential, the unreacted
azides are commonly quenched with nitrite under acidic condi-
tions, upon which nitrogen gas and nitrous oxide are released.44
Unfortunately, if some trace dialkylamines also exist as either
residuals from upstream steps or contaminants in other reagents,
they can readily react with nitrite under acidic conditions to gen-
erate nitrosamines. Consequently, when all the contributing fac-
tors to nitrosamine formation coincide in ARB syntheses,
previously unforeseen risks of carcinogen contamination emerge
and have caused major worldwide recalls as described previously.
In case of valsartan, its tetrazole ring was reportedly formed using
Bu3SnN3 as the azide source,45 whereas N,N-dimethylformamide
3121
(DMF) was also employed in the synthetic route. Being both a com-
mon precursor and a possible degradation product of DMF, dimethyl-
amine plausibly exists as an impurity in this solvent and serves as an
amine source for NDMA, the first nitrosamine that led to valsartan
recalls. Under certain circumstances, triethylamine is intentionally
used as an amine source in valsartan synthesis. If this reagent is con-
taminated with diethylamine, it could give rise to NDEA, the second
nitrosamine detected in valsartan. Considering the root cause of
NDMA and NDEA contamination in valsartan, it is expected that
other tetrazole-containing ARBs are prone to similar risks. Several
synthetic routes of losartan involve the use of DMF and require NaN3
for tetrazole formation.46 In addition, the synthetic pathways of
irbesartan47,48 and candesartan49 also rely on azide−nitrile coupling
to form tetrazole, and thus, similar risk factors for nitrosamine con-
tamination have been suggested.
Apart from NDMA and NDEA, ARB raw materials could also
contain other nitrosamine species, depending on the dialkylamine
sources. Further examinations by drug manufacturers and regula-
tory authorities have revealed possible contamination with N-
nitrosodiisopropylamine (NDIPA), N-nitrosoethylisopropylamine
(NEIPA) and, as mentioned previously, NMBA. Although NDIPA
and NEIPA have been suggested to originate from N-ethyl-N,N-dii-
sopropylamine (also called N,N-diisopropylethylamine or DIPEA), a
tertiary amine reportedly used in valsartan synthesis and possibly
contaminated with N,N-diisopropylamine and N-ethyl-N-isopro-
pylamine, the emergence of NMBA contamination is conversely
not as straightforward. While the reason could be N-methyl pyrro-
lidinone (NMP), an organic solvent reported in the synthetic routes
of some ARBs such as losartan,50 NMP itself is not reactive. How-
ever, its hydrolysis product, N-methyl amino-N-butyric acid, as
described in Fig. 3B, is a secondary amine that can react with
nitrite under acidic conditions to form NMBA. With current infor-
mation regarding the risks of process-related contamination, addi-
tional nitrosamine species can be sensibly expected due to the
diversity of amines used in ARB syntheses.
3122 B. Tuesuwan, V. Vongsutilers / Journal of Pharmaceutical Sciences 110 (2021) 3118−3128

Figure 3. Formation of nitrosamines in pharmaceuticals. Common factors contributing to nitrosamine formation (A) and examples of nitrosamine formation in ARBs (B) are provided.

Ranitidine and Nizatidine involves nucleophilic substitution of monochloramine (NH2Cl) by the

Unlike ARBs, the issue of nitrosamine contamination in ranitidine
remains unclear,8 and unfortunately, cannot be directly explained by
a careful analysis of its synthetic routes. Nevertheless, this H2-blocker
has been regarded as a possible culprit of nitrosamine contamination
in water supplies by serving as an NDMA precursor during chlora-
mine disinfection of water.51 The mechanism of NDMA formation
dimethylamine moiety in ranitidine to yield a hydrazine intermedi-
ate, which is further oxidized to produce NDMA.51,52 Several studies
have reported ranitidine as the most reactive precursor of
NDMA.51,53,54 In addition, oral intake of this drug appeared to cause
elevated levels of NDMA in urine.55 When the stability of some raniti-
dine products was tested by the U.S. FDA, a correlation existed
between the NDMA levels and expiration dates.56 Based on these

Figure 4. Nitrosamine generated during tetrazole formation in ARB synthesis (A) along with some examples of losartan synthetic routes prone to nitrosamine formation (B).
 B. Tuesuwan, V. Vongsutilers / Journal of Pharmaceutical Sciences 110 (2021) 3118−3128
reported findings both in vitro and in vivo, the degradation process of
ranitidine is thus a potential root of NDMA contamination. However,
the main question still remaining is the source of nitrosating agents
that react with ranitidine. One possibility is that, during its synthesis,
ranitidine might be contaminated with a trace amount of nitrosating
agents which then react with drug molecules over time to yield
nitrosamines. Alternatively, the intrinsic instability incurred by the
nitro functionality in ranitidine is also suspected to cause nitrosation.
This speculation relies on the report by Bauer and Fung57 that nitric
oxide was released from nitro-containing compounds upon light
exposure, in conjunction with the NO/O2 triggered nitrosation
described by Itoh et al.41 On the contrary, some nitro compounds,
such as bromonitromethane58 and nitromethane,59 have been dem-
onstrated to mediate direct nitrosation of secondary and tertiary
amines, albeit with the requirement of catalysts or high temperatures
to yield nitrosamines. Nizatidine, an isostere of ranitidine with simi-
lar dimethylamine and nitro substituents, was also recalled following
the detection of NDMA,60,61 further substantiating the presumption
that the instability of H2-blockers with a nitro group could lead to
nitrosamine formation. In-depth investigations into the mechanism
behind NDMA formation will be required to elucidate the cause of
nitrosamine contamination in both ranitidine and nizatidine.
NMDA Contamination in Other Medicines
Due to the detection of nitrosamines in ARBs, substantial investi-
gations by regulatory authorities and drug manufacturers have been
expanded to other suspected medicines. The EMA has reported the
detection of NDMA at acceptable trace levels in a few batches of pio-
glitazone manufactured by Hetero Labs in India.62 In contrast, Health
Canada has issued a voluntary recall of metformin from several com-
panies led by Apotex Inc. as a result of NDMA contamination above
its acceptable limit in many batches of finished products.63 Although
such problem was not detected in any batches of metformin investi-
gated by the U.S. FDA64 and EMA,12 this issue has been under close
monitoring by both authorities. In the presence of alkali or heat, as
well as under high-pressure oxidative conditions, metformin can
decompose via oxidation to give rise to NDMA.65 Therefore, assess-
ment of nitrosamine contamination in pharmaceuticals beyond ARBs
is now expected to be a common practice to ensure their safety and
minimize the consumer’s risks.
Quantitative Analysis of Trace Nitrosamines in Pharmaceuticals
Since the events of N-nitrosamine contamination in sartans
emerged, various regulatory authorities and research groups have
established a number of analytical methods to quantify the N-nitro-
sodialkylamines potentially contaminated in both drug substances
and drug products by using gas chromatographs-tandem mass spec-
trometers (GC−MS/MS) with either a headspace system66−68 or
direct injection,69 liquid chromatographs-tandem mass spectrome-
ters (LC-MS/MS) ,70−72 and liquid chromatographs-UV detectors.73
One of the greatest challenges is to obtain accurate and reliable
results as these impurities are present in such minute amounts. Anal-
ysis of ppm-level, low-molecular-weight contaminants like nitros-
amines in pharmaceuticals without meticulous quantitative
analytical methods could come up with inaccurate results. For
instance, Keire’s team at the U.S. FDA demonstrated in detail that
when NDMA in metformin was analyzed, DMF co-eluted with NDMA.
Without sufficient mass accuracy in data acquisition or sufficient
mass tolerance in data analysis, the quantity of NDMA could be
overcalculated.74
Among more than forty currently available methods, only those
providing a Limit of Quantification (LOQ) at or less than the technical
limit of 0.03 ppm are discussed herein as summarized in Table 1.
3123
The LC-MS methods published with an LOQ at or below the technical
limit all employed reversed-phase LC coupled with a high-resolution
mass spectrometer (HRMS) or MS/MS. Mass detection was achieved
by using a quadrupole-ion trap (QTRAP) or a triple quadrupole (QQQ)
mass analyzer with either electrospray ionization (ESI) or atmo-
spheric pressure chemical ionization (APCI) in positive mode (except
only some methods for NMBA that used negative mode). For GC−MS
procedures with qualified LOQs, samples were analyzed via either
direct injection or headspace sampling, followed by triple quadrupole
or ion trap mass detection.
Overall, the lowest LOQs in sub-ppb range have been achieved by
€
SOrgel et al. who employed LC-MS/MS to quantify NDMA and NDEA
in the finished products of 8 sartans.75 For this class of drugs, other
LC-MS/MS-based methods have been released by the European
Directorate for the Quality of Medicines & HealthCare (EDQM) and
Taiwan FDA.70,76,77 Alternatively, a number of GC−MS methods pub-
lished by the U.S. FDA,78 Health Canada,68 and EDQM (by Swiss-
medic)79 are also available and offer LOQs for various nitrosamines in
the range of LOQs of 0.005−0.025 ppm. Similarly, the quantification
of NDMA in metformin could be performed by either LC-MS76,80 or
GC−MS81−83 with the LOQs less than or equal to 0.03 ppm.
In case of NDMA contamination in ranitidine, LC-MS/MS is a pre-
ferred method due to thermal decomposition of this drug to form
NDMA. The U.S. FDA presented two NDMA analytical procedures for
ranitidine API and products with an LOQ at 0.033 ppm; one uses
LC HRMS (QTRAP)84 while the other is based on LC-MS/MS (triple
quadrupole with APCI) .85 However, by effective sample clean-up, for
instance, solid phase extraction (SPE) and solid phase microextrac-
tion (SPME), to remove interferences and to avoid exposure of raniti-
dine to high temperature, GC−MS may be used to accurately quantify
NDMA in ranitidine. A headspace-SPME-GC−MS method,86 as well as
an SPE GC−MS/MS method with isotope dilution (LOQ 0.0009 ppm)87
have been shown to produce comparable results to those from LC-
MS/MS.
In the near future, the United States Pharmacopeia (USP) will pub-
lish a new General Chapter h1469i Nitrosamine Impurities, which is
currently in the Pharmacopeial Forum PF 46(5) .88 There are four ana-
lytical procedures, including one LC HRMS (QTRAP with APCI+), one
HPLC-MS/MS (QQQ with APCI), and two GC−MS/MS (triple quad)
methods, for ARB drug substances. While verification usually suffices
for these standard and published methods since they have already
been validated, all newly developed procedures and modified stan-
dard methods require appropriate validation. Both method validation
and verification need to be performed by using the matrix compo-
nents most relevant to sample. In some cases, additional steps of
sample clean-up may be required to avoid matrix interferences and
ion suppression. Use of isotope-labeled internal standard has also
been considered in some methods. In addition, every laboratory must
establish their own LOQs that comply the technical limits for all ana-
lytes to be tested by each method.
Impact and Control Measures for Nitrosamine Contamination
The ARB crisis, ranitidine withdrawal, and subsequent drug recalls
worldwide due to nitrosamine contamination have caused such a
huge impact that not only accountability but also significant
improvement of pharmaceuticals are expected from all stakeholders,
including regulatory agencies, pharmaceutical manufacturers, and
healthcare professionals.
Risk evaluation
Currently available information regarding nitrosamine contami-
nation in pharmaceuticals suggests that this issue can stem from
such a very early stage of drug production as in the procurement of
3124 B. Tuesuwan, V. Vongsutilers / Journal of Pharmaceutical Sciences 110 (2021) 3118−3128

Table 1
Summary of Analytical Methods for Nitrosamine Determination in Pharmaceuticals.

Nitrosamine Drug Sample Type Analytical Method LOQ (ppm) Authority/ Ref.
Research group

NDMA Valsartan DPa LC-MS/MS 0.00026 (NDMA) €

SOrgel 84
NDEA Losartan (APCI+, QTRAP, MRM) 0.00013 (NDEA)
Irbesartan
Candesartan
Telmisartan
Olmesartan
Eprosartan
Azilsartan
NDMA All Sartans DSb, DP LC-MS/MS 0.02 Taiwan FDA 76
NDEA Metformin (APCI+, MRM)
NDEA Irbesartan DS, DP LC-MS/MS 0.0195 EDQM 77
(APCI+, QTRAP, MRM)
NMBA Losartan DS LC-MS/MS 0.0286 EDQM 70
(APCI, QTRAP, MRM)
NDMA Metformin DS, DP LC HRMS 0.03 U.S. FDA 80
(ESI+, QTRAP, PRM)
NDMA Valsartan DS, DP GC−MS/MS DS: U.S. FDA 78
NDEA (EI, QQQ, MRM) 0.008 (NDMA)
NEIPA 0.005 (NDEA)
NDIPA 0.005 (NEIPA)
NDBA 0.005 (NDIPA)
0.025 (NDBA)
DP:
0.013 (NDMA)
0.008 (NDEA)
0.008 (NEIPA)
0.008 (NDIPA)
NDMA Valsartan DS, DP GC−MS/MS 0.015 EDQM 79
NDEA Losartan (QQQ, MRM) (by Swissmedic)
NEIPA Irbesartan
NDIPA Olmesartan
NDBA Candesartan
NDPA
NDMA All Sartans DS, DP GC−MS/MS 0.0054 (NDMA) Health Canada 68
NDEA (EI, QQQ, MRM) 0.0073 (NDEA)
NDMA Sartans DS, DP SPE GC−MS/MS 0.0009 (NDMA) H.-S. Shin 87
NDEA Metformin (ESI+, QQQ, MRM) 0.0003 (NDEA)
Ranitidine
NDMA Metformin DS, DP GC−MS 0.025 EDQM 81
(Quadrupole, SIM)
NDMA Metformin DS, DP GC−MS/MS 0.015 EDQM 82
(EI, QQQ, MRM) (by CVUA Karlsruhe)
NDMA Metformin DP HRAM-GCMS 0.02 Singapore HSA 83
(EI, Orbitrap, SIM)
a
drug product.
b
drug substance.

starting materials, reagents, and solvents for API synthesis through-
out the shelf-life of finished products in the market, during which
degradation may occur under storage conditions. Based on the prob-
lem’s nature, it is most likely that several nitrosamine issues remain
to be discovered in other drugs, possibly with different root causes
from what we have observed so far. Therefore, a preemptive risk
evaluation of potential issues is considered mandatory.
Risk assessment of API production represents a practical starting
point for pharmaceutical industries to evaluate the risks and levels of
nitrosamine contamination potentially occurring during the process.
As mentioned previously, tetrazole formation has been pinpointed as
the critical step in the synthetic routes of ARBs. Unfortunately,
detailed synthetic methodologies are not always readily available
depending on the regulatory policy in each country. Based on the
ASEAN Common Technical Dossier,89 synthetic routes are not
required for generic drug registration. Therefore, there is no obliga-
tion for drug manufacturers to obtain API synthesis information. To
avoid delays in dealing with drug contamination issues, synthetic
routes should under no circumstances be omitted from drug registra-
tion. Apparently, this essential piece of information needs to be ana-
lyzed by drug manufacturers in order for both identification of
potential drug impurities and subsequent justification of product
specifications.
Nevertheless, evaluation of synthetic routes may prove to be
insufficient to serve as a sole means for risk minimization. For
instance, even when an ARB synthetic pathway shows low risk of
nitrosamine generation, these impurities can still be cross contami-
nated from some other manufacturing processes concurrently ongo-
ing at the same production site. Furthermore, it is often challenging
to identify trace amines, a mandatory piece of jigsaw in nitrosamine
formation, especially when their existence in various reagents and
solvents is not strictly controlled in common logistic processes.
Therefore, additional control measures may be necessary, including
the establishment of impurity limits for chemicals and reagents to be
used in synthesis. For example, a limit of dimethylamine in DMF, a
solvent commonly used in chemical synthesis, would be beneficial as
this impurity is a precursor of NDMA. This notion is unavoidably
applicable to finished products since the sources of nitrosamine for-
mation may derive from excipients, which should also be controlled
and included in risk evaluation. In addition, degradation of drug mol-
ecules to nitrosamines or nitrosamine precursors is another concern
that needs to be evaluated.
 B. Tuesuwan, V. Vongsutilers / Journal of Pharmaceutical Sciences 110 (2021) 3118−3128
That having been said, risk prioritization is very important since it
is highly possible that nitrosamine contamination also afflicts several
other drugs. In September 2019, the EMA advised drug companies to
evaluate the possibility of nitrosamine contamination in all con-
cerned medicines within 6 months.90 Tetrazole-containing drugs
rank in the top list due to the probability to have nitrosamine con-
tamination from tetrazole formation as observed with ARBs. Any
nitrosamine precursors that might be present in drug substances,
either the drug molecule itself or impurities, with possible exposure
to nitrosation conditions would pose high concern. In addition, vari-
ous determinants of patients’ exposure should also be taken into con-
sideration, e.g., maximum daily dose of drug, duration of treatment,
therapeutic indications, and volume of drug usage. Once all the risks
have been identified, laboratory testing to investigate the levels of
nitrosamine contamination is a subsequent step. Afterwards, some
process changes, such as control of materials, API synthesis, as well
as drug product formulation, manufacturing process, packaging, and
storage conditions may be necessary to eliminate or at least minimize
nitrosamine contamination. Stepwise scientific approaches to tackle
nitrosamine issue in pharmaceuticals is described in Fig. 5.
While current recommendations from regulatory authorities
serve as useful guidance for pharma industries on how to deal with
nitrosamine contamination, risk identification remains one of the
biggest challenges in the risk assessment towards this global concern.
At a minimum, extensive knowledge in organic chemistry, to be spe-
cific − nitrosation of amines, would be required to probe the poten-
tial of nitrosamine formation in individual processes. Although
various sources exist to address the chemical reactions that could
lead to formation of nitrosamines in pharmaceuticals,91,92 a well-
rounded and ready-to-use database would be invaluable to predict
nitrosamine generation. Furthermore, some other mutagenic species
are also described in ICH M7 and require similar measures to those
suggested for nitrosamines, thus emphasizing the need for the pre-
diction tool to be comprehensive for various chemical species.
Determination of nitrosamines in drug substances, excipients, and
drug products provides solid evidence for nitrosamine risk. Nitrosa-
mine detection mainly relies on mass spectrometry coupled with var-
ious chromatography techniques as described in the previous section.
Although the established mass spectrometry-based techniques have
proven to be selective, sensitive, and sufficiently effective for known
nitrosamines, screening of potential unknown contaminants can be
challenging. In addition, due to the destructive nature of mass spec-
trometry, false positive results or overestimation of nitrosamines can
be observed, especially during method development, and may cause
delayed lead time in the assessment process. Lastly, the affordability
and accessibility of LC-MS or GC−MS instruments could be an issue
for pharmaceutical companies worldwide. Therefore, a more cost-
friendly analytical tool with at least equally powerful screening
potential represents an unmet need for nitrosamine analysis.
Research and development into this area will greatly simplify nitrosa-
mine risk assessment.
Figure 5. Scientific approaches to tackle nitrosamine contamination in pharmaceuticals.
3125
Control of Nitrosamine Impurities
Control of impurities in drug substances, excipients, and drug
products usually follow the specifications published in official phar-
macopeias, e.g., the United States Pharmacopeia (USP), British Phar-
macopoeia (BP), European Pharmacopoeia (pH. Eur.), or International
Pharmacopoeia (pH. Int.), depending on regional or country policies.
In addition, the International Council for harmonization of Technical
Requirements for Pharmaceuticals for Human Use (ICH) quality
guidelines Q3A, Q3B, Q3C, and Q3D provide comprehensive guidance
and scientifically cover an array of impurities, including both organic
and inorganic impurities, as well as residual solvents, the concept of
which has been adopted by most pharmacopeias. However, the speci-
fications published in pharmacopeial monographs are far from per-
fect, especially in terms of impurity control since most monographs,
even those recently released ones, have been designed to control
organic impurities by using conventional techniques like HPLC with-
out the capability to detect trace amounts of nitrosamines. Although
ICH has published M7 guidelines37 to assess and control mutagenic
impurities, including nitrosamines in pharmaceuticals, there is a still
great challenge for drug manufacturers and regulatory authorities to
establish control measures for mutagens in pharmaceuticals. Identifi-
cation and quantification of the previously described mutagen spe-
cies are not straightforward and require exhaustive investigations as
well as state of the art analytical facilities that can quantify trace
amounts of impurities. In order to control the contamination of
nitrosamines and actually any other types of impurities in pharma-
ceuticals, drug manufacturers need to understand the limitations of
pharmacopeial monographs and start to implement their own scien-
tific evaluation of the control measures.
Analysis of nitrosamines in both APIs and drug products serves as
a conclusive measure to accurately and precisely determine their
contamination levels. In this regard, validity of the analytical meth-
ods for detecting trace amounts of the carcinogens would be the
greatest challenge. During analysis, both in situ generation of nitros-
amines and their contamination from reagents, e.g., water, must be
prevented. As stated in the U.S. FDA’s letter to Valisure LLC,56 unsuit-
able test methods can cause artifactual levels of NDMA found in ranit-
idine. Analytical methods that can detect diverse nitrosamine species
will be an asset to tackle any risks associated with the synthetic
routes. Then, if toxicity data of the detected nitrosamines is not avail-
able, the toxicity levels for their mutagenicity and carcinogenicity
can be characterized and used to assign their acceptable limits. Addi-
tionally, the permissible limits of nitrosamines reported by the EMA
and U.S. FDA are now based on individual chemicals. If their synergis-
tic effects have to be considered as described in ICH M7, it is possible
that such limits may be strengthened in the future in case that more
than one species of nitrosamines are detected. Due to the severity of
carcinogen contamination issues, analysis of N-nitrosodialkylamines
may need to be eventually integrated into drug specifications for rou-
tine quality control. We may see monograph revision to add
3126 B. Tuesuwan, V. Vongsutilers / Journal of Pharmaceutical Sciences 110 (2021) 3118−3128
nitrosamine analysis in high risk drugs, such as those of tetrazole-
containing ARBs in pH. Eur. .8 Nevertheless, risk evaluation to assign
specific criteria for individual manufacturers should be practiced
since there could be different types of contaminated nitrosamines,
which cannot be thoroughly addressed in any pharmacopeial
monographs.
For regulatory authorities, the incidence of ARBs has challenged
their crisis management related to drug regulation system. Investi-
gations for the root cause against time have been conducted in
collaboration with drug manufacturers to identify the origin of
nitrosamine contamination. The fact that ARB drug products have
been recalled worldwide has resulted in a great deal of concern
over possible drug shortage. Therefore, the regulatory agencies are
responsible for regulating the intricate balance between patients’
risks of exposure to probable human carcinogens and blood pres-
sure control failure due to ARB shortage. Obviously, the benefit of
ARB discontinuation is outweighed by the risk, urging the regula-
tors to find suitable means to manage the circumstance. As a short
term strategy, on February 2019, the U.S. FDA extended the
interim limit of NMBA in losartan from 0.96 ppm to 9.82 ppm for
a six-month period to avoid losartan shortage.93 Nevertheless,
their ultimate goal is to ensure that the ARBs marketed in the
United States must be free of nitrosamine contamination. In case
of ranitidine with a more complicated root of contamination simi-
larly affecting most products in the market, withdrawal or at least
suspension of ranitidine products are current actions until the
problem is resolved.
Healthcare providers have also played a crucial role during the
nitrosamine crisis to guide their patients through unsettling drug
recalls by using clear and professional communications to keep
patients’ concerns in check. The most important task for physicians
and pharmacists is to ensure that patients adhere to their treatment.
If necessary, switching to another molecule or class of medications
can also be an alternative under supervision of related healthcare
professionals.
Conclusion
From the previously unforeseen causes leading to nitrosamine
contamination in ARBs, ranitidine, and other reported drugs, all
stakeholders have experienced and learnt to tackle the crisis. Consid-
ering the nitrosamines detected in ARBs and ranitidine, this impurity
issue can also occur to or has already been observed with other phar-
maceuticals. Thus, similar challenges can be reasonably predicted to
arise in the near future, and a number of control strategies could be
implemented as preventive measures. For instance, the industries
can perform preemptive risk assessment of potential sources of
nitrosamines and any other mutagenic impurities that may exist in
their products, as well as thorough analysis, and risk mitigation if any
contaminants are present at concerning levels. In parallel, the regula-
tory agencies themselves must also be prepared to deal with the
diversity of these mutagens. To assist both the review process and
crisis management, risk analysis and control measures of mutagenic
contaminants should be included in drug registration dossiers. Unfor-
tunately, the synthetic routes and in-process controls of drug sub-
stances, which may be one of the root causes of contamination, are
not required for either generics registration or variation submission
in some countries. Pharmaceutical manufacturers and regulatory
agencies will have to exercise these proactive measures to evaluate
the risk of mutagenic impurities in medicines as worldwide societies
now expect better assurance on drug quality. In fact, any policies to
be enforced should be applied to not only new submissions, but also
the medicinal products already approved without any mutagenic
impurity data.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
This work was supported by Chulalongkorn University (CU-
GIF_62_05_33_01).
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