Official: Á1469Ñ Nitrosamine Impurities

Printed on: Mon Jun 06 2022, 09:39:37 AM(EST) Official Status: Currently Official on 06-Jun-2022 DocId: 1_GUID-C97F817C-A383-4693-8E0C-2F0A0A371977_2_en-US
Printed by: Basoalto Javier Official Date: Official as of 01-Dec-2021 Document Type: GENERAL CHAPTER @2022 USPC
DOI Ref: peb3u DOI: https://doi.org/10.31003/USPNF_M15715_02_01
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á1469ñ NITROSAMINE IMPURITIES
1. INTRODUCTION
2. NITROSAMINE IMPURITIES
3. SOURCES OF NITROSAMINES
3.1 Nitrosamine Formation Reaction
4. NITROSAMINE RISK ASSESSMENTS—DEVELOPMENT OF A CONTROL STRATEGY
5. LIMITS OF NITROSAMINES
5.1 Derivation of AI Limits
5.2 Example Calculations of Nitrosamine Limits
6. TESTING FOR THE PRESENCE OF NITROSAMINES
6.1 Presence of Two or More Nitrosamines
7. TEST METHOD PERFORMANCE CHARACTERISTICS OF NITROSAMINE METHODS
7.1 Considerations for Sample Preparation
8. ANALYTICAL PROCEDURES
8.1. Quantitative Procedures
8.2. Limit Test Procedures
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9. ADDITIONAL SOURCES OF INFORMATION
10. USP REFERENCE STANDARDS
REFERENCES
1. INTRODUCTION
The presence of nitrosamine impurities has been detected recently in several drug substances and drug products. In 2018,
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N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) were detected in some valsartan drug substances and
the drug products manufactured from drug substances using specific synthetic routes. This observation triggered extensive
synthetic route assessments and development of analytical procedures to quantify these two nitrosamine impurities. As
additional pharmaceuticals were evaluated and, in some cases tested, other nitrosamines beyond NDMA and NDEA were
added as impurities of concern. Given the potentially broad implications of the presence of carcinogenic members of this
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class of chemicals, this chapter has been developed to provide a science- and risk-based approach for the control of
nitrosamine impurities to ensure that the potential presence of nitrosamines in drug substances and drug products is
identified, assessed, and controlled.
Recommendations are provided regarding: a) the establishment of controls of nitrosamine levels in order to ensure their
elimination or reduction; and b) analytical procedure performance characteristics for procedures used to monitor nitrosamine
levels.
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2. NITROSAMINE IMPURITIES
Nitrosamines addressed in this general chapter are listed in Table 1 by their common names and chemical names. This list is a
compilation of the information shared by multiple global health authorities. As additional nitrosamines are identified as
potential concerns, the principles described herein should be applied for the assessment of these nitrosamines. If a
manufacturer finds a nitrosamine not listed in Table 1, the appropriate regulatory authority should be contacted for
determining appropriate AI limits. The potential presence of any one or more of these impurities is dependent on the reaction
chemistries and processes. The list of nitrosamines is not intended to be exhaustive but represents those that have been
observed and communicated by regulators and manufacturers as being potentially present or observed.
N-nitroso compounds are among the structural groups of high potency mutagenic carcinogens in several animal species, and
some are classified as probable or possible human carcinogens referred to as the “cohort of concern" in ICH M7: Assessment
and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk (1), a designation
that carries with it a recommendation to control the impurities at or below the acceptable cancer risk. As a result of the
potential toxicity associated with these impurities, it is recommended to take steps to control and limit their presence in
pharmaceutical materials.
Table 1. Nitrosamines Found as Contaminants in Drug Substances and Drug Products

Common Name and
Chemical Name Acronym CAS # Structure Chemical Formula Molecular Weight
Nitrosodimethylamine
N-Methyl-N-nitrosome-
thanamine NDMA 62-75-9 C2H6N2O 74.08
https://online.uspnf.com/uspnf/document/1_GUID-C97F817C-A383-4693-8E0C-2F0A0A371977_2_en-US 1/15
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Table 1. Nitrosamines Found as Contaminants in Drug Substances and Drug Products (continued)
Common Name and
Chemical Name Acronym CAS # Structure Chemical Formula Molecular Weight
Nitrosodiethylamine
N-Ethyl-N-nitrosoe-
thanamine NDEA 55-18-5 C4H10N2O 102.14
Nitrosodiisopropylamine
N-Isopropyl-N-nitrosoi-
sopropylamine NDIPA 601-77-4 C6H14N2O 130.19
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Nitrosoethylisopropyla-
mine
N-Ethyl-N-nitroso-
2-propanamine NEIPA 16339-04-1 C5H12N2O 116.16
Nitrosodibutylamine
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N-Butyl-N-nitroso-1-bu-
tanamine NDBA 924-16-3 C8H18N2O 158.25
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Nitrosomethylphenyla-
mine
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N-Methyl-N-nitroso-
phenylamine NMPA 614-00-6 C7H8N2O 136.15
Nitrosomethylaminobu-
tyric acid
4-[Methyl(nitroso)ami-
no] butanoic acid NMBA 61445-55-4 C5H10N2O3 146.15
3. SOURCES OF NITROSAMINES
There are a number of pathways by which nitrosamines can be introduced into or generated as impurities in pharmaceutical
drug products. Specifically, nitrosamines are formed by chemical reaction of secondary or tertiary amines with nitrites (the
latter via intermediate degradation) under acidic conditions (see 3.1. Nitrosamine Formation Reaction). Some examples of the
reported sources or pathways leading to the generation of nitrosamines identified empirically or reported in the literature
(2–3) include (but are not limited to) the following:
• Drug substance processing under specific conditions and in the presence of certain reagents, solvents, raw materials,
and processing aids. There is evidence that despite processing and purification steps, reactive species, whether
intentionally added to or formed during the process/reaction sequence (e.g., nitrites and secondary amines in the
presence of acidic conditions), can carry over to subsequent steps (see 3.1 Nitrosamine Formation Reaction). Special
attention should be given to the formation of nitrogen-containing heterocycles by employing azide followed by
quenching with nitrous acid to remove excess azide.
• The drug substance itself, which may degrade under some conditions resulting in the formation of nitrosamines (e.g.,
ranitidine).
• Degradation of solvents (e.g., dimethylformamide [DMF]) leading to the formation of dialkyl amines.
• Impurities in raw materials, solvents (including recycled solvents), reagents, or catalysts.
• Impurities in materials and intermediates, reagents, and solvents used to prepare the starting materials or intermediates.
• Impurities in water, excipients, or processing aids used in the production of the finished drug product.
• During drug product manufacture under certain reaction conditions and in the presence of requisite precursors
necessary for the formation of nitrosamines.
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• Impurities in the container–closure system for the finished drug product, which may include impurities capable of
forming nitrosamines, especially if associated with materials containing amines and potential sources of a nitrosating
agent (e.g., nitrite, nitrocellulose).
A risk assessment should be conducted to determine the materials that contribute to the potential for inclusion of nitrosamines
in the drug product. All potential sources for the introduction of nitrosamines should be considered in the risk assessment
including, for example, the drug substance, excipients, water, solvents, the manufacturing process, packaging components,
and formation on stability. See Figure 1 for a diagram of some potential sources to be considered.
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Figure 1. Potential sources of nitrosamine impurities in drug product.
Ongoing assessments and evaluations have identified risks associated with several of the potential sources of nitrosamines. Some
of the examples identified are summarized in Table 2.
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Table 2
Potential Source of Nitrosamines Observed Riska
• Presence of residual dialkyl amines or tri-substituted amines that can de-
grade to form intermediates that can further react with nitrosating agents
• Presence of nitrites or other nitrosating agents
• Presence of acid
• Limited controls/specification limits for recycled solvents
Solvents • Poor quality solvents
• Presence of residual dialkyl amines or impurities that can degrade to form

dialkyl amines
Water • Presence of acid and nitrosating agents
• Presence of nitrites or other nitrosating agents and/or nitrosamine impuri-

Excipients ties (if applicable)
• Use of sodium azide in the synthesis followed by use of nitrites in acidic

medium (nitrous acid) for quenching excess azides
• Use of di- or tri-alkylamines and amides (e.g., dimethylformamide [DMF],
dimethylamine [DMA], triethylamine [TEA], N-methylpyrrolidone [NMP])
in the presence of nitrites and acid media
• Use of recycled solvents that may contain nitrosamines or their precursors
• Use of sanitized water (e.g., chloramines)
• Insufficient purification
• Degradation of drug substances containing functional groups that can then
Drug substance participate in nitrosation reactions
• Contamination
• Use of poor quality or recycled solvents that may contain nitrosamines or
their precursors
• Presence of nitrous oxides in air used to dry the drug substance or drug
product
Manufacturing process • Carryover of relevant reactive species into subsequent steps
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Table 2 (continued)
Potential Source of Nitrosamines Observed Riska
• Secondary, tertiary, or quaternary amine group in molecule of drug sub-
stance
• Presence of nitrate counter ions (potentially containing nitrite as an impur-
ity)
• Potential reactions within the formulation matrix during stability/shelf life
Drug product (including stability) (e.g., presence or generation of acidic conditions, moisture, and heat)
• Packaging materials containing vulnerable amines that might react with ni-
trosating agents present in the packaging material itself (e.g., amines in inks
Container–Closures reacting with nitrocellulose print base)
a General chemical reactions leading to formation of nitrosamines can be found in 3.1 Nitrosamine Formation Reaction.
3.1 Nitrosamine Formation Reaction

The general schematic representation of the chemical reaction responsible for the formation of nitrosamines from
secondary amines is described in Figure 2. Examples of representative reactions are described in the scientific literature
(2–3).
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Figure 2. General example of formation of nitrosamines, where R1 and R2 are alkyl or functionalized alkyl groups (and only
one of them could be an aryl or functionalized aryl group).
If the potential for the presence of nitrosamines is identified, where appropriate, a control strategy should be developed.
If nitrosamines are identified as impurities in ingredients, they may be controlled as appropriate in the ingredients (e.g.,
manufacture of the drug substance or controls placed on the drug substance). If nitrosamines are identified as
degradation products (i.e., being formed during manufacturing of the drug product or formed during product storage),
they should be controlled as appropriate in the drug product. In some cases, changes to the manufacturing process(es)
or ingredients may be required to achieve acceptable levels or the elimination of nitrosamine impurities in the drug
product.
4. NITROSAMINE RISK ASSESSMENTS—DEVELOPMENT OF A CONTROL STRATEGY
In order to determine the level of control, if any, which may be required for ensuring that levels of nitrosamines are at or below
the acceptable intake (AI) if their presence cannot be avoided, the components of drug products should be assessed by the
drug product manufacturer for the potential to form nitrosamines or to be contaminated with nitrosamines. Although one
of the sources with the highest potential for nitrosamines is the drug substance synthetic route, the drug substance
manufacturing process, drug product manufacturing process, and excipients and raw materials should also be included in a
risk assessment to establish if controls or additional controls are needed. An example of high-level process flow for evaluating
materials is shown in Figure 3.
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Figure 3. High level process for development of a nitrosamine impurity control strategy. (a Refer to Table 2; P1, P2, P3 = Process
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1, 2, 3; D1, D2 = Decision 1, 2)
In all cases, if nitrosamines are predicted by the risk assessment or confirmed to be present through testing of the drug substance,
drug product, or other materials, a control strategy should define an approach to ensure that the nitrosamine levels comply
with the established AIs. The control strategy should be aligned with the current regulatory requirements in place.
5. LIMITS OF NITROSAMINES
Nitrosamine impurities identified in this chapter have potential and established toxicity with no therapeutic value. Because
nitrosamines are among the structural groups of high potency mutagenic carcinogens of the “cohort of concern” in ICH M7
(1), the threshold of toxicological concern (TTC) does not apply. Instead, the available safety data should be used to
establish a material-specific AI on a case-by-case basis. The AI is defined as an intake level that poses a negligible health risk.
5.1 Derivation of AI Limits
There are a number of methodologies that toxicologists have applied in establishing AIs. In this case, the median
tumorigenic dose (TD50) of NDMA, NDEA, and other nitrosamines was used as representative data in a linear
extrapolation to establish an acceptable risk level. The limits have been published in the FDA Guidance for Industry:
Control of Nitrosamine Impurities in Human Drugs (4).
5.2 Example Calculations of Nitrosamine Limits
The AIs in nanograms per day and the maximum daily dose (MDD) of the drug substance (DS) from the drug label in
milligrams per day can be used to calculate the maximum nitrosamine concentration limits, in ppm, for individual drug
products using the following equation:
Concentration = AI/DSdose
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Since the exposure to nitrosamines is related to the MDD of the DS, different concentrations of nitrosamines (ng/g) may
be acceptable for each material evaluated. The acceptable concentration in the material can be calculated using the
following equation:
Acceptable nitrosamine content = AI/MDD

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AI = acceptable intake of the nitrosamine (μg/day)
MDD = maximum daily dose of the drug substance (g/day)
Table 3. Example Using an AI of 96 ng/day for the Target Nitrosamine

Name Acceptable Concentration (μg/g)
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0.050 0.100 0.250 1.00
(50-mg dose) (100-mg dose) (250-mg dose) (1000-mg dose)
Nitrosamine 1 1.920 0.960 0.384 0.096
[NOTE—If multiple nitrosamines are identified in the material and the total exceeds 26.5 ng/day, the appropriate regulatory
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authority should be consulted to determine an acceptable approach.]

6. TESTING FOR THE PRESENCE OF NITROSAMINES
Upon completion of the risk assessment, exploratory testing may need to be performed to confirm the conclusions of the risk
assessment and proposed control strategy. On the basis of the controls identified (e.g., incoming material testing or
specification limit; drug substance or drug product specification limit), it may be necessary to implement routine testing for
nitrosamines. If testing is applied to ensure that the nitrosamine(s) concentration(s) do not exceed the AI, methods should
be established following the recommendations detailed in this chapter. Example analytical procedures can be found in 8.
Analytical Procedures.
6.1 Presence of Two or More Nitrosamines
The current published AIs reflect limits for the presence of a single nitrosamine. If multiple nitrosamines are possible and
are determined analytically to be present at levels exceeding the maximum amount permitted by the regulatory
authority, the relevant health authority should be consulted to determine a specific path forward. Manufacturers should
contact FDA for determining the AI limits if multiple nitrosamine impurities are detected in a drug substance or drug
product in which the total nitrosamine level exceeds 26.5 ng/day based on MDD.
7. TEST METHOD PERFORMANCE CHARACTERISTICS OF NITROSAMINE METHODS
The AIs associated with nitrosamines require the application of sensitive analytical procedures. In many cases, the most reliable
procedures take advantage of the sensitivity and selectivity of chromatographic separation techniques coupled with
quantitation by mass spectrometry (e.g., HPLC–MS/MS, GC–MS/MS). For additional guidance on validation of alternative
methods for nitrosamines, see Validation of Compendial Procedures á1225ñ.
7.1 Considerations for Sample Preparation
Appropriate sample preparation is a critical step in trace impurity analyses such as those required to evaluate the levels of
nitrosamines in drug substances and drug products. This is particularly critical to prevent the loss or generation of
nitrosamines as artifacts of the analytical procedure itself, as in the following circumstances.
• The presence of dialkyl amine (dimethylamine) as a process impurity or counter ion of the salt form of the active
ingredient in the presence of nitrite and acid can lead to in situ formation of nitrosamines as an artifact, especially
in GC analyses.
• Total solubilization versus selective extraction: If the active ingredient contains a dimethylamino group, total
dissolution of the drug substance should be avoided when applying GC techniques. High concentration of the
active ingredient, when injected in the GC instrument can generate nitrosamines in the injection port if a
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nitrosating agent is present. In these situations, sample extractions should be modified to prevent the solubilization
of the active ingredient while maintaining the extraction efficiency for nitrosamines present in the material.
The recommended method performance characteristics that need to be evaluated for quantitative analysis of nitrosamines
include range, accuracy, repeatability, intermediate precision, and limit of quantitation. If a limit test is intended for
use, the recommended method performance characteristics to be assessed include specificity, recovery, detectability,
and solution stability. The performance criteria for these parameters should be properly set and confirmed through
validation to ensure that the method is suitable for its intended use based on the specific analytes, matrices, and required
precision and accuracy of the analytical procedures. Precision and recovery depend highly on concentration and matrix
complexity, and the final proposed acceptance criteria need to be justified in the procedure validation documentation.
Higher variability may be tolerated or acceptable at concentrations approaching the limit of quantitation of the
procedure while lower variability (higher precision) would be expected at higher concentrations. Examples of
quantitative analytical procedures are included in 8. Analytical Procedures. Additional example methods may be provided
as they become available for these additional nitrosamines.
8. ANALYTICAL PROCEDURES
The following procedures have been established as suitable for their intended (specified) purpose. Users should validate these
methods while considering the effect of sample solubility and extraction efficiency on the test results for other materials for
which they are intended to be applied (see á1225ñ and Verification of Compendial Procedures á1226ñ).
8.1 Quantitative Procedures
Procedure 1: Quantitation of NDMA, NDEA, NDIPA, NEIPA, NMBA, NMPA, and NDBA in selected sartans (valsartan,
irbesartan, and losartan potassium) by HPLC–HRMS
Diluent: Methanol
Solution A: 0.1% formic acid in water
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Solution B: 0.1% formic acid in methanol
Mobile phase: See Table 4.
Table 4
Time ci Solution A Solution B
(min) (%) (%)
0 90 10
1.5 90 10
7.0 45 55
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17.0 45 55
17.1 10 90
21.0 10 90
21.1 90 10
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25.0 90 10
Sensitivity solution: 1.0 ng/mL each of USP N-Nitrosodimethylamine RS, USP N-Nitrosodiethylamine RS, USP
N-Nitrosoethylisopropylamine RS, USP N-Nitrosodiisopropylamine RS, USP N-Nitrosodibutylamine RS, USP
N-Nitrosomethylphenylamine RS, and USP N-Nitrosomethylaminobutyric Acid RS in Diluent
Standard solution: 6.0 ng/mL each of USP N-Nitrosodimethylamine RS, USP N-Nitrosodiethylamine RS, USP
N-Nitrosomethylphenylamine RS, and USP N-Nitrosomethylaminobutyric Acid RS in Diluent
Sample solution: 20 mg/mL of drug substance prepared as follows. Transfer 100 mg of drug substance into a suitable
container. Add 5.0 mL of Diluent and vortex until fully dispersed or dissolved. Pass the solution through a suitable
filter of 0.2-µm pore size. Use the filtrate for analysis.
Chromatographic system
(See Chromatography á621ñ, System Suitability.)
Mode: LC
Detector: High resolution mass spectrometer
MS conditions
Ionization: Electrospray ionization (ESI)
Scan settings: See Table 5.
Table 5
Nitrosamine Im-
purity NDMA NMBA NDEA NEIPA NDIPA NMPA NDBA
Scan type SIMa

SIM PRM b
SIM SIM SIM PRM
Polarity positive negative positive positive positive positive positive
Scan
start–end
(min) 1.0–3.5 3.5–5.5 5.5–7.0 7.0–8.5 8.5–10.0 8.5–10.0 13.0–15.5
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Table 5 (continued)
m/zc isolated
for PRM N/A N/A 103.0866 N/A N/A N/A 159.1492
NCE d
N/A N/A 25 N/A N/A N/A 20
Scan range
(m/z) 74.3–75.8 144.3–145.8 50.0–114.0 116.4–117.9 130.4–131.9 136.3-137.8 50.0–170.0
Microscans 3 3 3 3 3 (1)e e
3 (1) 3
Resolution 30,000 60,000 30,000 60,000 60,000 60,000 30,000
AGC target value

(%)f 250 250 250 250 250 250 250
a SIM = selected ion monitoring.

b PRM = parallel reaction monitoring.
c m/z = mass to charge ratio.
d NCE = normalized collision energy.
e 1 microscan must be used only if both NDIPA and NMPA are present.
f AGC = automatic gain control.
[NOTE—Divert the drug substance from the MS source during the elution.]
Data processing: Peak areas in the extracted ion chromatograms (EIC) with an m/z extraction window of
±15 ppm are used for quantitation. The m/z values extracted are listed in Table 6.
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Table 6
Nitrosamine Im-
purity NDMA NMBA NDEA NEIPA NDIPA NMPA NDBA
m/z to be ex-
tracted 75.0553 145.0619
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75.0553,
103.0866 117.1022 131.1179 137.0709
57.0704,
103.0872,
159.1492
Column: 4.6-mm × 10-cm; 2.6-µm packing L43

Temperatures
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Autosampler: 4°
Column: 40°
Flow rate: 0.6 mL/min
Flow rate to ion source: 0.6 mL/min
Injection volume: 3 µL
System suitability
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Samples: Sensitivity solution and Standard solution

[NOTE—The relative retention times for NDMA, NMBA, NDEA, NEIPA, NDIPA, NMPA, and NDBA are 0.20, 0.31,
0.46, 0.57, 0.66, 0.67, and 1.00, respectively.]
[NOTE—NMBA and NEIPA exist as syn- and anti-conformers due to the restricted rotation of the N–N bond. These
conformers are partially separated by the method’s chromatographic conditions. The NMBA peak is observed
as a doublet. Integrate both of the NMBA peaks and use the combined peak areas for calculation of the NMBA
concentration. The NEIPA peak may appear as a doublet or a single peak with a tailing shoulder. For NEIPA, if
the conformers are resolved, integrate both peaks and combine the peak areas for the calculation of the NEIPA
concentration. If the NEIPA conformers are not fully resolved (e.g., evidence of a shoulder is present), integrate
the main peak and shoulder as a single peak and use the combined peak area to calculate the NEIPA
concentration.]
Suitability requirements
Relative standard deviation: NMT 20.0% from 6 replicate injections, Standard solution
Signal-to-noise ratio: NLT 10, Sensitivity solution
Analysis
Samples: Standard solution and Sample solution
Calculate the concentration, in ppm, of each specified nitrosamine impurity in the portion of drug substance taken:
Result = (rU/rS) × (CS/CU) × 106
rU = peak response of the individual specified nitrosamine impurity from the Sample solution
rS = peak response of the corresponding nitrosamine impurity from the Standard solution
CS = concentration of USP N-Nitrosodimethylamine RS, USP N-Nitrosodiethylamine RS, USP
N-Nitrosomethylphenylamine RS, or USP N-Nitrosomethylaminobutyric Acid RS in the Standard solution
(µg/mL)
CU = concentration of the drug substance in the Sample solution (µg/mL)
Report the nitrosamine impurity concentration in the drug substance in ppm (µg/g).
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Procedure 2: Quantitation of NDMA, NDEA, NDIPA, and NEIPA in selected sartans (valsartan, irbesartan, losartan
potassium, olmesartan medoxomil, candesartan cilexetil, and telmisartan) by headspace GC–MS
Diluent: Methanol
Internal standard stock solution: 0.4 µg/mL of NDMA-d6 in Diluent
Internal standard solution: 0.016 µg/mL of NDMA-d6 prepared as follows. Transfer 2.0 mL of Internal standard stock
solution into a 50-mL volumetric flask, and dilute with Diluent to volume.
Nitrosamine RS stock solution: 0.4 µg/mL each of USP N-Nitrosodimethylamine RS, USP N-Nitrosodiethylamine RS,
USP N-Nitrosoethylisopropylamine RS, and USP N-Nitrosodiisopropylamine RS prepared as follows. Transfer an
appropriate amount of USP N-Nitrosodimethylamine RS, USP N-Nitrosodiethylamine RS, USP
N-Nitrosoethylisopropylamine RS, and USP N-Nitrosodiisopropylamine RS into a suitable volumetric flask, and dilute
with Diluent to the volume.
Standard stock solution: 0.016 µg/mL each of USP N-Nitrosodimethylamine RS, USP N-Nitrosodiethylamine RS, USP
N-Nitrosoethylisopropylamine RS, and USP N-Nitrosodiisopropylamine RS prepared as follows. Transfer 2.0 mL of
Nitrosamine RS stock solution and 2.0 mL of Internal standard stock solution into a 50-mL volumetric flask, and dilute
with Diluent to volume.
Standard solution: Transfer 1 mL of Standard stock solution to an appropriate headspace vial containing about 100 mg
of imidazole and 1.0 mL of acetonitrile. Apply the stopper, cap, and crimp tightly.
Sensitivity stock solution: 0.004 µg/mL each of USP N-Nitrosodimethylamine RS, USP N-Nitrosodiethylamine RS, USP
N-Nitrosoethylisopropylamine RS, and USP N-Nitrosodiisopropylamine RS in Diluent prepared as follows. Transfer
0.5 mL of the Nitrosamine RS stock solution and 2.0 mL of Internal standard stock solution into a 50-mL volumetric
flask and dilute with Diluent to volume.
Sensitivity solution: Transfer 1 mL of Sensitivity stock solution to an appropriate headspace vial containing about
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100 mg of imidazole and 1.0 mL of acetonitrile. Apply the stopper, cap, and crimp tightly.
Sample solution: Transfer 200 ± 10 mg of drug substance and about 100 mg of imidazole into a headspace vial, and
then add 1.0 mL of Internal standard solution and 1.0 mL of acetonitrile. Apply the stopper, cap, and crimp tightly.
Blank: Transfer about 100 mg of imidazole into a headspace vial, and then add 1.0 mL of Internal standard solution
and 1.0 mL of acetonitrile. Apply the stopper, cap, and crimp tightly.
Mode: GC
Injector: Headspace (see Table 7 for parameters)
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Table 7
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Equilibration temperature 95°–110°
Loop temperature 150°
Rate 1 10°/min
Transfer line temperature 160°

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Pressurizing gas pressure 20.00 psi
Equilibration time 10.00 min
Pressurizing time 2.00 min
Load time 2.00 min
Injection time 1.00 min
Vial size 20 mL
Injection type: (Split, Split ratio 1:1 or 1:3)

[NOTE—Split ratio can be modified to optimize sensitivity.]
Detector: Mass spectrometer
Column: 0.32-mm × 30-m fused-silica, coated with a 1.0-µm layer of phase G16
Column temperature: See Table 8.
Table 8
Hold Time at
Initial Temperature Final Final
Temperature Ramp Temperature Temperature
(°) (°/min) (°) (min)
45 0 45 3
45 10 130 3
130 15 190 —
190 40 240 10
Carrier gas: Helium
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Flow rates
Gas: Constant flow at 1.8 mL/min (adjustment and verification are necessary for other carrier gases)
Purge: 3.0 mL/min or default value
MS conditions: See Table 9.
Table 9
Ionization mode Electron impact (EI)
Polarity positive
Event 1
Name NDMA
Start time 10.0 min
End time 12.0 min
Acquisition mode multiple reaction mode (MRM)
Ch 1 m/z 74.00 ˃ 44.00
CH 1 collision energy 4.00 V
Ch 2 m/z 74.00 ˃ 42.00
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Event 2
Name NDMA-d6
Start time 10.0 min
End time 12.0 min
Acquisition mode
Ch 1 m/z
ci MRM
80.00 ˃ 50.00

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Event 3
Name NDEA
Start time 12.00 min
End time 12.75 min

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Acquisition mode MRM
Ch 1 m/z 102.00 ˃ 85.1
Ch 2 m/z 102.00 ˃ 56.1
Event 4
Name NEIPA
End time 13.35 min
Ch 1 m/z 116.00 ˃ 99.10
Ch 2 m/z 99.00 ˃ 44.10
Event 5
Name NDIPA
End time 14.00 min
Ch 1 m/z 130.00 ˃ 42.00
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Table 9 (continued)
Ch 2 m/z 130.00 ˃ 43.10
[NOTE—Ch 1 m/z in multiple reaction mode (MRM) is used for quantitation.]

System suitability
Samples: Standard solution, Sensitivity solution, and Blank
[NOTE—The relative retention times for NDMA, NDMA-d6, NDEA, NEIPA, and NDIPA are 0.80, 0.80, 0.90, 0.96,
and 1.00, respectively.]
Relative standard deviation: NMT 20.0% for the ratio of the impurity standard peak response to the internal
standard peak response from 6 replicate injections, Standard solution
Signal-to-noise ratio: NLT 10 for the impurity peak, Sensitivity solution
Interference: There should be no interfering peak in the Blank.
Analysis
Samples: Standard solution and Sample solution
Calculate the concentration, in ppm, of each specified nitrosamine impurity in the portion of drug substance
taken:
Result = 1/W × (RU/RS) × CS
W = weight of the drug substance in the Sample solution (g)
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RU = peak response ratio of the specified nitrosamine impurity to that of the internal standard from the Sample solution
RS = peak response ratio of the specified nitrosamine impurity to that of the internal standard from the Standard solution
CS = concentration of USP N-Nitrosodimethylamine RS, USP N-Nitrosodiethylamine RS, USP
N-Nitrosoethylisopropylamine RS, and USP N-Nitrosodiisopropylamine RS in the Standard stock solution (µg/mL)
ci
Procedure 3: Quantitation of NDMA, NDEA, NDIPA, NEIPA, NMBA, and NDBA in selected sartans (valsartan,
losartan potassium, olmesartan medoxomil, candesartan cilexetil, and telmisartan) by HPLC–MS/MS
Diluent: 1% formic acid in water
Solution A: 0.1% formic acid in water
ffi
Solution B: 0.1% formic acid in methanol
Mobile phase: See Table 10.
Table 10
Time Solution A Solution B
(min) (%) (%)
O
0 97 3
1.5 97 3
4.0 50 50
7.0 25 75
8.1 15 85
9.2 5 95
12.0 5 95
12.1 97 3
Internal standard solution: 10 µg/mL each of NDMA-d6 and NMBA-d3, 1 µg/mL each of NDEA-d10 and NDBA-d18 in
water
Nitrosamine standards stock solution mixture: Prepare a mixture of 200 ng/mL each of N-nitrosodimethylamine,
N-nitrosoethylisopropylamine, N-nitrosodiisopropylamine, N-nitrosodibutylamine, and
N-nitrosomethylaminobutyric acid by mixing appropriate volumes of the respective USP Reference Standards and
dilute with water.
[CAUTION—Prepare Nitrosamine standards stock solution mixture in amber vials and store at −18° to −20°).]
NDEA standard stock solution: Prepare a solution of 132 ng/mL of N-nitrosodiethylamine by diluting USP
N-Nitrosodiethylamine RS with water.
Standard solutions: Depending on the targeted nitrosamine concentration in the sample, prepare a set of 5
consecutive linearity solutions from Table 11 from the Nitrosamine standards stock solution mixture and NDEA standard
stock solution by mixing specified volumes of each solution as indicated. [NOTE—Table 11 represents an example for
preparing solutions for constructing the calibration curve. Other dilution schemes may be used for preparing the set
of 5 linearity solutions covering the range of interest. L1 is used only for NDEA when applicable. For others, linearity
starts with L2.]
12
Table 11
Concentration Content of
of NDMA, NDMA, NMBA, Volume of Ni- Volume of
NMBA, NDBA, NDBA, NEIPA, trosamine NDEA Stand- Volume of In-
NEIPA, NDIPA NDIPA Standard ard Stock Solu- Volume of Wa- ternal Stand-
Linearity Solu- Concentration (ng/mL)/ (ppb)/NDEA Stock Solution tion ter ard Total Volume
tion # Level NDEA (ng/mL) (ppb) Mixture (μL) (μL) (μL) (μL) (μL)
1 L1 1.33/0.66 19.95/10 8 6 1174 12 1200
2 L2 2/0.88 30/13.5 12 8 1168 12 1200
3 L3 5/3.3 75/49.5 30 30 1128 12 1200
4 L4 7.5/4.95 112.5/74.25 45 45 1098 12 1200
5 L5 10/6.6 150/99 60 60 1068 12 1200
6 L6 15/9.9 225/148.5 90 90 1008 12 1200

7 L7 30/19.8 450/297 180 180 828 12 1200
8 L8 60/39.6 900/594 360 360 468 12 1200
9 L9 90/59.4 1350/891 540 540 108 12 1200
Sample solution: Transfer about 80 mg of the drug substance into a 2-mL lidded centrifuge tube. Add 1188 µL of
al
Diluent and 12 μL of the Internal standard solution. Vortex at 2500 rpm for 20 min (except for losartan potassium,
which should be vortexed NMT 5 min). Centrifuge at about 10,000 rpm for 10 min, and filter into a vial using a
hydrophilic polytetrafluoroethylene (PTFE) filter of 0.45-μm pore size.
Mode: LC
ci
Detector: MS/MS (triple quadrupole mass spectrometer)
MS conditions
Ionization: Atmospheric pressure chemical ionization (APCI)
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Table 12
MRM Transitions (m/z)
Nitrosamine Impurity Acquisition Mode Polarity MRM-1a MRM-2

+75.0 → +75.0 →
O
NDMA MRM Positive +43.0 +44.1
+81.2 → +81.2 →
NDMA-d6 MRM Positive +46.0 +64.1
+103.1 → +103.1 →
NDEA MRM Positive +75.1 +47.1
+113.2 → +113.2 →
NDEA-d10 MRM Positive +34.2 +49.1
+147.1 → +147.1 →
NMBA MRM Positive +44.1 +117.1
+150.1 → +150.1 →
NMBA-d3 MRM Positive +47.1 +120.2
+159.2 → +159.2 →
NDBA MRM Positive +41.1 +29.1
+177.3 → +177.3 →
NDBA-d18 MRM Positive +66.2 +46.2
+117.1 → +117.1 →
NEIPAb MRM Positive +75.1 +47.2
+131.2 → +131.1 →
NDIPAb MRM Positive +89.1 +47.1
a MRM-1 is used for quantitation.

b NDEA-d10 is used as internal standard for NEIPA and NDIPA.
Column: 3.0-mm × 15-cm; 2.7-µm packing L1

Temperatures
Autosampler: 18°
Column: 60°
Flow rate: 0.5 mL/min
13
Flow rate to ion source: 0.5 mL/min

System suitability
Samples: Standard solutions
Generate the peak response ratio of the specified impurity to that of the internal standard versus the concentration
standard curve for each nitrosamine impurity under test using the corresponding selected Standard solutions and
perform the linear regression analysis.
[NOTE—The relative retention times for NDMA, NMBA, NDEA, NEIPA, NDIPA, and NDBA are 0.20, 0.31, 0.46, 0.57,
0.66, and 1.00, respectively.]
Correlation coefficient: NLT 0.99
y-Intercept: NMT 25% of the response of the medium concentration solution used in standard curve generation
Analysis
Samples: Standard solutions and Sample solution
Calculate the concentration, in ppm, of each specified nitrosamine impurity in the Sample solution using the
corresponding calibration curve:
Result = [(RU − yint)/a] × (1/CU) × 103
yint = y-intercept of the calibration curve for the specified nitrosamine impurity from the Standard solutions
a = slope of the calibration curve for the specified nitrosamine impurity from the Standard solutions [(μg/mL)−1]
al
CU = concentration of the drug substance in the Sample solution (mg/mL)
Procedure 4: Quantitation of NDMA, NDEA, NDIPA, NEIPA, NMPA, and NDBA in selected sartans (valsartan,
losartan potassium, and candesartan cilexetil) by GC–MS/MS (triple-quad)
Internal standard solution: 50 ng/mL of NDMA:C13-d6 in methylene chloride
ci
Standard solution: Prepare a mixture of 0.1 µg/mL each of N-nitrosodimethylamine, N-nitrosodiethylamine,
N-nitrosoethylisopropylamine, N-nitrosodiisopropylamine, N-nitrosomethylphenylamine and
N-nitrosodibutylamine by mixing appropriate volumes of respective USP Reference Standards and diluting with
Internal standard solution.
Calibration solutions: Depending on the targeted nitrosamine concentration in the sample, prepare a set of 5
ffi
consecutive solutions from Table 13 that are used for generating the calibration curve by following the preparation
scheme shown in the table. Volumes may be adjusted to prepare larger quantities of the calibration solutions as
needed, maintaining final concentrations of the nitrosamines. For each calibration solution, transfer the designated
aliquot of Standard solution to the designated volumetric flask, and adjust the volume with the Internal standard
solution.
O
Table 13
Final Nitrosamine Concen- Equivalent Nitrosamine Con-
Standard Solution Aliquot Final Volume tration centration
Calibration Solution ID (μL) (mL) (μg/mL) (μg/g)
Cal 1 50 10 0.0005 0.005
Cal 2 100 10 0.001 0.010
Cal 3 200 10 0.002 0.020
Cal 4 300 10 0.003 0.030
Cal 5 400 10 0.004 0.040
Cal 6 500 10 0.005 0.050
Cal 7 1000 10 0.010 0.100
Cal 8 1500 10 0.015 0.150
Sample solution: Transfer 500 mg of the drug substance into a disposable 10- to 15-mL glass centrifuge tube. Add
5.0 mL of the Internal standard solution. Cap the tube. Vortex the sample for 1 min, and then place in the centrifuge.
Centrifuge the sample at 4000 rpm for 2.5 min. Transfer 2 mL of the bottom methylene chloride layer to a 5-mL
syringe fitted with a 0.45-μm nylon filter. Filter 1 mL of sample extract into a 2-mL GC autosampler vial and cap.
Mode: GC
Injector: Split/splitless
Injection type: Splitless with purge
Purge time: 0.5 min
Detector: MS/MS (triple quadrupole mass spectrometer)
MS conditions
Ionization: Electron impact
14
Table 14
MRM Transitions (m/z)
Nitrosamine Impurity Acquisition Mode Polarity MRM-1 a

MRM-2
74 → 74 →
NDMA MRM Positive 44 42
82 →
NDMA:c13-d6 MRM Positive 48 –
102 → 102 →
NDEA MRM Positive 85 56
116 → 71 →
NEIPA MRM Positive 99 56
130 → 130 →
NDIPA MRM Positive 88 42
106 → 77 →
NMPA MRM Positive 77 51
158 → 84 →
NDBA MRM Positive 99 56
al
a MRM-1 is used for quantitation.
MS1 and MS2 resolution: Q1: normal; Q3: wide (1.5)

Minimum window: 1 min
Emission current: 50 μA ci
Column: 0.25-mm × 30-m; fused-silica coated with a 1.0-µm layer of phase G16
Temperatures
Injector: 250°
Transfer line to MS detector: 250°
Ionization source: 250°
Column: See Table 15.
ffi
Table 15
Hold Time at
Initial Temperature Final Final
Temperature Ramp Temperature Temperature
(°) (°/min) (°) (min)
O
40 0 40 0.5
40 20 200 0
200 60 250 3
Carrier gas: Helium

Flow rate: Constant flow at 1.0 mL/min (adjustment and verification are necessary for other carrier gases)
System suitability
Samples: Calibration solutions
[NOTE—The relative retention times for NDMA, NDEA, NEIPA, NDIPA, NDBA, and NMPA are 0.73, 0.78, 0.81, 0.82,
0.99, and 1.00, respectively.]
Generate the peak response ratio of the specified impurity to that of the internal standard versus concentration standard
curve for each nitrosamine impurity under test using the corresponding Calibration solutions and perform the linear
regression analysis.
Correlation coefficient: NLT 0.98
Signal-to-noise: NLT 10 for the impurity peak of the lowest concentration Calibration solutions used in the
calibration curve
Analysis
Samples: Calibration solutions and Sample solution
Calculate the concentration, in ppm, of each specified nitrosamine impurity in the Sample solution using the
corresponding calibration curve:
Result = 5 × (1/W) × [(RU − yint)/a]
W = weight of the drug substance in the Sample solution (g)

yint = y-intercept of the calibration curve for the specified nitrosamine impurity from the corresponding Calibration
solutions
15
a = slope from the calibration curve for the specified nitrosamine impurity from the corresponding Calibration
solutions [(μg/mL)−1]
8.2. Limit Test Procedures
While a limit test analytical procedure for nitrosamines content is not currently available, recommended sample
preparation procedures are shown in Table 16.
Table 16
Solutions Solution Preparation
Prepare a suitable Internal standard solution that, when added to the Sample
solution, will have the resultant peak response at the highest appropriate target
Internal standard solution limit of the nitrosamines of interest in the sample.
Prepare a solution of the article to be examined, spiked with the internal stand-
ard, and prepared as described in the sample preparation. The amount of sub-
stance to be examined is chosen in such a way that the amount, in ppm, of a
target N-nitrosamine, if present at its limit concentration for that substance,
Sample solution would be equal to the contribution of the respective spiking solution.
A solution of target N-nitrosamine(s) of a concentration that, if added to the

amount of article used for the preparation of the Sample solution, would result
Spiking solution in the acceptance limit.
Prepare a solution of the article to be examined spiked with a) appropriate
al
Spiking solution(s) and b) Internal standard solution prepared as described in the
Spiked sample solution Sample solution.
Analysis
Samples: Spiked sample solutions and Sample solution ci
Determine the peak response ratio of the respective target N-nitrosamine to the internal standard from the Sample
solution: RU(i)
Determine the peak response ratio of the respective target N-nitrosamine to the internal standard from the Spiked sample
solution: RST(i)
Acceptance criteria: RU(i)/RST(i) is NMT 0.5
ffi
9. ADDITIONAL SOURCES OF INFORMATION
Several test procedures have been developed for the specific testing of nitrosamines in sartans and/or other official articles based
on different scientific principles and are publicly available from many regulatory agencies. The hyperlinks in this section direct
the user to the respective regulatory agencies’ procedures. These can be used as alternative procedures and must be validated
under actual use for the respective performance characteristics recommended in 7. Test Method Performance Characteristics
of Nitrosamine Methods.
O
1. FDA-published testing methods to provide options for regulators and industry to detect NDMA and NDEA impurities:
https://www.fda.gov/drugs/drug-safety-and-availability/
fda-updates-and-press-announcements-angiotensin-ii-receptor-blocker-arb-recalls-valsartan-losartan/?
utm_campaign=UPDATE on angiotensin II receptor blocker (ARB) recalls - FDA publishes LC-HRMS and RapidFire-MS/
MS&utm_medium=email&utm_source=Eloqua#testingmethods.
2. Ph. Eur. 2.5.42 N-Nitrosamines in active substances: https://www.edqm.eu/sites/default/files/medias/fichiers/
European_Pharmacopoeia/News/european_pharmacopoeia_n-nitrosamines_in_active_substances.pdf.
3. EDQM projects on sampling strategies and testing methods with the Official Medicines Control Laboratory (OMCL)
Network: https://www.edqm.eu/en/ad-hoc-projects-omcl-network.
10. USP REFERENCE STANDARDS á11ñ
USP N-Nitrosodibutylamine RS
USP N-Nitrosodiethylamine RS
USP N-Nitrosodiisopropylamine RS
USP N-Nitrosodimethylamine RS
USP N-Nitrosoethylisopropylamine RS
USP N-Nitrosomethylaminobutyric Acid RS
USP N-Nitrosomethylphenylamine RS
REFERENCES
1. International Council for Harmonisation of Technical Requirements of Pharmaceuticals for Human Use. ICH M7:
Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk,
2017. https://www.ich.org/page/multidisciplinary-guidelines.
2. Williams DLH, Chapter 1: Reagents effecting nitrosation. In: Nitrosation Reactions and the Chemistry of Nitric Oxide.
Amsterdam, Netherlands: Elsevier Science; 2004:1–34.
3. Ogata Y, Sawaki Y, Kuriyama Y. The reaction of trialkylamine with nitric acid in a mixture of acetic acid and acetic
anhydride. Tetrahedron. 1968;24(8):3425–3435.
4. Food and Drug Administration. Control of Nitrosamine Impurities in Human Drugs-Guidance for Industry, 2021. https://
www.fda.gov/media/141720/download.▲ (USP 1-Dec-2021)

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Printed on: Mon Jun 06 2022, 09:39:37 AM(EST) Official Status: Currently Official on 06-Jun-2022 DocId: 1_GUID-C97F817C-A383-4693-8E0C-2F0A0A371977_2_en-US

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Table 1. Nitrosamines Found as Contaminants in Drug Substances and Drug Products

• Presence of residual dialkyl amines or impurities that can degrade to form

• Presence of nitrites or other nitrosating agents and/or nitrosamine impuri-

• Use of sodium azide in the synthesis followed by use of nitrites in acidic

3.1 Nitrosamine Formation Reaction

Acceptable nitrosamine content = AI/MDD

Table 3. Example Using an AI of 96 ng/day for the Target Nitrosamine

Nitrosamine 1 1.920 0.960 0.384 0.096

authority should be consulted to determine an acceptable approach.]

Scan type SIMa

Polarity positive negative positive positive positive positive positive

Resolution 30,000 60,000 30,000 60,000 60,000 60,000 30,000

AGC target value

a SIM = selected ion monitoring.

Column: 4.6-mm × 10-cm; 2.6-µm packing L43

Samples: Sensitivity solution and Standard solution

Result = (rU/rS) × (CS/CU) × 106

Loop temperature 150°

Transfer line temperature 160°

Pressurizing gas pressure 20.00 psi

Equilibration time 10.00 min

Pressurizing time 2.00 min

Load time 2.00 min

Injection time 1.00 min

Injection type: (Split, Split ratio 1:1 or 1:3)

Carrier gas: Helium

Start time 10.0 min

End time 12.0 min

Acquisition mode multiple reaction mode (MRM)

Ch 1 m/z 74.00 ˃ 44.00

CH 1 collision energy 4.00 V

Ch 2 m/z 74.00 ˃ 42.00

CH 2 collision energy 15.00 V

Start time 10.0 min

End time 12.0 min

CH 1 collision energy 5.00 V

Start time 12.00 min

End time 12.75 min

Acquisition mode MRM

Ch 1 m/z 102.00 ˃ 85.1

CH 1 collision energy 6.00 V

Ch 2 m/z 102.00 ˃ 56.1

CH 2 collision energy 15.00 V

Start time 12.75 min

End time 13.35 min

Acquisition mode MRM

Ch 1 m/z 116.00 ˃ 99.10

CH 1 collision energy 6.00 V

Ch 2 m/z 99.00 ˃ 44.10

CH 2 collision energy 9.00 V

Start time 13.35 min

End time 14.00 min

Acquisition mode MRM

Ch 1 m/z 130.00 ˃ 42.00

CH 1 collision energy 10.00 V

CH 2 collision energy 18.00 V