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BRIEFING
〈 1469〉 Nitrosamine Impurities. Starting in July 2018 the World Health Organization (WHO), the FDA, the European Directorate for the Quality of Medicines (EDQM),
and other regulatory and global health agencies issued guidance documents and public health alerts regarding the presence of nitrosamine impurities in several drug
products. To protect patients from the adverse effects of nitrosamines as impurities in drug products, USP’s General Chapters–Chemical Analysis Expert Committee,
Chemical Medicines Monographs 2 Expert Committee, and Chemical Medicines Monographs 3 Expert Committee are proposing this new general chapter. This chapter
is aligned with current scienti c and regulatory approaches developed to ensure the appropriate control of nitrosamine impurities in drug substances and drug
products. The objective of this standard is to provide a science-based approach for the control of nitrosamine impurities, eliminating or reducing their presence in drug
products. The approach described thereby ensures the quality of the product as it relates to safety.
1. The 1. Introduction presents the concern of nitrosamine presence and summarizes the current industry and regulatory thinking. It is followed by a compilation of
the possible sources of nitrosamines.
2. The 2. Nitrosamine Impurities section provides a list of nitrosamines of concern in the pharmaceutical industry, compiled from the information shared by
multiple global health authorities and personal experience of members of the joint subcommittee. It includes additional chemical information for each entry. It
also positions nitrosamines from the ICH M7 perspective, which includes the N-nitroso compounds in the “Cohort of Concern”.
3. The 3. Sources of Nitrosamines section provides a general overview on how nitrosamines are formed and could end up in pharmaceutical products. It includes a
comprehensive table that lists each potential source of nitrosamines and the associated observed or assessed risk for that source.
4. The 4. Nitrosamine Risk Assessments—Development of a Control Strategy section provides recommendations on how the risk assessment for nitrosamines is
performed and the development of a relevant control strategy to ensure that the nitrosamine presence is avoided or limited to levels below acceptable intake.
5. The 5. Limits of Nitrosamines section details the approach that is used for establishing speci c nitrosamines daily acceptable intake (AI) and how the
concentration limits are calculated based on it and the maximum daily dose of the drug substance.
6. The 7. Test Method Performance Characteristics of Nitrosamine Methods section provides guidance on the veri cation process, the procedures being
implemented in the laboratory, and the validation of alternative procedures.
7. The 8. Analytical Procedures section contains procedures that have been validated or veri ed in the USP laboratories.
Procedure 1 is based on analyses performed with the Phenomenex Kinetex brand of 2.6-μm F5 100Å, 100 × 4.6-mm column with L43 packing. The
Orbitrap Fusion Lumos Tribrid brand of mass spectrometer (ThermoFisher Scienti c) was used during validation.
Procedure 2 is based on analyses performed with the following fused-silica columns with phase G16: a) Supelcowax 10 (Supelco #24211) (30 m × 0.32
mm × 1.0 µm); b) SH-Stabilwax (Shimadzu) (30 m × 0.32 mm × 1.0 µm); or c) DB-Wax (Agilent #123-7032) (30 m × 0.32 mm × 0.25 µm). The MS detectors
used were TSQ9000 VPI-HS 500 Triple Quadrupole GC–MS (ThermoFisher Scienti c), TQ8040 NX mass spectrometer (Shimadzu), or the 7010B Triple
Quadrupole GC/MS (Agilent).
Procedure 3 is based on analyses performed with the Restek Raptor brand of 2.7-μm ARC-18, 150-mm × 3.0-mm column with L1 packing. The LCMS-8050
Triple Quadrupole brand of liquid chromatograph mass spectrometer (Shimadzu) was used during method validation. The Xevo TQD Triple Quadrupole
brand of mass spectrometer (Waters) was used during method veri cation.
Procedure 4 is based on analyses performed with the J&W VF-WAXms GC brand of fused-silica column (30 m × 0.25 mm × 1 μm) with phase G16. The
TSQ 9000 GC-MS/MS brand of spectrometer (ThermoFisher Scienti c) was used during method validation, and the 7000 Series Triple Quad GC/MS brand
of spectrometer (Agilent) was used during method veri cation.
8. The 9. Additional Sources of Information section presents a compilation of references to analytical procedures that are currently on the websites of regulatory
agencies in the United States (FDA) and Europe (EDQM). Users can verify alternative procedures by meeting the requirements recommended in this chapter.
(GCCA: E. Biba)
Correspondence Number—C263095

Add the following:

▲ 〈 1469〉 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 the Interim Limits for AI

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

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, N-nitrosodimethylamine (NDMA)
and N-nitrosodiethylamine (NDEA) were detected in some valsartan drug substances and the drug products manufactured from drug substances using speci c
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 potential broad impact of the presence of this class of carcinogenic chemicals, this chapter has been developed to provide guidance in the
assessment of materials to ensure that the potential presence of nitrosamines is identi ed, provide recommendations regarding the establishment of controls,
and to provide initial guidance on analytical procedure performance criteria for procedures used to monitor nitrosamine levels.

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. The potential presence of any one or more of these impurities is dependent on the reaction chemistries and
processes. It is unlikely that all of the listed nitrosamines will be anticipated or observed as impurities in any single material. The list is not intended to be
exhaustive but represents the most commonly expected or observed nitrosamines.
N-nitroso compounds are listed as Class 1 mutagens in ICH M7: Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit
Potential Carcinogenic Risk (1). ICH M7 also includes N-nitroso compounds in the cohort of concern, a designation that carries with it a recommendation to
control the impurities at or below the acceptable cancer risk. In addition, some N-nitroso compounds are listed as Class 2 compounds. As a result of the
signi cant 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-
nitrosomethanamine NDMA 62-75-9 C2H6N2O 74.08

Nitrosodiethylamine
N-Ethyl-N-
nitrosoethanamine NDEA 55-18-5 C4H10N2O 102.14

Nitrosodiisopropylami
ne
N-Isopropyl-N-
nitrosoisopropylamine NDIPA 601-77-4 C6H14N2O 130.19

Nitrosoethylisopropyla
mine
N-Ethyl-N-nitroso-2-
propanamine NEIPA 16339-04-1 C5H12N2O 116.16

Nitrosodibutylamine
N-Butyl-N-nitroso-1-
butanamine NDBA 924-16-3 C8H18N2O 158.25

Nitrosomethylaminob
utyric
4-
[Methyl(nitroso)amino]
butanoic acid NMBA 61445-55-4 C5H10N2O3 146.15

3. SOURCES OF NITROSAMINES
 In acidic conditions, secondary or tertiary amines react with nitrites to form nitrosamines. There are a number of pathways by which nitrosamines can be
introduced into or generated as impurities in pharmaceutical drug products. Examples of sources/pathways identi ed empirically or reported in the literature (2–
3) include (but are not limited to) the following:
API (active pharmaceutical ingredient) processing under speci c conditions and in the presence of certain reagents, solvents, raw materials, and
processing aids. There is evidence that despite processing and puri cation 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 nitrite to remove excess azide.

The API 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 nished drug product.
During drug product manufacture under certain reaction conditions and in the presence of requisite precursors necessary for the formation of
nitrosamines.
Impurities in the container–closure system for the nished 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.

Figure 1. Potential sources of nitrosamine impurities in drug product

Ongoing assessments and evaluations have identi ed risks associated with several of the potential sources of nitrosamines. These are summarized in Table
2.

Table 2

Potential Source of Nitrosamines Observed Riska

Presence of residual dialkyl amines or tri-substituted amines that can

degrade to form dialkyl amines (e.g., triethylamine)
Presence of nitrites or other nitrosating agents
Presence of acid
Limited controls/speci cation limits for recycled solvents
Solvents Poor quality water or solvents

Presence of residual dialkyl amines or impurities that can degrade to

form dialkyl amines
Presence of nitrites or other nitrosating agents
Water Presence of acid

Excipients Presence of nitrites or other nitrosating agents

Use of sodium azide and nitrite for azide quenching in the synthesis
in acid media
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)
Drug substance Need of additional puri cation steps (e.g., crystallization)
 Potential Source of Nitrosamines Observed Riska

Contamination
Use of poor quality or recycled solvents that may contain
nitrosamines or their precursors
Poor quality solvents
Manufacturing process Presence of nitrous oxides in air used to dry the API or drug product

Secondary or tertiary amine group in molecule

Presence of nitrate counter ions (potentially as an impurity)
Potential reactions within the formulation matrix during stability/shelf
life (e.g., presence or generation of acidic conditions, moisture, and
Drug product (including stability) heat)

Thermal decomposition of nitrocellulose to produce nitrites followed

by migration to the drug product
Biodegradation of nitrocellulose to produce nitrites followed by
Container–Closures migration to the drug product

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 reaction responsible for the formation of nitrosamines is described in Figure 2. Examples of representative reactions are described in the scienti c
literature (2–3).

Figure 2. General example of formation of nitrosamines.

If the potential for the presence of nitrosamines is identi ed, an appropriate control strategy should be developed. If nitrosamines are identi ed 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 identi ed 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 requires ensuring that levels of nitrosamines are at or below the provisional acceptable intake (AI) if their
presence could not be avoided, the components of drug products should be assessed 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 API synthetic route, the API manufacturing process, drug product
manufacturing process, and excipients and raw materials should also be subjected to a risk assessment to establish the level of control needed. The high level
process ow for evaluating materials is shown in Figure 3.
 Figure 3. High level process for development of a nitrosamine impurity control strategy. (aRefer to Table 2; P1, P2, P3 = Process 1, 2, 3; D1, D2 = Decision 1, 2)

In all cases, if nitrosamines are predicted in the risk assessment or con rmed to be present through testing in the drug product, the control strategy should
de ne the approach to ensure that the nitrosamine levels comply with the established interim AIs.

5. LIMITS OF NITROSAMINES
Nitrosamine impurities identi ed in this chapter have signi cant toxicity with no therapeutic value. Because nitrosamines are classi ed as Class 1 mutagenic
impurities, rather than applying a Threshold of Toxicological Concern (TTC), the available safety data should be used to establish a material-speci c AI. The AI is
de ned as an intake level that poses a negligible health risk.
5.1 Derivation of the Interim Limits for AI
There are a number of methodologies that toxicologists have applied in establishing AIs. Considering the toxicity of NDMA and NDEA as representative
nitrosamines and the available non-clinical (animal model) data that are available, the interim limits published by FDA (at the time this document was submitted
for publication) were based on the dose giving a 50% tumor incidence (TD50) with a 1:100,000 safety factor applied (decreasing the potential cancer risk to 1 in
100,000).
For the o cial AI values, refer to FDA Updates and Press Announcements on Angiotensin II Receptor Blocker (ARB) Recalls (Valsartan, Losartan, and
Irbesartan).
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 then be used to
calculate the maximum nitrosamine concentration limits, in ppm, for individual drug products using the equation below.

Concentration = AI/DSdose
Since the exposure to nitrosamines is related to the MDD of the drug, different concentrations of nitrosamines (ng/g) may be acceptable for each material
evaluated. The acceptable concentration in the material can be calculated using the equation below.

Acceptable nitrosamine content = AI/MDD

AI = acceptable intake of the nitrosamine (ng/day)

MDD = maximum daily dose of the API (g/day)

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

Name Acceptable Concentration (ng/g)

Nitrosamine 1 0.050 0.100 0.250 1.00

(50 mg dose) (100 mg dose) (250 mg dose) (1000 mg dose)

1920 960 384 96

6. TESTING FOR THE PRESENCE OF NITROSAMINES

Upon completion of the risk assessment, exploratory testing may need to be performed to con rm the conclusions of the risk assessment and proposed
control strategy. On the basis of the controls identi ed (e.g., incoming material testing or speci cation limit; API or drug product speci cation 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 section. Example analytical procedures can be found in 7. Test Method Performance
Characteristics of Nitrosamine Methods.

6.1 Presence of Two or More Nitrosamines

The current published recommended AIs re ect limits for the presence of a single nitrosamine. If multiple nitrosamines are possible and are determined
analytically to be present at levels above the limit of quantitation (LOQ) and below the AI, the relevant health authority should be consulted to determine a
speci c path forward.

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 〈1225〉.
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 nitrosating agent is present. In these situations, sample extractions should be modi ed to prevent the solubilization of the active
ingredient while maintaining the extraction e ciency for nitrosamines present in the material.
In selecting the appropriate analytical procedure, the performance characteristics for a quantitative analytical procedure shown in Table 4 are recommended to
ensure that the method is suitable for its intended purpose. Examples of quantitative analytical procedures are included in 8. Analytical Procedures.

Table 4. Recommended Quantitative Analytical Procedure Performance Criteria

Parameter Recommended Range

Range 50%–150% of the limit corresponding to AI

Accuracy Recovery 70%–130%

Repeatability Relative standard deviation (%RSD) < 25% (n = 6)

Intermediate precision %RSD < 30% (n = 12)

 Parameter Recommended Range

Limit of quantitationa (See 〈1225〉.) Dependent on material MDD and AI

a
  If LOQ is the concentration limit corresponding to AI, then a system suitability test should be added to the procedure for sensitivity veri cation.

If a limit test is selected for the analytical procedure, the performance criteria shown in Table 5 are recommended.

Table 5. Recommended Limit Test Analytical Procedures Performance Acceptance

Parameter Acceptance Criteria

Results RU(i)/RST(i) = NMT 0.5a

The procedure must be able to unequivocally assess (see 〈1225〉 each

target compound in the presence of components that may be expected to
Speci city be present, including other target compounds and matrix components.

Recovery 70%–130%

The minimum concentration at which the analyte can reliably be detected is

Detectability established (signal-to-noise ratio 10:1).

The detectability should meet the requirements throughout the testing

Solution stability period.

a
  RU(i) = Peak response ratio of the respective target N-nitrosamine to the internal standard from the Sample solution; RST(i) = Peak response ratio of the respective target N-nitrosamine to the
internal standard from the Spiked sample solution

8. ANALYTICAL PROCEDURES
The following procedures have been established as suitable for their intended (speci ed) purpose. Use of these methods should be veri ed by the user for the
speci c materials for which they are intended to be applied. The objective of veri cation is to demonstrate the suitability of a test procedure under actual
conditions of use (see Veri cation of Compendial Procedures 〈1226〉).
8.1 Quantitative Procedures
Procedure 1: Quantitation of NDMA, NDEA, NDIPA, NEIPA, NMBA, and NDBA in selected sartans by HPLC–HRMS
Diluent: Methanol
Solution A: 0.1% formic acid in water
Solution B: 0.1% formic acid in methanol
Mobile phase: See Table 6.

Table 6

Time Solution A Solution B

(min) (%) (%)

0 90 10

1.5 90 10

7.0 45 55

17.0 45 55

17.1 10 90

21.0 10 90

21.1 90 10

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, 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-Nitrosoethylisopropylamine RS, USP N-
Nitrosodiisopropylamine RS, USP N-Nitrosodibutylamine 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 lter of 0.2-µm pore size. Use the ltrate for analysis.
Chromatographic system
 (See Chromatography 〈621〉, System Suitability.)
Mode: LC
Detector: High resolution mass spectrometer
MS conditions
Ionization: Electrospray ionization (ESI)
Scan settings: See Table 7.

Table 7

Nitrosamine
Impurity NDMA NMBA NDEA NEIPA NDIPA NDBA

Scan type SIMa SIM PRMb SIM SIM PRM

Polarity positive negative 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 13.0–15.5

m/zc isolated for

PRM N/A N/A 103.0866 N/A N/A 159.1492

NCEd N/A N/A 25 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 50.0–170.0

Microscans 3 3 3 3 3 3

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

AGC target value

(%)e 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
  AGC = automatic gain control.

[NOTE—Divert the API from the MS source during the elution.]

Data processing: Peak areas in the extracted ion chromatograms (EIC) with an m/z tolerance of 15 ppm are used for quantitation. The m/z values extracted
are listed in Table 8.

Table 8

Nitrosamine
Impurity NDMA NMBA NDEA NEIPA NDIPA NDBA

57.0704,
m/z to be 75.0553, 103.0872,
extracted 75.0553 145.0619 103.0866 117.1022 131.1179 159.1492

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

Temperatures
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
Samples: Sensitivity solution and Standard solution
[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.]

[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 speci ed nitrosamine impurity in the portion of drug substance taken:

Result = (rU/rS) × (CS/CU) × 106

rU = peak response of the individual speci ed 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-Nitrosoethylisopropylamine RS, USP N-
Nitrosodiisopropylamine RS, USP N-Nitrosodibutylamine 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).
Procedure 2: Quantitation of NDMA, NDEA, NDIPA, and NEIPA in selected sartans by 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 ask, 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 ask, 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 ask, 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 ask and dilute with Diluent to volume. Transfer 1 mL of this solution to an appropriate headspace vial containing about 100
mg of imidazole. Apply the stopper, cap, and crimp tightly.
Sensitivity solution: Transfer 1 mL of Sensitivity 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.
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.
Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
Mode: GC
Injector: Headspace (see Table 9 for parameters)

Table 9

Equilibration temperature 95°–110°

Loop temperature 150°

Rate 1 10°/min

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

Vial size 20 mL

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

[NOTE—Split ratio can be modi ed 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 10.

Table 10

Hold Time at
Initial Temperature Final Final
Temperature Ramp Temperature Temperature
(°) (°/min) (°) (min)

45 0 45 3

45 10 130 3

130 130 15 —

190 40 240 10

Carrier gas: Helium

Flow rates
Gas: Constant ow at 1.8 mL/min (adjustment and veri cation are necessary for other carrier gases)
Purge: 3.0 mL/min or default value
MS conditions: See Table 11.

Table 11

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

CH 2 collision energy 15.00 V

Event 2

Name NDMA-d6

Start time 10.0 min

End time 12.0 min

Acquisition mode MRM

 Ch 1 m/z 80.00 ˃ 50.00

CH 1 collision energy 5.00 V

Event 3

Name NDEA

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

Event 4

Name NEIPA

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

Event 5

Name NDIPA

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 1 m/z 130.00 ˃ 43.10

CH 2 collision energy 18.00 V

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.]

Suitability requirements
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 speci ed 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)

RU = peak response ratio of the speci ed nitrosamine impurity to that of the internal standard from the Sample solution

RS = peak response ratio of the speci ed 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 solution (µg/mL)

Report the nitrosamine impurity concentration in the drug substance in ppm (µg/g).
Procedure 3: Quantitation of NDMA, NDEA, NDIPA, NEIPA, NMBA, and NDBA in selected sartans by HPLC–MS/MS
Diluent: 1% formic acid in water
Solution A: 0.1% formic acid in water
Solution B: 0.1% formic acid in methanol
Mobile phase: See Table 12.

Table 12

Time Solution A Solution B

(min) (%) (%)

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 standard stock solution 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 13 from the
Nitrosamine standards stock solution mixture and NDEA standard stock solution by mixing speci ed volumes of each solution as indicated. [NOTE—Table 13
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.]

Table 13

Concentratio Content of
n of NDMA, NDMA,
NMBA, NMBA, Volume of Volume of
NDBA, NDBA, Nitrosamine NDEA
NEIPA, NEIPA, Standard Standard Volume of
NDIPA NDIPA Stock Stock Volume of Internal
Linearity Concentratio (ng/mL)/NDE (ppb)/NDEA Solution Solution Water Standard Total Volume
Solution # n Level A (ng/mL) (ppb) Mixture (μL) (μL) (μL) (μL) (μL)

1 L1 1.33/0.66 19.95/10 8 6 1174 12 1200

 Concentratio Content of
n of NDMA, NDMA,
NMBA, NMBA, Volume of Volume of
NDBA, NDBA, Nitrosamine NDEA
NEIPA, NEIPA, Standard Standard Volume of
NDIPA NDIPA Stock Stock Volume of Internal
Linearity Concentratio (ng/mL)/NDE (ppb)/NDEA Solution Solution Water Standard Total Volume
Solution # n Level A (ng/mL) (ppb) Mixture (μL) (μL) (μL) (μL) (μL)

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 Diluent and 12 μl of the Internal standard
solution. Vortex at 2500 rpm for 20 min, except for losartan (vortex losartan NMT 5 min). Centrifuge at about 10,000 rpm for 10 min, and lter into a vial using a
hydrophilic polytetra uoroethylene (PTFE) lter of 0.45-μm pore size.
Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
Mode: LC
Detector: MS/MS (triple quadrupole mass spectrometer)
MS conditions
Ionization: Atmospheric pressure chemical ionization (APCI)
Scan settings: See Table 14.

Table 14

Transitions

Nitrosamine Impurity Acquisition Mode Polarity MRM-1 MRM-2

+75.0 amua → +75.0 amu →

NDMA MRM Positive +43.0 amu +44.1 amu

+81.2 amu → +81.2 amu →

NDMA-d6 MRM Positive +46.0 amu +64.1 amu

+103.1 amu → +103.1 amu →

NDEA MRM Positive +75.1 amu +47.1 amu

+113.2 amu → +113.2 amu →

NDEA-d10 MRM Positive +34.2 amu +49.1 amu

+147.1 amu → +147.1 amu →

NMBA MRM Positive +44.1 amu +117.1 amu

+150.1 amu → +150.1 amu →

NMBA-d3 MRM Positive +47.1 amu +120.2 amu

+159.2 amu → +159.2 amu →

NDBA MRM Positive +41.1 amu +29.1 amu

+177.3 amu → +177.3 amu →

NDBA-d18 MRM Positive +66.2 amu +46.2 amu
 Transitions

Nitrosamine Impurity Acquisition Mode Polarity MRM-1 MRM-2

+117.1 amu → +117.1 amu →

NEIPAb MRM Positive +75.1 amu +47.2 amu

+131.2 amu → +131.1 amu →

NDIPAb MRM Positive +89.1 amu +47.1 amu

a
  The abbreviation amu is atomic mass unit.
b
  NDEA-d10 is used as internal standard for NDIPA and NEIPA.

Column: 3.0-mm × 15-cm; 2.7-µm packing L1

Temperatures
Autosampler: 18°
Column: 60°
Flow rate: 0.5 mL/min
Flow rate to ion source: 0.5 mL/min
Injection volume: 20 µL
System suitability
Samples: Standard solutions
Generate the peak response ratio of the speci ed impurity to that of the internal standard versus 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.]
Suitability requirements
Correlation coe cient: 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 speci ed nitrosamine impurity in the Sample solution using the corresponding calibration curve:

Result = [(RU − yint)/a] × (1/CU) × 103

RU = peak response ratio of the speci ed nitrosamine impurity to that of the internal standard from the Sample solution

yint = y-intercept of the calibration curve for the speci ed nitrosamine impurity from the Standard solutions

a = slope of the calibration curve for the speci ed nitrosamine impurity from the Standard solutions

CU = concentration of the drug substance in the Sample solution (mg/mL)

Report the nitrosamine impurity concentration in the drug substance in ppm (µg/g).
Procedure 4: Quantitation of NDMA, NDEA, NDIPA, NEIPA, and NDBA in selected sartans by GC–MS/MS (triple-quad)
Internal standard solution: 50 ng/mL of NDMA:C13-d6 in methylene chloride
Standard solution: Prepare a mixture of 0.1 µg/mL each of N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosoethylisopropylamine, N-
nitrosodiisopropylamine, 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 ve consecutive solutions from Table 15 that are
used for generating the calibration curve by following the preparation scheme shown in Table 15. Volumes may be adjusted to prepare larger quantities of the
calibration solutions as needed, maintaining nal concentrations of the nitrosamines. For each calibration solution, transfer the designated aliquot of Standard
solution to the designated volumetric ask, and adjust the volume with the Internal standard solution.

Table 15

Final Nitrosamine Equivalent Nitrosamine

Standard Solution Aliquot Final Volume Concentration Concentration
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

 Final Nitrosamine Equivalent Nitrosamine
Standard Solution Aliquot Final Volume Concentration Concentration
Calibration Solution ID (μL) (mL) (μg/mL) (μg/g)

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 tted with a 0.45-μm nylon lter. Filter 1 mL of sample extract into a 2-mL GC autosampler vial and cap.
Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
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
Scan settings: See Table 16.

Table 16

Transitions

Nitrosamine Impurity Acquisition Mode Polarity MRM-1a MRM-2

74 amu → 74 amu →
NDMA MRM Positive 44 amu 42 amu

82 amu →
NDMA:c13-d6 MRM Positive 48 amu –

102 amu → 102 amu →

NDEA MRM Positive 85 amu 56 amu

116 amu → 71 amu →

NEIPA MRM Positive 99 amu 56 amu

130 amu → 130 amu →

NDIPA MRM Positive 88 amu 42 amu

158 amu → 84 amu →

NDBA MRM Positive 99 amu 56 amu

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
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 17.

Table 17

Hold Time at
Initial Temperature Final Final
Temperature Ramp Temperature Temperature
(°) (°/min) (°) (min)

40 0 40 0.5

40 20 200 0
 Hold Time at
Initial Temperature Final Final
Temperature Ramp Temperature Temperature
(°) (°/min) (°) (min)

200 60 250 3

Carrier gas: Helium

Flow rate: Constant ow at 1.0 mL/min (adjustment and veri cation are necessary for other carrier gases)
Injection volume: 2 µL
System suitability
Samples: Calibration solutions
Generate the peak response ratio of the speci ed 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.
[NOTE—The relative retention times for NDMA, NDEA, NEIPA, NDIPA, and NDBA are 0.74, 0.80, 0.83, 0.85, and 1.00, respectively.]
Suitability requirements
Correlation coe cient: 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 speci ed 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)

RU = peak response ratio of the speci ed nitrosamine impurity to that of the internal standard from the Sample solution

yint = y-intercept of the calibration curve for the speci ed nitrosamine impurity from the corresponding Calibration solutions

a = slope from the calibration curve for the speci ed nitrosamine impurity from the corresponding Calibration solutions

Report the nitrosamine impurity concentration in the drug substance in ppm (µg/g).

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 18.

Table 18

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
Internal standard solution target limit of the nitrosamines of interest in the sample.

Prepare a solution of the article to be examined, spiked with the internal

standard, and prepared as described in the sample preparation. The amount
of substance 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, would be equal to the contribution of the respective spiking
Sample solution 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
Spiking solution result in the acceptance limit.

Prepare a solution of the article to be examined spiked with a) appropriate

Spiking solution(s) and b) Internal standard solution prepared as described in
Spiked sample solution the Sample solution.

9. ADDITIONAL SOURCES OF INFORMATION

Several test procedures have been developed for the speci c testing of nitrosamines in sartans and/or other o cial articles based on different scienti c
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 to meet the respective performance characteristics
acceptance criteria set forth in 7. Test Method Performance Characteristics of Nitrosamine Methods.
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%20on%20angiotensin%20II%20receptor%20blocker%20%28ARB%29%20recalls%20-%20FDA%20publishes%20LC-
HRMS%20and%20RapidFire-MS%2FMS&utm_medium=email&utm_source=Eloqua#testingmethods.
2.
 Ph. Eur. 2.4.36 N-Nitrosamine impurities in active substances: https://pharmeuropa.edqm.eu/app/phpa/content/issue32-2/20436E.htm?
3. EDQM projects on sampling strategies and testing methods with the O cial Medicines Control Laboratory (OMCL) Network: https://www.edqm.eu/en/ad-
highlight=on&terms=2.4.36.
hoc-projects-omcl-network.

10. USP REFERENCE STANDARDS 〈11〉

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

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:
ElsevierScience; 2004:1–34.
Ogata Y, Sawaki Y, Kuriyama Y. The reaction of trialkylamine with nitric acid in a mixture of acetic acid and acetic anhydride. Tetrahedron.
3. 1968;24(8):3425–3435.
▲ (USP 1-May-2022)

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Topic/Question Contact Expert Committee

<1469> NITROSAMINE IMPURITIES Edmond Biba GCCA2020 General Chapters - Chemical Analysis
Senior Scienti c Liaison 2020

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