Your Nitrosamine Impurity Testing FAQ: Answers for Regulatory Readiness and Confirmatory Analysis

Nitrosamine impurity testing is the analytical process of detecting and quantifying nitrosamines—carcinogenic compounds often formed as by-products in drug synthesis or storage. These impurities can originate from amines, nitrites, or specific reaction conditions involving solvents and catalysts. Testing is essential to comply with international guidelines and protect patient safety.
At ResolveMass, our nitrosamine impurity testing workflows involve LC-MS/MS, GC-MS, and high-resolution mass spectrometry to detect trace levels (ng/g or ppb range) of known and unknown nitrosamines.
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Some of the most frequently encountered nitrosamines include:
NDMA (N-Nitrosodimethylamine)
NDEA (N-Nitrosodiethylamine)
NMBA (N-Nitroso-N-methyl-4-aminobutyric acid)
DIPNA (N-Nitroso-diisopropylamine)
EIPNA (N-Nitroso-N-ethylisopropylamine)
NDIPA (N-Nitrosodiisopropylamine)
NDBA (N-Nitrosodibutylamine)
ResolveMass routinely performs nitrosamine impurity testing for all listed compounds and provides custom method development for client-specific targets.

The following instrumentation is typically employed:
LC-MS/MS for polar and thermally labile nitrosamines
GC-MS for volatile nitrosamines
High-resolution MS for unknown impurity screening
Our Nitrosamine Analysis Services combine these techniques with risk-based workflows and orthogonal confirmation for regulatory accuracy.

Regulatory agencies have established AI (acceptable intake) levels, usually expressed in nanograms per day:
NDMA: 96 ng/day
NDEA: 26.5 ng/day
NMBA: 96 ng/day
DIPNA & EIPNA: 26.5 ng/day
These limits form the basis of specification setting during nitrosamine impurity testing. If an impurity exceeds the AI, it must be either justified, mitigated, or the product must be reformulated or withdrawn.

Common root causes include:
Use of nitrite-containing excipients
Secondary or tertiary amines in APIs or reagents
Acidic pH environments
Recycled solvents or catalysts
Manufacturing equipment residues
Our Nitrosamine Risk Assessment Services include route analysis and synthetic process reviews to identify and mitigate these risks.

Confirmatory nitrosamine impurity testing refers to the validated, sensitive reanalysis of a sample following an initial detection. It ensures result reproducibility and supports regulatory submissions.
ResolveMass offers:
Validation per ICH Q2(R1) and FDA guidelines
Isotope dilution and internal standards
LOD/LOQ determination
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With techniques like LC-MS/MS operating at detection limits below 10 ng/g, nitrosamine impurity testing at ResolveMass is both precise and reproducible. We perform linearity, accuracy, and robustness checks to ensure method reliability.

Yes. Using HRMS (High-Resolution Mass Spectrometry), our experts can identify and characterize unknown or novel nitrosamines—critical for regulatory response in the case of non-targeted impurity findings.
Learn about advanced impurity profiling

Typical timelines are:
Risk Assessment: 5–7 business days
Confirmatory Testing: 7–10 business days
Custom NDSRI Synthesis: 3–6 weeks
Visit our nitrosamine testing service page for expedited options.

Yes. For investigational or confirmatory work, we provide custom synthesis of Nitrosamine Drug Substance-Related Impurities (NDSRIs).
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Nitrosamine impurity testing typically involves advanced analytical methods such as LC-MS/MS and GC-MS. These techniques allow for sensitive detection down to the parts-per-billion (ppb) or nanogram per gram levels. At ResolveMass Laboratories Inc., we use validated methods with isotope dilution, matrix-matched calibration, and internal standards to ensure accuracy and reproducibility across all test runs. This ensures regulatory-compliant quantification of nitrosamines such as NDMA, NDEA, and other NDSRIs.

Calculating nitrosamine content involves comparing the peak area (or intensity) of a sample against that of a known standard curve generated using authentic nitrosamine standards. The response factor, sample weight, and dilution are factored into a formula:
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Nitrosamine Content (ng/g) = (Peak Area_sample / Slope_standard curve) × Dilution Factor / Sample Weight
ResolveMass provides precise calculation of impurity concentrations as part of our standard nitrosamine impurity testing reports.

Nitrosamines are a class of genotoxic and potentially carcinogenic compounds that may form in drug products through several pathways, including:
Secondary amine reactions with nitrites under acidic conditions
Residual solvents or catalysts during synthesis
Cross-contamination from excipients or equipment
Understanding the basics of nitrosamine formation is critical for effective nitrosamine impurity testing and risk assessment. This includes knowledge of structure-reactivity relationships and potential synthetic routes of impurity formation.

Detecting nitrosamines requires highly sensitive and selective analytical techniques due to their low permissible limits and structural diversity. The most widely used methods in nitrosamine impurity testing include:
LC-MS/MS (Liquid Chromatography–Tandem Mass Spectrometry): Ideal for polar and thermally unstable nitrosamines like NMBA. Allows accurate quantification in the sub-ng/g range.
GC-MS (Gas Chromatography–Mass Spectrometry): Preferred for volatile nitrosamines such as NDMA and NDEA. Suitable for detecting low molecular weight impurities.
High-Resolution Mass Spectrometry (HRMS): Used for identifying and confirming unknown nitrosamines, including novel NDSRIs (Nitrosamine Drug Substance-Related Impurities).
At ResolveMass Laboratories Inc., our validated nitrosamine impurity testing platform integrates all three techniques to provide orthogonal detection, confirmation, and quantification in accordance with FDA, EMA, and Health Canada guidelines.
Explore our validated nitrosamine analysis services →

While nitrate testing is distinct from nitrosamine impurity testing, it is often relevant due to the role of nitrites/nitrates in forming nitrosamines. Common methods include:
Ion Chromatography (IC): The most accurate method for quantifying nitrate (NO₃⁻) and nitrite (NO₂⁻) ions in pharmaceutical excipients and water.
UV Spectrophotometry: Applicable for nitrate detection using diazotization or Griess reagent for colorimetric analysis.
Capillary Electrophoresis: An alternative technique for nitrate/nitrite speciation, especially in complex matrices.
Monitoring nitrate content in raw materials is part of a broader nitrosamine risk assessment strategy, which complements our nitrosamine impurity testing workflows.

Products most likely to contain high concentrations of nitrosamines include:
Pharmaceuticals: Especially those containing secondary or tertiary amines in their API structure or synthesis pathway. For instance, some batches of ranitidine, sartans, and metformin have been found to contain elevated nitrosamine levels during nitrosamine impurity testing.
Cured Meats and Processed Foods: Bacon, sausages, smoked fish, and preserved meats often contain nitrosamines formed from nitrite preservatives during cooking.
Tobacco Products: Cigarettes and chewing tobacco release nitrosamines upon combustion or metabolic activation.
Rubber and Cosmetic Products: Nitrosamines may also be present in rubber nipples, condoms, or cosmetic ingredients derived from nitrosatable precursors.
At ResolveMass Laboratories Inc., our advanced nitrosamine impurity testing methods help detect trace nitrosamine levels even in complex matrices, supporting consumer safety and regulatory compliance.
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Determination of nitrate (NO₃⁻) concentrations is crucial in both pharmaceutical and environmental analysis. Standard methods include:
Ion Chromatography (IC): The gold standard for accurate nitrate quantification in raw materials, APIs, and excipients. IC is highly selective and sensitive.
UV/Vis Spectrophotometry (Griess Method): Commonly used for nitrate and nitrite in food and water testing. Involves reduction of nitrate to nitrite followed by colorimetric detection.
Potentiometric Analysis (Ion-Selective Electrodes): Useful for rapid field testing, though it has lower specificity than chromatographic methods.
Capillary Electrophoresis (CE): Offers high resolution for nitrate/nitrite speciation, especially in multicomponent formulations.
These nitrate detection methods are often integrated into our nitrosamine risk assessment strategy, helping clients proactively prevent nitrosamine formation. This complements our confirmatory nitrosamine impurity testing protocols.
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Foods that naturally contain or can form high levels of nitrosamines include:
Cured meats (e.g., bacon, hot dogs, ham): Due to nitrite preservatives that can form nitrosamines during cooking at high temperatures.
Smoked and grilled fish: Especially species like smoked salmon and mackerel.
Cheese and dairy: Some aged cheeses may accumulate trace nitrosamines during fermentation.
Beer and malt beverages: Malt drying processes may contribute to nitrosamine presence.
Pickled vegetables and fermented foods: Under certain pH and bacterial conditions.
While food exposure is monitored by regulatory agencies, nitrosamine impurity testing is more stringent in pharmaceuticals, where even trace levels can trigger recalls or reformulations.
Understand more on our nitrosamine testing protocols →

Several high-profile drug recalls have occurred due to the detection of nitrosamine impurities above acceptable intake limits. Common examples include:
Ranitidine (Zantac®) – Recalled globally after detection of NDMA under storage and heat conditions.
Sartans (e.g., Valsartan, Losartan) – Recalled due to contamination with NDMA, NDEA, and NMBA during synthesis.
Metformin ER formulations – Recalled in various regions due to NDMA detection above safe limits.
Rifampin and Rifapentine – Under surveillance due to risk of N-nitrosamine formation.
Our validated nitrosamine impurity testing services help prevent such outcomes through early detection, confirmatory analysis, and NDSRI synthesis.
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Nitrosamines are classified as probable human carcinogens, but they do not cause immediate or acute symptoms. However, long-term exposure—even at trace levels—has been linked to:
Liver damage and fibrosis
DNA alkylation and mutation
Gastrointestinal cancer risks
Tumor development in various organs (observed in animal studies)
Since symptoms develop over prolonged exposure, nitrosamine impurity testing in pharmaceuticals is critical to avoid chronic ingestion risks.
If your product is at risk, our team at ResolveMass can provide comprehensive confirmatory testing and risk assessment services to ensure regulatory compliance and patient safety.
Explore our advanced testing capabilities →