Nitrosamine Impurities Testing and Analysis

Nitrosamine impurities are tested using highly sensitive analytical techniques such as LC-MS/MS (Liquid Chromatography-Tandem Mass Spectrometry), GC-MS (Gas Chromatography-Mass Spectrometry), and Headspace GC-MS. At ResolveMass Laboratories, we follow a multi-step confirmatory workflow that includes:
Sample preparation (solid, liquid, or tablet form)
Extraction and concentration of nitrosamines
Quantitative detection using validated LC-MS/MS or GC-MS protocols
We comply with FDA, EMA, and Health Canada guidance on nitrosamine impurity testing in pharmaceutical APIs and finished drug products.
✅ Also see: Nitrosamine Analysis Service

The commonly recognized 7 nitrosamine impurities of concern in pharmaceuticals are:
NDMA (N-Nitrosodimethylamine)
NDEA (N-Nitrosodiethylamine)
NMBA (N-Nitroso-N-methyl-4-aminobutyric acid)
NDIPA (N-Nitrosodiisopropylamine)
NEIPA (N-Nitrosoethylisopropylamine)
NDBA (N-Nitrosodibutylamine)
NPIP (N-Nitrosopiperidine)
These nitrosamines are classified as probable human carcinogens and are regulated by international agencies such as ICH, EMA, and FDA.

Nitrosamine impurity levels are typically calculated by:
Running standard calibration curves using known concentrations of the nitrosamine standards
Analyzing the sample using validated LC-MS/MS or GC-MS
Measuring the peak area or intensity of the analyte and interpolating the value from the calibration curve
The result is reported as ng/g or ng/day, and compared against acceptable intake (AI) limits defined by regulatory bodies (e.g., 96 ng/day for NDMA).

The acceptable intake (AI) limits for nitrosamine impurities are defined based on lifetime cancer risk. Examples include:
NDMA: 96 ng/day
NDEA: 26.5 ng/day
NMBA: 96 ng/day
NEIPA/NDIPA/NDBA/NPIP: Between 26.5–96 ng/day depending on the compound
These limits are derived by regulatory agencies such as the FDA, EMA, and Health Canada, and are used to ensure the safety of pharmaceuticals over long-term use.

The maximum contaminant level (MCL) for nitrates in drinking water, as set by the U.S. Environmental Protection Agency (EPA), is 10 mg/L (or 10 ppm) measured as nitrogen (NO₃⁻-N). Exceeding this level can pose serious health risks, especially for infants (i.e., “blue baby syndrome”) and immunocompromised individuals.
💡 Nitrates are precursors to nitrosamine formation, especially during pharmaceutical synthesis or in contaminated water systems.

The root causes of nitrosamine impurities in pharmaceuticals include:
Use of secondary or tertiary amines in drug synthesis or as residuals in APIs and excipients
Contaminated raw materials, solvents, or reagents used in the manufacturing process
Reactions with nitrite under acidic conditions, especially during synthesis or storage
Cross-contamination in shared manufacturing lines
Degradation of certain drug substances (e.g., sartans, ranitidine, metformin)
✅ Risk assessment and impurity identification should be conducted using validated workflows such as LC-MS/MS and GC-MS, as offered by our Nitrosamine Analysis Service.

Nitrosamines can be reduced or eliminated from pharmaceutical products and manufacturing processes through several approaches:
Process Optimization: Modify synthetic routes to avoid nitrosating agents or secondary amines.
Alternative Reagents: Replace nitrite salts or amine precursors with non-nitrosating alternatives.
Purification Techniques: Use solid-phase extraction (SPE), distillation, or chromatographic separation to isolate and remove nitrosamines.
Control of Storage Conditions: Limit exposure to heat, humidity, and acidic environments that promote nitrosamine formation.
Routine Testing: Implement LC-MS/MS and GC-MS screening with validated Nitrosamine Impurity Testing workflows, like those available at ResolveMass Laboratories.

Over the last few years, numerous pharmaceuticals have been recalled globally due to unacceptable levels of nitrosamine impurities, including:
Valsartan, Losartan, Irbesartan (sartan-class antihypertensives)
Ranitidine and Nizatidine (H2 blockers for gastric reflux)
Metformin (for type 2 diabetes)
Chlorzoxazone (muscle relaxant)
Quinapril (ACE inhibitor)
These recalls were triggered by detection of NDMA, NDEA, and other nitrosamines beyond acceptable intake limits. Testing and confirmatory analysis is now critical in all stages of pharmaceutical development.
Explore our Impurity Profiling Service for deeper insights on compound-specific risk.

Vitamin C (ascorbic acid) is well-documented to inhibit the formation of nitrosamines, especially in acidic environments like the human stomach. It works by:
Reacting with nitrite before it can form nitrosating species
Acting as a reducing agent to prevent the conversion of amines into nitrosamines
This antioxidant effect is especially relevant in food chemistry and dietary supplementation but also informs nitrosamine risk assessments during oral drug formulation.