Nitrosamine Testing of Pharmaceuticals: Detection of Carcinogenic Contaminants and Nano-Test Solutions
In 2018, the pharmaceutical industry experienced a major crisis due to contamination with N-nitrosodimethylamine (NDMA), which was detected in blood pressure medications in the “sartan” class. This incident led to the recall of millions of medications worldwide and prompted regulatory agencies to fundamentally change their approach to nitrosamine testing in medications. Today, monitoring for nitrosamine contamination has become an integral part of drug development and manufacturing processes.
Nitrosamines are compounds whose chemical structure contains an N-nitroso group (R1N(-R2)-N=O). Many of them have shown strong carcinogenic effects in animal studies. Their formation in pharmaceuticals can depend on numerous factors, including the synthesis of the starting materials, the manufacturing process, packaging materials, and even storage conditions. Therefore, continuous monitoring using precise and reliable analytical methods is essential to prevent patients from being exposed to these harmful compounds.
Sources and Formation Mechanisms of Nitrosamine Contaminants
Nitrosamines can enter medicinal products in various ways or form spontaneously within them. The most common mechanism of formation is the reaction of secondary or tertiary amines with nitrous acid (HNO₂) or nitrite salts. This reaction is accelerated under acidic conditions.
- Derived from raw materials: Solvents, catalysts, or starting materials used in the synthesis of the active pharmaceutical ingredient (API) may contain nitrosamine impurities.
- Process-related: Certain excipients used during manufacturing may contain nitrites and react with the amines in the API.
- Due to packaging: In particular, blister packaging containing nitrocellulose-based printing inks can cause the migration of volatile nitrosamines into the drug.
- Storage conditions: High humidity and temperature are factors that accelerate the formation of nitrosamines.
Regulatory limits and current legal provisions
The European Medicines Agency (EMA), the U.S. Food and Drug Administration (FDA), and the Turkish Medicines and Medical Devices Agency (TİTCK) have established extremely low limits for nitrosamine impurities. These limits were calculated based on a “lifetime exposure scenario” and are intended to keep the cancer risk at a negligible level.
For NDMA, one of the most common nitrosamines, the acceptable daily intake (ADI) has been set at 96 ng/day. This means that the maximum amount of NDMA permitted in a daily dose of a drug is one part per billion (ppb). Highly sensitive analytical techniques are required to detect such low levels.
Regulatory authorities require marketing authorization holders (MAHs) to conduct a risk assessment for all their products and to perform confirmatory testing for products in which a risk has been identified. This process is carried out in three steps: “Step 1: Risk Assessment,” “Step 2: Confirmatory Testing,” and “Step 3: Change Management.”
Commonly Monitored Nitrosamine Contaminants
- NDMA (N-nitrosodimethylamine): One of the most common and potent carcinogenic nitrosamines.
- NDEA (N-nitrosodiethylamine): Also detected in medications of the sartan class and in some antihistamines.
- NMBA (N-nitroso-N-methyl-4-aminobutyric acid): Has been detected in medications such as valsartan.
- NIPEA (N-nitrosoethyl-isopropylamine): Found in some generic drugs.
- NDSRI (nitrosamine drug substance-related impurities): These are new-generation nitrosamines that originate from the active pharmaceutical ingredient itself and have been the focus of attention in recent years.
Common Nitrosamines and Acceptable Daily Intake (ADI) Limits
The following table summarizes the nitrosamine impurities most commonly detected in drugs, as well as the limits for acceptable daily intake established by regulatory authorities:
| Name of the nitrosamine | Chemical Formula | Acceptable Daily Intake (AI) | Carcinogenicity Class |
|---|---|---|---|
| NDMA (N-nitrosodimethylamine) | C₂H₆N₂O | 96 ng/day | IARC Group 2A (probably carcinogenic) |
| NDEA (N-nitrosodiethylamine) | C₄H₁₀N₂O | 26.5 ng/day | IARC Group 2A (probably carcinogenic) |
| NMBA (N-nitroso-N-methyl-4-aminobutyric acid) | C₅H₁₀N₂O₃ | 96 ng/day | IARC Group 2B (possibly carcinogenic) |
| NIPEA (N-nitrosoethyl isopropylamine) | C₅H₁₂N₂O | 26.5 ng/day | IARC Group 2B (possibly carcinogenic) |
Analytical Methods for Nitrosamine Analysis
Detecting nitrosamines requires extremely sensitive analytical techniques due to the low detection limits in the ppb range (parts per billion). Conventional HPLC methods often cannot achieve this level of sensitivity.
1. LC-MS/MS (Liquid Chromatography–Mass Spectrometry)
This is currently the gold standard for nitrosamine analysis. Thanks to its high selectivity and sensitivity, nitrosamines can be reliably detected even in complex pharmaceutical matrices at very low concentrations. The Multi-Reaction Monitoring (MRM) mode minimizes false-positive results by tracking specific ion transitions.
2. GC-MS/MS (Gas Chromatography–Mass Spectrometry)
Suitable for volatile nitrosamines (such as NDMA and NDEA). Can be used with headspace or direct injection techniques. However, the method may not be suitable for some thermally unstable nitrosamines.
3. HRMS (High-Resolution Mass Spectrometry)
A powerful tool for identifying and elucidating the structure of unknown nitrosamine contaminants. It plays a crucial role, particularly in the discovery of NDSRIs.
GLP-Compliant Nitrosamine Testing Services with Nano-Test
Conducting nitrosamine risk assessments and confirmatory testing for pharmaceutical manufacturers requires extensive expertise and an appropriate infrastructure.
With its GLP-compliant analytical services in this field, Nano-Test is the most reliable solution partner for the pharmaceutical industry.
Description of Nano-Test Services
Nano-Test offers the pharmaceutical industry comprehensive services in the field of nitrosamine impurity analysis. In our laboratories, which are accredited according to GLP (Good Laboratory Practice) and ISO/IEC 17025, we perform analyses that comply with all current limit values established by the EMA, the FDA, and the TİTCK.
Our scope of services includes the detection and quantitative determination of all common nitrosamines, including NDMA, NDEA, NMBA, NIPEA, and NDSRI. We use state-of-the-art equipment such as LC-MS/MS, GC-MS/MS, and HRMS in our analyses. For each analysis, we apply fully validated methods that comply with the relevant pharmacopoeia monographs and current regulatory guidelines.
As Nano-Test, we not only provide test results, but also prepare comprehensive and well-founded GLP-compliant reports that you can use for your regulatory submissions and that are accepted for inspections. We support you throughout all processes, from risk assessment to confirmatory testing.
The advantages of Nano-Test in nitrosamine analysis:
- Extremely low detection limits: Detection performance below the ppb range (ng/g) using LC-MS/MS.
- Full validation: Method validation in accordance with ICH Q2(R1) and the relevant guidelines.
- Fast Turnaround: Results are delivered within 10 business days for standard analyses.
- Expert Team: A team of chemists specializing in mass spectrometry and impurity profiles.
- Compliance with current regulatory requirements: Immediate adaptation to the latest changes in limit values published by the EMA and the FDA.
Nitrosamine contamination is one of the most serious quality and safety issues facing the pharmaceutical industry. To ensure patient safety and meet regulatory requirements, nitrosamine testing of pharmaceuticals is no longer an option but a requirement. Selecting the right analytical methods, ensuring thorough validation, and reporting results in accordance with GLP standards are of critical importance.
Nano-Test supports pharmaceutical manufacturers in managing nitrosamine risks with its state-of-the-art technological infrastructure and team of experts. Have your nitrosamine analyses performed by Nano-Test to ensure your products have unimpeded access to global markets.