Nitrosamine testing (NDMA, NDEA, NMBA) for Pharmaceutical products
Do you have any request or concern about nitrosamine testing of pharmaceutical APIs and formulations?
- To assess the level of nitrosamine in APIs or formulations is below detection limit.
- To perform nitrosamine testing other than NDMA and NDEA.
Eurofins Analytical Science Laboratories, Inc. is equipped with GMP-complied analytical equipment for nitrosamine testing. We can provide method development, validation, quantitative testing and detection limit testing services.
Also, Eurofins companies offer wide variety of screening services of Nitrosamine.
Traces of N-nitrosodimethylamine (NDMA) found in some sartans active pharmaceutical ingredients (APIs). In a later phase, detection of these impurities in other classes of medicine, such as ranitidine and metformin, raised additional concerns about patient safety.
The FDA, the EMA, and the PMDA are defining new requirements: temporary limits for most common nitrosamines are currently being set based upon the Maximum Daily Intake. And they further recommend that manufactures ensure nitrosamines impurities are below the limits.
Control of nitrosamine impurities
What are nitrosamines?
Nitrosamines are compounds of the chemical structure where hydrogen of the amine nitrogen is substituted with a nitroso group (Figure 1). They are generally formed from the reaction of nitrous acid and secondary amines.
(Figure 1)
N-nitrosodimethylamine (NDMA) having -CH3 in R1 and R2 and N-nitrosodiethylamine (NDEA) having -CH2CH3 in R1 and R2 are considered as carcinogenic nitrosamines.
The carcinogenicity of NDMA was confirmed in 1957 in Norway, through the report of an increase of liver cancers in domestic animals.
This was caused by herring meal preserved with sodium nitrite. Its sodium nitrite reacts with dimethylamine in herring to produce NDMA.
N-nitrosamines are present in some food, water, cosmetic products, cigarettes etc.
Backgrounds of Nitrosamines in Pharmaceuticals from its awareness to regulations
NDMA and NDEA were found in Valsartan API, an angiotensin II receptor blocker (sartan drugs) in July 2018.
The nitrosamine forming pathway in sultan-based drugs is considered to be due to the reaction of the solvent dimethylformamide (DMF) during synthesis with nitrous acid used to quench residual azide used in tetrazole ring formation.
[References: WHO; Information Note Nitrosamine impurities, FDA; Control of Nitrosamine Impurities in Human Drugs]
In response to this event, investigation and recalls of sartan drugs were done in the United States and Europe.
Also in Japan, the Ministry of Health, Labor and Welfare has set the control indicators for NDMA and NDEA in sartan drugs.
Manufacturers of sartan drugs which has a risk of generating NDMA and NDEA need to manage according to this control indicators.
In 2019, the FDA and EMA reported that NDMA was detected in formulations and APIs of ranitidine hydrochloride, the histamine H2 receptor antagonist.
The forming pathway of NDMA in Ranitidine is considered to be due to temperature and the structure of ranitidine itself. Ranitidine has an N-dimethyl structure, which is the source of NDMA, and a nitro group (Figure 2).
(Figure 2)
NDMA levels increase in ranitidine even under normal storage conditions, and NDMA has been found to increase significantly in samples stored at higher temperatures, including temperatures the product may be exposed to during distribution and handling by consumers.
[References: FDA; FDA Requests Removal of All Ranitidine Products (Zantac) from the Market]
According to the assessment by the third party testing institution, it has been shown that ranitidine can react with itself in standard analysis conditions with temperature of 130 °C to produce NDMA.
[References: Valisure; Valisure Citizen Petition on Ranitidine]
Following the results, the Ministry of Health, Labor and Welfare has set an acceptable intake limit of NDMA for ranitidine hydrochloride or nizatidine, which is chemically similar to ranitidine.
Manufactures which manufacture these APIs and drug products should take appropriate control measures to ensure the acceptable intake levels.
In addition, the Health Sciences Authority of Singapore (HSA) reported that metformin hydrochloride medicine, a biguanide oral hypoglycemic agent has been found to contain trace amounts of a nitrosamine impurity, N-nitrosodimethylamine (NDMA).
The pathway of NDMA formation in metformin has not yet been identified.
Following this report, the Ministry of Health, Labor and Welfare has set an acceptable intake levels of for metformin medicines.
Control of nitrosamines in pharmaceutical products in Japan
Control of nitrosamines in pharmaceutical products is based on the guideline "ICH M7 Guideline on assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk", which emphasizes considerations of both safety and quality risk management in establishing levels of mutagenic impurities that are expected to pose negligible carcinogenic risk.
Once setting the acceptable intake levels of the impurity from the toxicity data, appropriate acceptable intake limits of mutagenic impurities in drug substances and drug products can be set so as not to exceed an acceptable dose.
A compound-specific acceptable intake limit of NDMA and NDEA is controlled as shown below.
| Name of compounds | A maximum daily dose (mg) | An acceptable intake limit of NDMA in API (ppm) | An acceptable intake limit of NDEA in API (ppm) |
| Valsartan | 160 | 0.599 | 0.166 |
| Irbesartan | 200 | 0.479 | 0.133 |
| Olmesartan | 40 | 2.39 | 0.663 |
| Losartan | 100 | 0.959 | 0.265 |
| Ranitidine | 300 | 0.32 | --- |
| Nizatidine | 300 | 0.32 | --- |
| Metformin | 2250 | 0.043 | --- |
The acceptable intake limits above are referred the following notice from the Ministry of Health, Labor and Welfare.
- https://www.mhlw.go.jp/content/11121000/000378171.pdf
- https://www.pmda.go.jp/files/000231528.pdf
- https://www.pmda.go.jp/files/000232925.pdf
Impurity assessment must be performed at an inspection level confirming that it is below each acceptable intake limit.
Approaches to testing nitrosamines in Eurofins Analytical Science Laboratories
A general workflow of testing nitrosamines in Eurofins Analytical Science Laboratories, Inc. is shown below (Figure 3).
(Figure 3)
First of all, we will develop a test method that can qualify the limits of NDMA and NDEA using LC-HRMS (HR: High Resolution) according to the properties of the API and formulation. We also offer method development by GC-MS as per your request.
We perform method validation according to ICH-Q2 guidelines.
And we perform quantitative testing and limit testing by using the validated methods.
Why choose Eurofins Analytical Science Laboratories for your Nitrosamine testing?
- Eurofins Analytical Science Laboratories, Inc. can analyze with high accuracy, high accuracy and high accuracy by using GMP-compliant LC-HRMS.
- Eurofins companies offer variety of screening services and Eurofins Analytical Science Laboratories, Inc. fully supports the testing.
Eurofins Analytical Science laboratories, Inc. is an organization in compliance with GMP Ministerial Ordinance.
Under its control and management, we create protocols, perform testing, store samples, and manage facilities, equipment, and systems.
We use LC-HRMS under its control.
GC-MS is normally used for Nitrosamine testing.
However, GC-MS is not suitable instrumentation as compounds such as ranitidine and nizatidine are sensitive to heat, and the compound itself may decompose to generate NMDA.
In addition, nitrosamines are quantified in the lower concentration range.
Therefore, we develop test method, perform validation and quantitative testing, or limit testing using LC-HRMS, which can measure heat-sensitive compounds and also can be measured with high sensitivity, high accuracy, and high resolution.
In Japan, we can perform testing of NDMA, NDEA and also NMBA（N-Nitroso-N-methyl-4-aminobutyric Acid that are considered impurity risks of Nitrosamines by FDA*.
[*References: FDA; FDA Updates and Press Announcements on Angiotensin II Receptor Blocker (ARB) Recalls (Valsartan, Losartan, and Irbesartan)- 3/1/2019: UPDATE - Torrent again expands its voluntary recall of losartan; Hetero also voluntarily recalls losartan]
These 3 are not the only nitrosamines need to be analyzed.
We can also perform screening of the following 6 types of nitrosamines at Eurofins global labs. We fully support the testing that is performed at our global labs.
- NDMA：N-Nitrosodimethylamine
- NDEA：N-Nitrosodiethylamine
- NMBA：N-Nitroso-N-Methyl-4-Aminobutyric Acid
- NDBA：N-Nitrosodi-n-butylamine
- NEIPA：N-Nitrosoethylisopropylamine
- NDIPA：N-Nitrosodiisopropylamine
Eurofins Analytical Science Laboratories, Inc. is equipped with GMP-complied analytical equipment for nitrosamine impurities testing. We can provide method development, validation, quantitative testing and detection limit testing services.
We support your nitrosamine impurities testing. Please feel free to contact us if you have any questions and requests.