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Nitrosamines problem: 

Nitrosamines belong to a well-known group of carcinogens known as N-nitroso compounds because of their reactivity with DNA metabolizing into potent carcinogen diazonium ions. Nitrosamine formation lies in the oxidative release of the known nitrosating agents and subsequent nitrosamine formation by reaction with secondary and tertiary amines or dealkylated ammonium salts under acidic conditions.  

These conditions can be found in manufacturing processes for small molecule drugs and therefore present a risk of nitrosamine formation. In addition, the use of recycled reagents may introduce nitrosamines through the recycling process, so that even if the specific synthesis routes do not include the at-risk components, there may be nitrosamine contaminants in the raw materials.

Regulatory Requirements

FDA and CHMP are requiring that manufacturers assess the risk of nitrosamine impurities in APIs, marketed products, and products under approved and pending applications. For licensed products, this step should be completed by 31 March 2021 for US medicines, by 31 March 2021 for EU chemical medicines and by 1July 2021 for EU biological medicines. Manufacturers do not need to submit the risk assessment but should retain it in the event that the regulatory bodies wish to review it.

To aid in the process, FDA has published a guidance document: Control of Nitrosamine Impurities in Human Drugs, this guidance recommends steps manufacturers of APIs and drug products should take to detect and prevent unacceptable levels of nitrosamine impurities in pharmaceutical products. The guidance also describes conditions that may introduce nitrosamine impurities.

Manufacturers need to conduct confirmatory testing when there is any risk for the presence of nitrosamine impurities using suitable analytical procedures as soon as risks are identified; for licensed products, this step should be completed by 1 Sept.2023 for US medicines and confirmatory testing results should be reported as soon as completed for EU medicines.

Limits

The acceptable limits on nitrosamine impurities have been set by regulatory bodies. They are based on the daily intake of the drug. Most are set at 26.5 ng/day, with NDMA and NMBA set at 96 ng/day.  Manufacturers must take into account the dosage form and administration profile. This data is used to calculate analytical limits that are suitable for the targets daily limits.

The limits are based on a patient cancer risk of 1:100,000 with 70 years of exposure to the daily dose of drug.

Nitrosamine Compounds Listed in USP Guidance

The USP guidance document Control of Nitrosamine Impurities in Human Drugs, February 2021, gives limits for the following nitrosamine compounds:

  • N-nitroso-dimethylamine (NDMA), 96 ng/day
  • N-nitroso-diethylamine (NDEA), 26.5 ng/day
  • N-nitroso-N-methyl-4-aminobutyric acid (NMBA), 96 ng/day
  • N-nitroso-methylphenylamine (NMPA), 26.5 ng/day
  • N-nitrosoisopropylethyl amine (NIPEA), 26.5 ng/day
  • N-nitroso-diisopropylamine (NDIPA), 26.5 ng/day
  • N-nitroso-N-methyl-4-aminobutanoic acid (NMBA), no limit listed

 

Methods

The primary approach to testing nitrosamines has been using extraction of the sample followed by GC-MS analysis, primarily with a triple quadrupole mass detector.  

There is a known exception in the compound ranitidine. It has been demonstrated that high temperatures, such as those used in GC injection ports and column ovens, will result in overestimation of NDMA in ranitidine samples due to the thermal degradation of ranitidine and subsequent formation of NDMA. The Ranitidine molecule contains both nitrite and dimethylamine groups, which have been shown to react to produce NDMA. It is believed that this pathway is the source for NDMA found in Ranitidine drug product. It has further been demonstrated that this degradation reaction is accelerated by heat, as evidenced by the very high levels of NDMA detected in Ranitidine tested under GC-MS conditions (130 °C oven). Therefore, LC-MS is usually used for determination of NDMA in Ranitidine drug substance and drug products. This exception does not apply generally to other compounds.

FDA published methods for GC-MS evaluation, including Combined Headspace N-Nitrosodimethylamine (NDMA), N-Nitrosodiethylamine (NDEA), N-Nitrosoethylisopropylamine (NEIPA), and N-Nitrosodiisopropylamine (NDIPA) Impurity Assay by GC-MS/MS, originally developed for Valsartan.

Instrument companies like Thermo, Sciex and Agilent have also published application/Technical notes by following FDA and European Medicine Agency (EMA) using GC-MS/LC-MS. 

United States Food and Drug Administration (FDA) FDA developed and validated LC-ESI-HRMS in conformance with ICH Q2(R1) for the detection and quantitation of eight nitrosamine impurities in metformin drug substance and drug product. 

Boston Analytical has developed and validated methods for nitrosamines screening and quantitation. The methods may be used to target NDMA specifically, or for a more sweeping screen to identify the 8 FDA listed nitrosamines along with 3 others.  

The first step in the process of analyzing for nitrosamines is to perform a risk assessment. It is a paper exercise that looks at the manufacturing paradigm used to see whether any process intermediate steps would carry risk of creating nitrosamines. Along with analysis of the process, the raw materials involved are also scrutinized. The source of raw materials can also add a risk factor, particularly if the raw materials are recycled. The outcome of the analysis is a risk-report that helps to direct whether the materials will require lab testing.

The analytical strategy begins with screens by GC-MS (using a triple quadrapole mass spec) or LC-MS (using an Orbitrap mass spec) as dictated by the nature of the samples to be analyzed. That analysis provides identification of nitrosamine compounds present in the sample and is semi-quantatative. The screen can be followed by precise quantitation of targeted nitrosamine compounds, either by GC-MS or LC-MS.

Potential Nitrosamine Compounds: 

While the FDA guidance lists 7 specific nitrosamines, there are others possible as well:

  • N-nitrosodimethylamine (NDMA)
  • N-nitrosodiethylamine (NDEA)
  • N-ethyl-N-nitroso-2-propanamine (NEIPA)
  • N-nitroso-diisopropylamine (NDIPA) 
  • N-nitroso-di-n-propylamine (NDPA)
  • N-nitroso-methylphenylamine (NMPA)
  • N-nitroso-di-n-butylamine (NDBA) 
  • N-nitroso-N-methyl-4-aminobutyric acid (NMBA)
  • N-Nitroso-4-methylpiperazin (MeNP)
  • N-nitroso-4-methyl-4-aminobutyric acid (NMBA)
  • N-nitrosoethylmethylamine (NMEA)
  • N-nitrosopyrrolidine (NPyR)
  • N-nitrosopiperidine (NPIP)
  • N-Nitrosomorpholin (NMOR)
  • N-methyl-N-nitrosoaniline (NMPhA)
  • N-isopropylmethyl nitrosamine (NMIPA)
  • N-tert-butyl-N-ethylnitrosamine (NTBE)

 

Applied GC-MS/LC-MS Techniques: 

The sensitivity of the instruments must be very high as the daily limit for nitrosamines by FDA and EMA is very low. Boston Analytical has the following equipment available to meet this need:

Nitrosamines Lab, Boston Analytical Ins. Current equipment:

  1. Agilent 8890 GC with 7010B triple quad MS
  2. LTQ-Orbitrap-XL with Vanquish UHPLC. 

Methods used to evaluate nitrosamines should be validated. The methods available from FDA are not considered to be validated. Each laboratory must demonstrate the methods, used in that specific laboratory with the equipment available there, to be suitable for the purpose of measuring nitrosamine impurities.