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In 2018, medication such as angiotensin II receptor blockers (ARBs) were found to contain high levels of nitrosamines. Since then, drug manufacturers have been on a sprint to perform product assessments, understand the risks and implement testing strategies to ensure patient safety is not compromised as a result of the presence of these genotoxic impurities.

As other medications (metformin, ranitidine, rifapentine) were then also confirmed to contain dangerously high levels of nitrosamines, the scale of the issue really started to hit home.

Of course, the first place to start is with final product testing, so you can determine the true size of the problem, but with quite a few methods published, where is the best place to start?

Navigating the technology choices

Due to the analytical sensitivity requirements and the complex nature of the drug product, mass spectrometry has very quickly become the gold standard. However, the variables do come with a degree of decision-making; methods have been produced using both liquid and gas chromatography coupled to either a triple quadrupole mass spectrometer, or a high-resolution mass spectrometer. There is no real wrong answer, but decisions still need to be made as to which setup is right for you.

High-resolution mass spectra showing nitrosamine NDMA, and closely related interference 15N DMF to within 0.002 amu. If not resolved by HRAM this would cause overestimation.

High-resolution mass spectra showing nitrosamine NDMA, and closely related interference 15N DMF to within 0.002 amu. If not resolved by HRAM this would cause overestimation.

Confidence in detection 

From the start, you want confidence in the result. If you are performing unknown analysis to assess which impurities are present, or you would like to make sure you do not fall foul of a dreaded false-positive result by measuring a closely related interference as described by A Cautionary Tale, or simply performing due diligence during method development to ensure your drug product does not contain anything it shouldn’t, then high-resolution accurate-mass (HRAM) mass spectrometry is an absolute must.

If you are a contract lab offering high-throughput targeted-only analysis, or you are confident in which nitrosamines are of concern and are simply monitoring their levels, the fastest, most robust technique is more suitable. Triple quadrupole mass spectrometry is typically a more cost-effective approach, proven to hit the attributes required by regulatory bodies in terms of sensitivity. Excellent robustness has been demonstrated on challenging samples.

These approaches are also excellent in combination: a high-resolution platform for method development and drug product assessment, while triple quadrupole being deployed for the bulk of the targeted screening, and if needed confirming any positive results by switching back to high resolution for absolute confidence. This will ensure zero false positives and, consequently, no unnecessary batch recalls.

GC separation of 15 nitrosamine compounds.

So what separates the separation?

As with detection, both LC and GC are applicable, and the choice may boil down to the nature and properties of drug products you have. Typically, GC has higher-efficiency separations, meaning better separation from unknown interferences and higher sensitivity due to narrower peak widths. However, GC has some limitations in this case; certain nitrosamine compounds are not volatile enough to work well by GC and so some upfront sample preparation may be required to derivatize the samples. In addition, there are known issues when looking at ranitidine drug products: during head space sampling the conditions are just right to cause the breakdown of ranitidine to NDMA, causing a false positive result. In this case, LC should be employed.

LC is a more universal approach, and although it suffers a little from some challenging separations, it can be more widely applied to this application type. Once methods are set, incredibly robust chromatography can be achieved and implemented for high-throughput analysis. As with detection, both LC and GC are applicable, and the choice may boil down to the nature and properties of drug products you have.

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Problem solved. Nothing else to think about?  

NDMA formation.

NDMA formation.

There is one final piece left in the puzzle: All that effort is wasted if we have the ingredients for nitrosamines in our cooking pot, but they do not form until later when they are sitting on the shelf. How can we assess the likelihood of this?

By measuring and monitoring for nitrosamine precursors in raw ingredients or in final product, we can get a handle on the potential of nitrosamines to form during storage and act before it gets expensive. Nitrite and amines can be easily monitored with ion chromatography and can aid in both the initial assessment of the risk, and ongoing monitoring for early warning signs of nitrosamine formation.

Find out the full story by visiting our dedicated resource site or, better still, have our experts talk you through their experience (including industry case studies) by registering for a 2021 short webinar series.

Additional resources

HRAM LC-MS method for the determination of nitrosamine impurities in drugs

Highly sensitive and robust LC-MS/MS solution for quantitation of nitrosamine impurities in metformin drug products

A validated method for the rapid determination of fifteen nitrosamine impurities in metformin drug substance

Determination of genotoxic nitrosamines in Valsartan with gas chromatography and mass spectrometry

Determination of nitrite in pharmaceuticals