Trends and Challenges in Nitrosamine Testing: Part Two – Analytical Challenges

What are the main analytical challenges involved in detecting nitrosamines?
 

Kevin Parker: As with any ultra-low level quantitation experiment, the inherent issue that is always present regards concentration. With sub ppm levels required for almost all analysis, it is necessary to push the instruments to the edge of their capabilities. Often mass spectrometry is used for low-level quantitation, with plenty of examples using both high resolution hybrid instruments and triple quadrupole mass analyzers. The sensitivity observed with modern triple quadrupole mass spectrometers mean they are usually the most ideal instruments to use for ultra-low-level quantitation.

With n-nitrosamines being such a ubiquitous topic in the field of analytical chemistry, there are a lot of manufacturer notes and methods published to use as starting points for analysis. However, these methods often don’t consider sample matrices – which can complicate things. Drug products often contain compounds that are not well ionized during mass spectrometry, and can therefore cause significant losses in sensitivity through ionization suppression. 

Another challenge in n-nitrosamine analysis comes when differentiating between n-nitrosamines and other isobaric compounds which share a similar neutral loss. It is here where chromatography comes into play and method development is key. Ensuring that you have n-nitrosamine specifically (as opposed to c- or o-nitrosamines) is a requirement for any triple quadrupole method which relies on the neutral loss of NO for detection. 

While less common, there are quite a few methods published which use gas phase chromatography for smaller n-nitrosamines such as NDMA, NDEA, NDIPA, etc. 

Jingyue Yang: In my opinion, sample preparation and matrix effect are the main analytical challenges in detecting and quantitating nitrosamines. Getting NDSRI reference standards also can be challenging for us, but I’m not sure whether that should be counted too! 

In the pharmaceutical industry, liquid chromatography coupled with mass spectrometry (LC-MS), including LC-MS/MS and LC-HRMS, is the most commonly used technique for nitrosamine detection.

Alan Thompson: I agree, the most common techniques in the pharmaceutical industry are GC-MS and LC-MS, as well as HPLC-UV. The eventual technique used is decided by a number of factors including solubility, volatility, workup procedure and required limit of detection or quantitation. 

The main challenge we have experienced in detecting nitrosamines thus far is ensuring we can achieve adequate method sensitivity. However, sensitivity is dependent on the solubility of the API in question. Using a more concentrated sample increases the possibility of reaching low detection and quantitation limits, but risks increasing the content of excipients in formulated drug product samples, potentially hindering the extraction process or suppressing the mass spectrometer signal.

When developing methods, we always try to ensure that excipients and the API are chromatographically resolved as much as possible from the nitrosamine under investigation. This is particularly important for the API since it often has a similar mass to the nitrosamine derived from it, resulting in adduct ions or isotopic interference from the API being captured by the nitrosamine MRM transition. We also try to ensure that only the nitrosamine is diverted to the MS source, to minimize the potential build-up of API or excipient on the source, which can lead to diminishing recovery as the run progresses. 

Previously, we have used internal standards (IS) in sample preparations as a way of correcting nitrosamine recovery. Isotopically labeled standards are often not commercially available, however, which means Almac would have to synthesize these. Nitrosamines generated from related API impurities can be used as internal standards, however it needs to be ensured that they too are also resolved from the API and excipients and enters the MS at a similar time to the nitrosamine under investigation.

Jessica Hoskins: One of the main challenges faced by the pharmaceutical industry is reaching the necessary limit of detection for NDSRIs. Compound specific acceptable intake levels set by regulatory agencies can be as low as 18 ng/day. The absolute detection limit needed analytically therefore depends on the combination of the potential maximum daily dosage (MDD) of the drug, the practical concentration you can prepare the drug product sample solution and the percentage of that drug product that is API. For example, with a MDD of 1 g per day, a moderate API drug load (15 percent of drug product by weight), and a prepared drug product sample concentration of 100 mg/mL (15 mg/mL on API basis), an acceptable intake limit of 26.5 ng/day translates to a 0.4 ng/mL method limit. As an analytical scientist, you'd prefer to develop your method with a limit of quantification (LOQ) below the acceptable intake limit – but sometimes this can be challenging to achieve! 

Since drug product matrices can be quite complex, sample preparation and chromatography are key parameters to achieve the necessary LOQ (in addition to mass spectrometry optimization, of course). For example, asymmetric N-nitrosamines exist in two stable rotameric forms, which can be separated chromatographically. Some excipients are not UV active and do not ionize well in MS, and yet may still interfere with the MS signal of a nitrosamine analyte if coeluting from the column.

Naiffer Romero: Detecting nitrosamines in pharmaceuticals presents several analytical challenges, including the need for trace-level analysis, as others have mentioned – nitrosamine analysis requires detection at very low levels, often in the parts-per-billion (ppb) range, requiring highly sensitive analytical methods. Specificity is another challenge, as the potential for false-positive identification due to background contamination or co-eluting compounds necessitates the use of highly specific detection techniques. Additionally, artifact formation during analysis requires careful method optimization, and in some cases the use of inhibitors can prevent unintended nitrosamine formation. Recovery poses yet another hurdle, as matrix interference and differences in solubilities make it challenging to achieve accurate recovery of nitrosamines when spiked in drug products.

As companies started to undergo “confirmatory testing,” they found how critical the quality of the reference material is to guarantee accuracy in their testing results.

Is structural determination important for nitrosamine analysis?
 

Naiffer Romero: In the pharmaceutical industry, isomeric structure determination is not typically a major concern for small nitrosamines analysis, but it does play a critical role during NDSRI characterization and analysis. Though the focus is primarily on detecting and quantifying specific, known nitrosamines, if necessary, techniques like nuclear magnetic resonance (NMR) can be employed for structural elucidation and even predicting nitrosamine formation in materials and finished products.

Knowing a nitrosamine’s precise molecular structure is crucial for reliable risk assessment and the determination of its safety profile. Nitrosamines can vary significantly in their toxicity, carcinogenic potency, and metabolic pathways based on their specific structural features – such as the substituents on the nitrogen or the overall size and shape of the molecule. Detailed structural characterization helps predict how these compounds are activated in the body (e.g., metabolic pathways leading to DNA-reactive intermediates), which in turn informs toxicological evaluations and regulatory thresholds. Moreover, selecting and validating an accurate analytical method depends heavily on recognizing the unique physical and chemical properties inherent to each structure. Ultimately, the better the structural understanding, the more accurately one can assess the potential risks posed by a nitrosamine impurity, enabling science-based strategies to mitigate exposure and ensure product safety.

Kevin Parker: Yes, structural determination of nitrosamines is particularly of interest in NDSRIs where determining the exact location of nitrosation is key, as n-nitrosamines are of concern, but not their c- and o-isomers. Often collision induced dissociation or other fragmentation methods are not enough to fully elucidate the structure of the nitrosamine. In some cases column chromatography can be used to generate a sample that contains a concentration of the nitrosamine in question, and subsequently can be analyzed by NMR, as Naiffer mentioned. This is not always possible, however. Recent publications have shown the use of HDX for localization of the nitrosamine to certain atoms on the molecule. 

I am a bit biased, but based on my graduate work in gas phase ion chemistry, one of my favorite examples of structural elucidation of n-nitrosamines has come from the Kenttamaa Lab at Purdue. By using gas phase ion-molecule reactions a molecule can be isolated in a linear ion trap, where it then selectively undergoes a reaction with volatile 2-methoxypropene, in which only an n-nitrosamine will provide a diagnostic fragment. This is a unique way to probe the presence of n-nitrosamines and help to rule out c- and -nitrosamines.

Jingyue Yang: The most common nitrosamine isomers are rotational isomers, which arise from the partial double-bond characteristic of the N-N bond. These isomers can be separated chromatographically, leading to multiple peaks. When performing quantitation, the areas of these peaks should be considered collectively. Other types of isomers likely require case-by-case discussion.

Alan Thompson: Isomeric structure can have an impact on nitrosamine analysis, especially if the API contains isomeric impurities. However, these isomeric impurities will have the same MRM transition as the nitrosamine under investigation and can therefore be identified and quantified should they be resolved from the main impurity. A second qualifier MRM transition using a second fragment of the nitrosamine can also be added to the method, to confirm if any other peaks in the chromatogram are isomers or co-incidental fragments of other peaks in the sample.

Jessica Hoskins: I agree! Since API molecules often have more than one potential site of NO addition, and addition of NO to a carbon or oxygen is of less concern than addition to a nitrogen atom to form N-nitrosamine (N-NO), isomer determination can be critical. Often MSⁿ analysis alone is not enough to determine the location of NO addition, particularly since N-NO bonds are easily broken during MSⁿ analysis. We’ve implemented a few techniques in our lab to help with this, including ion-molecule reactions (based on work from the Kenttämaa group) and HDX experiments. These techniques sometimes help us to avoid isolating enough material for NMR analysis, which can be a time and resource intensive process. 

What about sample preparation – what are the main challenges there?
 

Jingyue Yang: Sample preparation is one of the most challenging aspects of nitrosamine analysis, as it often is for many types of analyses. The main challenge, in my opinion, is selecting an appropriate extraction solvent for efficient extraction, as well as demonstrating that efficiency. Additionally, evaluating and preventing artifacts – such as the formation or degradation of NDSRIs during the preparation process – presents another significant challenge.

Alan Thompson: Yes, the chosen solvent should be compatible with mass spectrometry, whilst also achieving maximum API solubility to ensure that better method sensitivity can be achieved. For the analysis of formulated drug products the dissolution of excipients is not critical, though interaction of the nitrosamine with undissolved excipients has previously been shown to impact sample recovery (especially in low dose formulations). The introduction of appropriate mixing, sonication and filtration steps is critical in these instances. The addition of internal standards and assessment of recovery via nitrosamine/IS ratio rather than response can help rectify poor recovery, as can sample addition to standard solutions.

Given their severe carcinogenic potential, appropriate risk assessments and handling procedures should be followed at all stages of sample preparation when using nitrosamines. 

Jessica Hoskins: I’ve encountered quite a few challenges with sample preparation. As I previously mentioned, we often try to increase the sample concentration as much as is practical to reach the necessary LOQ for the nitrosamine. Since we want to ensure that the sample preparation step does not generate any additional nitrosamine, I usually try to avoid sonication or heating of the sample. Solubility considerations mean that sometimes the best sample preparation method is a liquid extraction from the solid, and in this case you must carefully consider how to appropriately evaluate recovery of your nitrosamine analyte (as mentioned by Jingyue). 

Finally, since many common excipients in the drug product matrix may cause ionization suppression or enhancement during detection, it’s also important to consider your MS analysis approach. In my experience, APCI can be an excellent alternative to ESI to overcome this issue if you don’t have appropriate internal standards available or need to avoid a standard addition approach to quantitation. Particularly for NDSRIs, stable label internal standards are often not readily available. 

Naiffer Romero: One of the biggest challenges for nitrosamine testing is the development of an effective and robust sample extraction protocol. Key considerations include matrix interference, as the complex nature of pharmaceutical formulations can hinder analyte detection, and analyte stability, since nitrosamines are susceptible to photolysis and degradation during preparation. In-situ nitrosamine formation is another concern, arising from the presence of amines and nitrosating agents in the sample matrix. The inclusion of nitrosation inhibitors, such as ascorbic acid or sulfamic acid, during preparation can prevent artifact formation. Proper extraction time and mixing are critical, as inefficient extraction affects the limit of detection (LOD), limit of quantitation (LOQ), accuracy, and precision of the analytical method. Strategies to compensate for matrix effects, such as using internal standards and implementing matrix-matched calibration or appropriate equilibration, further enhance the robustness of the sample preparation process.

Now that we’ve explored the analytical challenges involved in nitrosamine testing, our experts will return to offer their top tips for building a method from scratch in part three – keep your eyes peeled! 

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