Sample Preparation
Some sample types can be added directly to the sample vial; others may require extraction into a solvent first. Once in the sample vial, the Reagent can be added, the vial sealed and then loaded into the autosampler.
Automated Chemical Reaction Process
The samples are vortexed and then heated. The chemical reaction causes the NO groups from any nitrosamines present to be released into the headspace of the vial.
Headspace sampled
The samples are allowed to cool to ensure vapours from the samples or reagent have condensed. A sample of the headspace containing the aleviated NO molecules is then injected into the TEA system.
Detection by TEA detector
The carrier gas sweeps the Headspace to the reaction chamber of the TEA. Here it reacts with Ozone to produce NO in an excited state. As this decays, a photon of light is generated and measured by a PMT.
The technique detects down to 1 ppb of NOx gas from the degradated nitrosamines. And I’m not sure it can distinguish nitrosamines from nitrites.
Dear @Yosukemino , while I’m not personally familiar with this specific automated analyser, I can provide you with some insights based on the published literature:
It is well-known that GC-TEA has been widely utilized as a fluorescent detector for certain nitrosamines, including: N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosopiperidine and N-nitrosopyrrolidine. In fact, GC-TEA has been adopted as the standard method in EN 12868:2017 for the analysis of these nitrosamines (Li et al., 2021).
However, it is important to note that the specificity of GC-TEA is somewhat limited. While it has demonstrated effectiveness in detecting the aforementioned nitrosamines, its application may not extend to distinguishing between nitrosamines and nitrites. Therefore, I think further investigation would be required to determine the capabilities of the automated analyser in this regard. Do you agree?
Thank you for asking me. I’m not familiar with this technique, but I guess that the advantage of this method is to measure total nitrosamines without reference standards as screening. We can determine the possible nitrosamine amount if the API or impurity is a certain secondary amine. On the other hand, nitrites may be included in the total amount of NOx. Even though API is the compound free from nitrosation under WHO NAP test, the total nitrosamines may count for the hypothetical nitrosamines.
I guess we can decide nitrosamine amounts due to GC separation and reference standards at GC-TEA. And I think the technique of avoiding overestimation due to unreacted nitrites in GC was discussed in the analysis example of metformin. Does it make sense?
Certainly @Yosukemino ! I agree with you. One thing is to look at total nitrosamines, and another is to search for possible nitrosamines specifically.
Therefore, I believe we can cautiously employ this automated technique, taking into consideration the need for separation and the use of reference standards (whenever available). By carefully considering these factors, we can ensure more accurate and reliable results in the analysis of nitrosamines.
Yes, you are right. In addition, we can determine the possible maximum amount of NAs if vulnerable amines are decided. It can be enough for a risk assessment. We don’t have to synthesize reference standards and develop chromatographic separation methods. It’s good for a screening method.
I had looked at this piece of kit, but hadn’t considered the possibility that nitrites may give you a false high nitrosamine result!! It would have to be investigated further, comparing LC/MS or GC/MS results for a tablet (with known presence of nitrite) to see the impact.
I had wondered about the use as a screening tool, more and more we are seeing that if there is a secondary amine and nitrites present then there will be nitrosamines formed, regardless almost of the format of the product and so the extra time and cost of screening is difficult to justify, compared to moving straight to developing a specific test, providing your risk assessment is robust.
Where I could see a potential use would be as a QC release technique, if testing of nitrosamine level is required upon release. Installing and staffing an LC/MS in every QC lab is not something that can be accommodated quickly and easily, and this could be an alternative. This would rely on there only being a single nitrosamine present in the products though, two or more nitrosamines requiring control would not be possible.
It seems an adaptation of what is used in the ISO of Cosmetics and rubbers. I remember that there they only ask for the classic nitrosamines… not the NNO-API. I think it does not contribute much… that it does not exist with a GC/MSMS or HR-GC
In my opinion there are no easy solutions to complex problems.
I believe that the world of pharma is going to have to evolve as has been done in the environment, forensics or food. Possibly the pharmacopoeias methods cease to have the classical structure and become new ISO’s. A new era…
If you are thinking of investing money, I would not spend it on anything that does not show results for my product. You are going to waste money.
As an analytical chemist I would tell you that the NNO-API is going to war. If you have a Katana like an HR_MS, which is my sword , you have to learn to use it (be a samurai not an inter rockie ) but it promises you victory . If you go with the butter knife … well, you could find yourself in serious trouble.
Greetings.
An additional thought on this - if it does detect nitrites then is it actually a better tool, with lower levels of detection, than the classical techniques used at the moment??
Hi a very interesting topic. The point of nitrites is interesting what is if a compound contains other nitro groups? i understood the system will finally measure NO conten in gas phase so it is no differentiating between NO already party of the molecule or maybe any nitration reaction which may occur instead of a nitrosation reaction. Maybe @Yosukemino you have some insights here?
Thank you again for the great and interesting post. Is there already a contract lab using such a system?
Sure @MarkS , I agree with you that we need accessible screening techniques, and I see this as a challenging point, too, especially when we have more than one nitrosamine.
Addition to above points, my opinion about total Nitrosamine limit_Option 2 as per EMEA when more than one nitrosamines presents, is to be usefull to analyse total Nitrosamines against the specificaion.
Thankyou @Yosukemino for the introduction. and to other members for their interest in the technique.
From our experience with how customers are using this approach, it is clearly not a replacement for GC/MS or LC/MSMS systems. However, they are finding it very useful as an approach for identifying where to focus their efforts with those systems, rather than having to fully develop methods for everything.
We have some clients that are now using this as part of their new product development to quickly identify which formulations may present future issues to help guide their efforts in that area.
The ability of the system to pick up potential unknown or unexpected nitrosamines is another factor that we think means it could be a useful tool as part of a Risk assessment.
The other big advantage of the system is as it gives a molar response to the NO Group rather than a specific nitrosamine molecule, it can be calibrated using any nitrosamine. So it is not a requirement to get specific standards created for every potential nitrosamine.
With regards to Nitrite content, the system will also give a response to the presence of these. We have actually now developed an SPE cartridge that a sample can be passed through which removes the nitrite content. This means a sample can be run once to give a total nitrosamine+Nitrite content then run again, having been passed through the SPE cartridge to give a result of just the nitrosamine.
Im more than happy to answer questions on here, or if there is enough interest I can get one of our applications chemists to give a presentation for members here if that would be of interest as we have some results and demos of the system we can share.
What level of detection is the analyser able to provide in terms of nitrite?
Current methodologies for nitrite determination in raw materials (ion chromatography, DAN derivatisation, Greiss reaction) are sensitive down to the ppm level, and sometimes lower, but there can be differences in the results seen for different techniques and sample preps, especially with some problematic, but common, pharma raw materials.
Understanding and control of nitrite levels in raw materials is but one tool available in resolving the nitrosamine issue, and far from the simplest, but when at times it is difficult to know where you are starting from it makes it even trickier. A robust, reliable, accurate methodology that could be adopted by all would be a (large) step in the right direction.
Just had a chat with our applications team, and they have been running standards of sodium nitrate at 10ppb and this is giving a response very similar to that of 10 ppb NDMA. So we would expect it to be able to see down to 1 ppb sodium nitrate without any issues.
As with the nitrosamines we are measuring the NO not the whole molecule so the molar weight of the nitrite will need to be taken into account.
Thanks Andrew, for the valuable information you shared. I am just wondering if you can share some application notes that cover API(s) and Finished Product(s).
I would tell you that the NNO-API is going to war. If you have a Katana 
like an HR_MS, which is my sword , you have to learn to use it (be a samurai 
not an inter rockie ) but it promises you victory . If you go with the butter knife … well, you could find yourself in serious trouble. 
