We are trying to develop a nitrosamine (NSDRI) method. However, we have a problem. We cannot get recovery from the sample we spiked. Our recovery remains at 40%. Despite this, we can go down to very low levels and we have no problem with this. We can see 1% of the AI limit.
I would like to ask this. Can I validate this method using a recovery factor? In the scenario above, I can make a recovery correction by dividing my results by 0.4. What is the accuracy of this? I looked at the guidelines but I could not see any comments on the recovery factor.
What is the validity of this process I did? Can you give me information?
From a purely analytical chemistry perspective I have never been in favour of applying recovery factors, as they imply that there is an unresolved issue with the method, normally either with the extraction or the detection (but other factors can be involved).
You haven’t said much about your methodology, but assuming that it is LC-MS based, to reach the required levels of sensitivity then the general rule for recovery that I have seen applied are limits of 70-130%, and so your 40% would fall outside of this. Were this to be a method that we were developing internally then we wouldn’t look to apply a recovery factor, we would investigate why we weren’t able to hit those prescribed recovery limits.
@Hasan_T Thanks for the post. To provide the best possible exchange with the community, I suggest including as much detail as possible about your current challenge. This helps guide the discussion and the advice you can receive from our analytical experts.
I wouldn’t recommend using a recovery correction factor. The reason is that it tends to mask an underlying issue with the method performance, which ideally should be addressed during method development (e.g., optimizing extraction or sample preparation).
When it comes to recovery, you’ll find some typical ranges in the literature, such as:
In your situation, the best course of action would be to investigate the root causes — for example, adsorption to glassware, losses during extraction, or matrix interferences.
Of course, this is just based on the information you’ve shared so far. Having more data could help clarify the situation further.
I would not use that data. We have often found that optimizing MS parameters using a direct infusion has greatly helped our efforts. This also could be more of a chromatographic problem. I would be interested to see the recovery with a smaller injection size.
What you could also do is prepare separate reference solutions, each containing one of the excipients from your product at the same concentration as in your test solution. It seems likely that you’re experiencing ion suppression caused by your matrix.
This approach could help you identify which excipient is interfering. Once identified, you might be able to adjust your sample preparation accordingly or simply use the standard solution containing that specific excipient as your reference.
Another option would be to use a T-connector and an infusion pump to continuously infuse your analyte while injecting your blank solution. You will observe a high baseline and some drops. If those drops appear to be at least partially separable chromatographically from your Nitroso-Peak, you have a chance to optimize your chromatographic conditions.
While developing a method that doesn’t require matrix-matched standards is always preferable, this could serve as a quick fix in the meantime.
Thank you very much for your help. I have been sick for a while and could not look.
All your comments are very valuable.
By the way, we are using a LC-MS/MS and working on MRM. The ionization mode is ESI+. Mobile phase %0.1FA in Water and methanol. Column is c18.
@MarkS I think it was a matrix-based interference. I made changes to the gradient and column, but my results did not improve much.
@Naiffer_Host thank you for your comment. You are right, I should have given more details.
@lucas10mauriz there is a matrix-based interference. Despite my MRM method work, a high peak is coming right next to my standard peak in the samples. I need to solve this interference.
@jason.brown The injection volume is 5 ul and quite low. As I mentioned above, a large peak is coming right next to my standard peak in the samples despite my MRM work.
@Phil Thank you for your excipient suggestion, I will try it. However, we do not have an infusion pump, so unfortunately we do not have the chance to do that part.
@dianalaluna thank you for your suggestions. I will think about them, but unfortunately I could not find the deuterated form of the relevant NSDRI.
hi, as you said that there is a large peak adjacent to analyte peak which indicates about degradation or side reaction. You should try change the solvent, and you can add a very small amount of ammonia in in the extraction solvent or change in the pH. It might help.
Ammonia helps in reducing side reactions or degradation. You can try other weak base if needed.
Suggestion:
First confirm whether the API Peak and NDSRIs Peak are effectively separated. If not, modify the chromatography method to make the two effectively separated.
If the two are effectively separated, formic acid can be added to the extract to increase the ionization degree of the analyte in ESI+.
Have you try to reduce de sample concentration?. In my experiencie, when we are having matrxi interferences, reducing sample conenctration helps a lot in increasing extraction from samples.
Also, if you have such a big interference, sometimes helps to change to APCI source in order to reduce matrix effects.
The speaker at the seminar I attended today said the same thing. Typically, we aim to increase the concentration of the sample solution to enhance sensitivity; however, in analysis using an MS detector, lowering the concentration can reduce interference and increase sensitivity. That’s very interesting.