Hello all,
I am developing an LC–MS/MS method for N-nitroso tadalafil. Based on structure, I expected the [M+H]⁺ precursor around m/z 419, but in my scan I am seeing a strong signal at m/z 389.
Has anyone else observed m/z 389 for N-nitroso tadalafil?
Is this a valid precursor ion or could it be an in-source fragment/adduct?
Any insights or shared experience would be very helpful.
Hi.
if I may intervene, I see that community’s attention and interest is increasing in relation to Nitroso-compound. I am wondering if this comes from regulatories’ requests…
This topic is discussed in Analytical challenge for determination of instable standard of other N-nitroso structure
Regards
Hello,
The ion 389 that you are working is the nitroso-tadalafil molecule without the nitroso (NO) group. We had the same issue and after some testing (Scans, etc.) and discussions with the standard supplier we were able to find the the compound is unstable in the LC-MS/MS source (probably due the temperature, as we find out later in the method development) and could not find the ion 419 with enough intensity to work with. So we use also the 389.
However, if you work with 389 you must be able to separate the nitroso-tadalafil from tadalafil with enough resolution, as tadalafil as the ion 390 as [M+H]⁺ and can interfere with the 389 ion (with neutral form 389 or isotopic profiles).
Regarding the stability of the compound I already talk a little in the topic Analytical challenge for determination of instable standard of other N-nitroso structure.
Best Regards,
Daniel
Hi everyone, here is a link where you can find more strategies on how to mitigate in-source fragmentation in NDSRI analysis using LC-MS. I believe this resource will be valuable to the community:
Best regards,
Lucas Maciel
Could you share how to separate N-nitroso tadalafil from tadalafil? We are also using the 389 ion, but tadalafil’s interference is too significant. Anyway, thanks very much.
Hello,
The separation had enough resolution between the tadalafil API and Nitroso-Tadalafil to ensure the analysis. We use as mobile phase of water (A) and acetonitrile (B), with minimum additive concentration (formic acid) to prevent the degradation of nitroso-tadalafil during elution. We use a F5 column, but I think that a C18 can do the work too. We tried several gradients and found a compromise between sensibility and elution with 50:50 H2O and ACN.
The sample concentration was not to high to ensure that the API peak was not to intense (to not interfering with Nitroso-Tadalafil) (around 0.3 mg/mL of API).
Hope this helps.
Best Regards,
Daniel Silveira
Hello, could you please tell us the concentration of formic acid you are using? Have you measured the specific pH value?
Hello,
For mobile phase A, we use H2O with 0.1% of formic acid and for mobile phase B we use ACN without additive. I think the method could work without additives if you need to. The additive was added due to the determination of another nitrosamine (together with nitroso-tadalafil).
No pH was verified.
Best Regards,
Daniel
Thank you so much for your answer.
Best Regards,
Shuhui Gu
Hi Daniel! Found the other discussion, thanks for the prompt response. Do you have any idea on the stability of this nitroso compound with your preparation?
Kind regards,
Figo
Hello, we studied until approximately 72 hours and it was stable at low temperatures. Just for you to know we studied the stock solution at -18ºC (we had to guarantee that no degradation happened) and the standard solutions at -18ºC and 5ºC. It helped a little ask to the supplier some information of the stability. Some suppliers have.
Best Regards,
Daniel
Hi Daniel, thanks for the information. I asked the supplier but have no answer yet. I tested the cooled preparation and indeed finally got some results. I lowered source fragmentor voltage and temperatures as well and actually got the M+H from the nitrosamine as a confirmatory test! My assumption is that the 72 hours is app. 80% recovery and at 5 degrees C?
kind regards,
Figo
Hello Figo,
You can assume that, yes. When we developed the method we keep always the same solutions also at -20ºC just to be sure. Regarding the MS parameters, because we had to quantified the Nitroso-Tamsulosin, the had to comprise between the tow nitrosamines (Nitroso-Tadalafil + Nitroso-Tamsulosin).
Best Regards,
Daniel