I am trying to develop an LC–MS/MS method for N‑nitroso‑ambroxol, but I am facing a recurring problem: I only observe the intact ambroxol ion, with no evidence of the nitrosated ion under any conditions tested. I have already varied source temperature and fragmentation energy, tested different mobile phases, several additives (both acidic and basic), and even other ionization conditions, but there is still no consistent signal for the nitrosated species.
This raises a fundamental question: Is N‑nitroso‑ambroxol actually stable enough to be detected by LC–MS?
Or does it simply decompose rapidly (e.g., by losing NO and reverting to the ambroxol m/z)?
I would like to ask the community: Has anyone ever attempted to analyze N‑nitroso‑ambroxol? Did you encounter similar difficulties? Was the nitrosated standard stable at all? What strategies worked (or did not work) for you? Is there any viable alternative if the nitrosated species is not stable for ESI analysis? (I have also tested APCI without success.)
Any insight, practical experience, or suggestions would be greatly appreciated.
We used negative ESI mode with normal eluents + formic acid. Method development was quite easy and straightforward.
No insource fragmentation was observed on our LC-MS.
However we did have problems later with a fresh standard purchased from a new supplier. No response at Nitrosamin m/z, maybe the same issue that you are facing. So keep that in mind.
Hello! No experience with that certain compound, but first thing I’d do would be to search/request data from standard supplier regarding standard batch identity, NMR and/or MS.
It is also worth checking fragments whether they are consistent with structure. Btw does this [presumable] nitrosoambroxol separate from ambroxol when analyzed on LC/MS?
We had develop a method LC-MS/MS in ESI in positive polarity. The MS source had to be at a temperature a little bit lower to ensure that the molecular ion could be detected (150 - 200 C) and a soft ionization was applied (lower CAD/CID energy).
The standard was quite stable. We had some difficulties with our first standard (it was not the compound) so we change the supplier and guarantee that the CoA was OK before purchase.
We develop the method for tablets and oral solutions. For Oral solution the addition of NaOH help in the extraction step be keeping the molecules in state that we needed.
Beside this, the process was quite straight forward. And yes, the API was completely separated from the nitrosamine.