Determination of N-nitroso desmethyl Tetracycline

Confirmatory Testing & Analytical Challenges
1

Dear specialists,
we are currently working on a drug product which contains tetracycline.
So far, it seems that the separation of N-Nitroso-N-Desmethyl Tetracycline impurity form Tetracycline as well as their detection in LC-MS/MS (System: UPLC-ESI(negative)-QQQ) are quite challenging. Co-elution and low signal for both compounds are the main problems that we are facing.

Does anyone have any experience on this analysis and could suggest something?

Thank you in advance!

Christos

2

Our group has no experience with this compound, but I’ll say what I would do if in your shoes. First, many standard manufacturers are happy to share their methods and it doesn’t hurt to ask. Next, I would begin talking to column manufacturers. @MTwohig from Waters was a great help to us, both in having an excellent discussion & providing a few methods that we used as starting points for our own.

3

Dear Jason,
first of all thank you for your feedback, it is much appreciated!!
Actually, the method which have been send to us from the API manufacturer was a little bit disappointing as it was developed without the reference standard.
The ref. standard which we have in the lab it is from another vendor and it is a good idea to ask for the method from them.
best regards
Christos

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4

Hi Christos,

I had no experience with the impurity you mentioned, but I previously performed detection of tetracyclines in biological objects.
First, I would attempt ESI+ instead of negative. The impurity should ionize well, and I’d expect you could reach rather low levels (will depend on the instrument, of course).
The separation will probably be a harder task. By the way, I wonder, how many peaks of the impurity you get in one run?

BR
Renat

P.S. Somehow the topic name contains “clarithromycin” (?)

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5
  1. Can you please confirm this impurity is available or not?
  2. Because, i communicate with multiple vendors but they are regret on it.
6

Hello,
We don’t have experience with that nitrosamine of Tigecycline. We had only analyze Nitroso-Tigecycline and no much problem was found. it could be analyze quite well by ESI+. We had tried by APCI+, with no must sensitivity, has we had to analyze also NDMA and NDEA.
At the moment we are waiting form our standard supplier if they could synthetize the standard successfully.

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  1. Can you please update? Are you avoided FDA listed two impurities (N-Nitroso des methyl- tigecycline-1 & N-Nitroso des methyl- tigecycline-2)?
8

Hello, At the moment only Nitroso-Tygecicline was required and I think submmited. The impurity 1 and 2 are still in syntesis process, with known outcome from it.

9

Has the method for N-nitrosodemethyldoxycycline been successfully developed? Our laboratory has encountered the same problem. Can you share your solution?

10

Dear Chris,

We have worked with Doxycycline which is structurally similar. Here are a few observations that may help you

  1. Ionization was instrument dependent. We got a better response on LCMS from one instrument make over another. If you have multiple instruments you can try on another make.
  2. Compound fragments even at low collision energy. We have optimized the energy to get an optimum response. We got better response using the ESI negative mode
  3. The peak shape observed with commonly used LCMS mobile phases was broad. Suggest using a smaller column with sub 2 micron particle size for improving peak shape.
  4. Try using a diluent system where the API is initially soluble and can be later precipitated out without impacting solubility of the nitrosamine. This may help to achieve better separation.
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11

Dear Amit,
thank you very much for your input, it is very much appreciated!!
definitely i will transfer them to our technical team,
best regards
Christos

12

just to refresh this case.

The usage of Doxycycline did not work with the matrix we have.

So we continued to work with the reference standard ‘‘N-nitrosodesmethyl tetracycline’’.

While we still face analytical problems with this development and validation, i found out that there is an impurity vendor who provides failure report for the N-nitrosodesmethyl tetracycline!!

So, i am wondering if finally the reference we have in our hands is not valid.

Did someone has any recent experience with Tetracycline nitrosamines and this reference standard?

thank you in advance,

Christos

13

Hello, I had the same problem (not related with our compound), but with anothers, where the standard that i had, others suppliers would not sell it, and have the fail report instead.
I also had seen the some suppliers quote me both: the standard and the fail report. Thats why is very difficult to me, still, to find the fail report fully reliable.
What I can say to you, is to push you standard suppliers with your questions and make them explain to you and evidence to you, the identidy of the compound of your standard. Usually, if they know what they doing, they will give you an awnser that hope will leave you with no doubts. I have done it before several times and it build more trust in the use of the standard.

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14

Daniel, thank you a lot for your reply.

The problem is that we have an offer only from one supplier (and this is wired) who provided us the usual characterization data (HPLC, MS, NMR, IR etc). Most probably you know that is very difficult to certified this kind of compounds without HR-MS/MS and/or 2D NMR spectra and of course you should have a staff very strong in spectrochemistry.

15

Hi, I totally agree with you. In some cases I also have one supplier (like Nitroso-Lacosamide, etc.), and in this cases I ask for more detailed evidence and more explanation (as I have my limitations in all the techniques of characterisation).
But usually I ask to evidence/explain the “peaks” or other evidence in each technique that is so specific (like the NO band in IR of the zone of the NRM spectra that is specific of this compound) of the compound, that make possible the identity.
If the supplier cannot give you any good information, then it is possible that something may be wrong with the standard.

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16

There was a recent article that used lithium adducts to confirm the presence of a nitroso group. This adduct forms predictable fragmentation, characterized by a consistent loss of 30 Da through the elimination of NO. You might consider trying this approach.

We’ve tested it on a variety of compounds and have consistently observed this transition. In contrast, without lithium, we definitely saw different fragmentation patterns.

I understand this isn’t definitive proof, but it’s nevertheless a quick and useful check.

You could also try other adducts, such as [M+Na]+, by using sodium formate or acetate as a modifier. For some compounds, this can lead to a significant increase in sensitivity.

Best regards,
Philipp

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17

Dear Philipp,

I will review it. Could you provide the reference for the article?

Thank you in advance.

Best Regards,