Demoxepam causing false positive for oxazepam/nordiazepam

Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.

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For anyone out there still doing benzodiazepine analysis by GCMS (specifically toxicology analysis), we recently became aware that samples containing demoxepam (metabolite of chlordiazepoxide) were showing false positive results for oxazepam diTBDMS and possibly nordiazepam TBDMS when derivatized with MTBSTFA. The problem was discovered when a standard of demoxepam was analyzed and we have looked back at years of data and found many test samples were incorrectly interpreted.

Has anyone else had this problem? Does anyone know the chemistry behind what is happening? When analyzed underivatized, we only see nordiazepam so the oxazepam is coming from the derivatization process somehow. We haven't had much luck with literature searching. We are currently evaluating our really old BSTFA method to see if happens with the TMS derivatives too.

I would suggest that any labs using GCMS should try running a demoxepam standard on their method to see what happens.
Note that i'm not active in pharmaceutics or toxicology but as a chemist I thought this was interesting.

First of all, are you sure that the false positives comes from the sample preparation? I don't know you detection settings, but seeing these very similar molecules (demoxepam/oxazepam) it is likely that EI-MS doesn't distinguish them depending on the ions you choose. If so, there's only the retention time that distinguishes them. How well are they separated? Furthermore, as there's a different derivatization, there's a chance of derivatization products to overlap.

That said it doesn't seem unlikely of demoxepam to form oxazepam. It is an intermediate in the synthesis of the latter. This reaction is called a Polonovski rearragnement.
The full mass spectrum we are seeing at the retention time is an exact match to oxazepam diTBDMS. There is no resolution between the two. Based on the slight structural differences, we don't think the spectra should be identical, therefore it seems that actual oxazepam diTBDMS is forming somehow.

We contacted Cerilliant (where we purchased the demoxepam) and they confirmed that by LCMS there was no oxazepam present, so the heat/derivatization seems to be the problem. It is too abundant to be an impurity in the synthesis, unfortunately. Thanks for the Polonovski tip. That's exactly the type of information I was looking for and will look into it a bit more.

It looks like we see a similar phenomenon with our really old BSTFA method that we just resurrected, so apparently the TMS derivative has the same problem.
Glad that it helped.

Doing the analysis on LCMS, without derivatization of course, would most likely make your problem/doubts disappear.

Is it a (legal?) requirement to run this analysis on GCMS, or is it because you don't have LCMS available?
No, not a legal requirement thankfully!

We just purchased our first two LC triple quads last year and have been working on validating a method for opiates. Our benzo method was working fine for now on GCMS so we weren't in a hurry to move it to the LC just yet, but it may be next on our list now!

We are wondering if the reaction with MTBSTFA is causing the Polonovski rearrangement. It looks like an acetic anhydride or acetic chloride is mentioned as part of the reaction, but perhaps the MTBSTFA can react the same way?

https://www.sigmaaldrich.com/catalog/product/aldrich/375934?lang=en&region=US&gclid=CjwKCAiA-KzSBRAnEiwAkmQ151WwZVQKjV1v81QbElzkRtaJgXjlOz1R4KOPMV-4nG4M0qi4yzZzmBoCUiMQAvD_BwE
Very hard to predict. I would say it's possible in the presence of water (maybe even trace amounts?).

In my opinion it's best to answer that question experimentally (which you actually did accidently). It would be wise, if you want to dig deeper, to gather more data possibly using different analytical equipment (NMR, LCMS,..).

Is this link interesting for you? Note that I only read the abstract, it mentions the formation of nordiazepam during the GCMS analysis of demoxepam.
The only two reagents present with the demoxepam are the MTBSTFA (with 1% TBDMSCl) and either ethyl acetate or acetonitrile. Our blood and urine methods have different co-solvents for the derivatization. Our GC wash vials are ethyl acetate and methanol, so there is a possibility of trace contact there as well. We try our best to limit water exposure as it interferes with the derivatization, however I'm sure it is there a little bit.

I have seen that article before but am unable to access the full text. We are well aware of the conversion to nordiazepam when it is analyzed underivatized as that seems to be well documented. It looks like the heat of the injector port is the problem, and other benzos have a similar issue which is why you have to derivatize. Interestingly, none of us can find any reference to someone reporting this conversion to oxazepam diTBDMS, hence my desperation post here.

Basically, we just need an educated possible explanation so we can train our analysts going forward on how to recognize the problem so they can interpret the data and understand what is happening (until we move the method to LC). I think your suggestion is the closest we have right now...
kvirkler wrote:
We are well aware of the conversion to nordiazepam when it is analyzed underivatized as that seems to be well documented. It looks like the heat of the injector port is the problem, and other benzos have a similar issue which is why you have to derivatize.


I thought derivatization in your case was necessary to make it volatile enough for GC, like in most cases where you derivatize.

Are you sure the amine oxide function group of demoxepam, where the magic happens, is derivatized? How many silyl groups are added during the derivatization? Is enough silation agent added? If this function is not, or only partially, protected it means that you could still have conversion in the injector port.

Something else I was thinking about (and maybe easier to check) is to substitute ethyl acetate for another solvent (acetonitrile, acetone,..). While ethyl acetate is not the same as acetic anhydride, it can still be a source of acetylation in some conditions. Or it could contain impurities as the source of your problems (acetic acid, water,..). In any case you should definitely stick to high purity solvents.
Yes, that is definitely the main purpose of derivatization, however it seems that the benzos as a group are more prone to degradation without it so there is just one more reason to do it.

Our theory was that the amine oxide was unstable and somehow rearranging so that a hydoxyl group was forming to match the structure of oxazepam, and then that hydroxyl group was getting derivatized (along with the NH) to form a di TBDMS. We don't think the amine oxide is getting derivatized as it exists because then the structure and mass spectrum would be distinguishable. So there are two silyl groups being added to oxazepam and one to nordiazepam. We do see noridazepam TBDMS as well when analyzing demoxepam, so it looks like some conversion is happening to that before the derivatization. However the oxazepam diTBDMS is the second most abundant peak next to another unknown compound/artifact.

I think we have some comparison data between ACN and ethyl acetate and we see the same problem in both co-solvents, but we always use ethyl acetate as a pre-wash in the syringe so it ends up being around regardless. This wouldn't be the first time we tried blaming a problem on ethyl acetate, however. I think we have some higher grade we can try too.
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