DMT N-oxide analysis

[Dozuki]

Taken From this thread, post #75:

Here's the mass spectra file of the N-Oxide analysis I tried to do (10% hydrogen peroxide mixed to DMT in IPA)

Regarding N-Oxides, they do appear in GC-MS, except part of it seems to convert back to the parent compound. In this paper, they say:

approximately one third of clozapine-N-oxide was determined to be reduced to clozapine under the described capillary GC conditions. On-column reductions of N-oxides to the corresponding tertiary amines find a precedent in the property of other tricyclic drug N-oxides subjected to the generally more reactive conditions of packed column GC [12-14]. (...) thermal degradation of this Noxide to the parent drug may result in an overestimation of clozapine concentrations

Now interestingly, when i tried to make an n-oxide with 10% hydrogen peroxide, it did seem to create the dmt n-oxide peak (eluted at around 5.7, with the mass peak at 204, in the file im attaching, same as with FASW file eluted at around 5.72). But if we take the above paper to be right also for DMT, then the n-oxide amount is probably being under-estimated because some of it is converting back to the parent compound. LC-MS will help settling this. The other interesting thing is that the supposed N-Oxide product I created has several other peaks appart from dmt n-oxide and DMT.. It also has a very large quantity of 2MTHBC and several other components.. In the paper mentioned above, when analyzing the pure n-oxide, they also came up with 3 peaks instead of one.. Here's what they said:

Formation of these latter components may represent thermally induced Meisenhiemer rearrangement [15] (C-N oxygen insertion) and/or enamine forming processes [13,14], both characteristic GC degradation pathways of N-oxides.

I wonder how much of this explanation is valid for the other compounds we're seeing in the n-oxide analysis, and how much are actually other degradation products of DMT .. ? Even in the TLC of the n-oxide there were other peaks appearing, so maybe its not only in the gc-ms but DMT can oxidize to other things with hydrogen peroxide, it seems (maybe also with air.. ?) Here's the TLC Plate:

(made with adding 20mg dmt to a petri dish, adding a few ml hydrogen peroxide to see some of it dissolve, then add some IPA and more hydrogen peroxide for it all to dissolve

Also compare it with last column in TLC plate in the first post of this thread


Maybe 3 = 2MTHBC ?


? Now take into account what infundibulum said to me by pm (hope you dont mind me sharing, inf) :

This is basic rule in chemistry and oxidation is such a generic term and used to describe an array of diverse reaction and products. Believe it or not, the cyclisation of dmt to 2MTHBC is actually an oxidation reaction!

Burning dmt is also an oxidation reaction, an extreme one; all organic substances burn (or oxidise) to CO2 and H2O. So an oxidation product of dmt is carbon dioxide and water, also some nitrogenous compounds due to the presence of nitrogen as well. But there are so many other oxidation intermediates and by-products, like 2MTHBC as mentioned

I also googled "indole oxidation" just to get a rough idea of what else can happen to dmt and damn, it is diverse! one can get indole fusion by products (like in here: http://www2.iq.usp.br/docente/lhc/lab/indole.pdf) or just addition of oxygen atoms such as here: http://en.wikipedia.org/...dole_NBS_Oxidation.png. And psilocin also is thought to be oxidising to a blue compound, maybe of similar structure to indigo (File:Indigo.svg - Wikipedia).

So when one employs H2O2 he's sure to oxidise the tertiary amine of dmt to n-oxide, but he may be oxidising the molecule in who knows in how many other different ways. We may also need to take into account that some oxidations may be fast (like the oxidation of n to n-oxide) but others may be slower. Once I tried to make some n-oxide using peracetic acid and yes, it turned deep red pretty quickly (maybe n-oxide?). I left it alone for few days and the oxidation appeared to be proceeding and the deep red started changing to clearer yellow (maybe hell knows further oxidised products?)

And with air oxidation, who really knows what may be happening; some n-oxide formation? some other oxidation by products? Still all too interesting and crying to be tested!

How interesting, that 2mthbc amount is increasing a lot when doing this oxidation test with hydrogen peroxide, as inf said, its also an oxidation reaction.... But wow, can it really be?

 

[Dozuki]

I had a look through the MS file and here what I've got from it. The first Image is the Chromatogram and the second image is the data for peak P6.

P1 - ? M+ 146 base 146
P2 - ? 4-Methylquinoline (4MeQ) - NIST interpretaion
P3 - ? 4-Methyl-1,5-Dihydroxyquinoline (4Me1,5DHQ) - NIST interpretaion
P4 - DMT
P5 - DMT N-oxide
P6 - Alk '206' M+ 206 base 120 [See attached image with tentative identification]
P7 - 2MeTHBC
P8 - ? Bufotenine- NIST interpretaion [Edit not a good interpretaion]
P9 - ? M+ 218 base 190

Quite the alkaloidal soup!!
Here are some more interesting very small peaks that might give some clues to the formation of these peaks, specifically P6. I'm listing them by their retention time (NIST interpretations) [Edit: these also need look at more closely]:

1.109 - Indole
2.489 - Indole
3.626 - Oxindole
4.463 - Close to 3-Propalindole according to NIST, M+171 base 143 [+115, 89, and 53]

I looked at both of the papers from the thread link to above, but need to really go through the first one to make any more out of it other than you what you posted above posted above. There are definitely some reactions going on.

From looking at the second paper, I drew in what the redox molecule would be for DMT according to their reaction. The weight of this matches up perfectly with P6. I also looked at the suggestions NIST gives for the peak and looked at the ones that have a similar structure to that of P6 and those compounds share a lot of the minor peaks. Makes me think this is probably a good match.

Did the DMT for the oxidation come from an extraction? How clean was it? How was it extracted? Were any metal utensils used? Even a spatula? I once contaminated some salicylic acid with a spatula giving some of it a nice purple tinge. I have some ideas about what may be happening, and the answers to these questions might help some.

There seems to be oxidation and cyclization both going on possibly.

 

[endlessness]

Excellent

Why do you think peak 8 is bufotenine? I dont think it is.. It has a very distinct fragment at 72 which bufotenine lacks... Also how sure are you of the other identifications (appart from 2mthbc, dmt and dmt n-oxide) ?

Yeah its possible there was some stainless steel utensil at some point.

Also regarding the source for oxidation, it should have been clean dmt, I thought I had sent a control test but I cant find it so apparently I didnt.. I could repeat the process again, any tips on what I should do? Better dissolve in solvent and then add h2o2, let react and evap, or straight away add h2o2 to dmt? I only have 10% h2o2.... Anything else?

 

[Dozuki]

Why do you think peak 8 is bufotenine? I dont think it is.. It has a very distinct fragment at 72 which bufotenine lacks... Also how sure are you of the other identifications (appart from 2mthbc, dmt and dmt n-oxide) ?

These were NIST interpretations. Feel free to re-analyze

 

[Infundibulum]

So to recap, when people say they make n-oxide from their dmt preparations by the H2O2 they are actually getting a soup of what-ever, including N-oxide and 2meTHBC? Interesting comparing to what people have been claiming thus far with regard to "making" and bioassaying n-oxide...

I also think we're safe (maybe not?) to say that most of the dmt shown in gcms is n-oxide converting back to its parental compound, but there's a way to tell for sure (as opposed to being unoxidised dmt).

Also regarding the source for oxidation, it should have been clean dmt, I thought I had sent a control test but I cant find it so apparently I didnt.. I could repeat the process again, any tips on what I should do? Better dissolve in solvent and then add h2o2, let react and evap, or straight away add h2o2 to dmt? I only have 10% h2o2.... Anything else?

Time-course oxidation is key here. You will also address whether any dmt shown in gcms is due to conversion of n-oxide to dmt.

First off, a preparation of dmt needs to be analysed just to get an idea about its profile. Then do a time-coursed oxidation (as typically described - dissolve dmt in methanol, add equal volume of H2O2, wait). Time-course means that you set up a reaction and then you sample it at t=0 hours, then 30 min, 1 hour, 2 hours, 3 hours, overnight, after 3 days (or similar). Intuition is important here....

The main problem is how to stop the oxidation reaction; somehow H2O2 neesd to be neutralised so that its oxidative power ceases. One way (the molecular biologist's way!) is to utilise the enzyme peroxidase which breaks down H2O2 to water and oxygen. Peroxide tablets can be bought at pharmacies and are commonly used to neutralise peroxide from people who sterilise and clean their contact lenses with peroxide. The downside is that you add in the reaction a very complex molecule (of a molecular weight of 45000...!) which can break down (either by peroxide itself or during the gassing) to what-ever small detectable organic by-products.

Maybe there's a chemist's way to neutralise H2O2 fast enough and with minimal impact to the reaction per se and products' detection? Zinc would most certainly reduce and neutralise H2O2, but at a cost of reducing any n-oxide as well...(but this can also be useful, read below).

So given that there are time-course products of dmt oxidation, one can see what forms when. Maybe one can get a preparation high on 2meTHBC and low on other stuff? Now the follow-up experiment (and part of almost half the fun) would be to not only analyse the H2O2 oxidation by-products, BUT ALSO to reduce a portion of each preparation (or maybe of the most interesting preparations) with zinc to see which, if any compounds are reduced and to what? Then N-oxide should revert to dmt, ok we may not get a good feeling of it by gcms, but how about the other guys? Say that for instance 2meTHBC is not reduced back to dmt (i.e. it is an oxidation dead-end) by successive oxidation/reduction reactions one can possibly enrich in one of these novel alkaloids, then maybe do more studies with respect to their solubility, isolation, bioactivity, etc etc. Ejaculation.

 

[endlessness]

LOL, sounds like a few interesting venues for experiment.. Im not sure how much of it I would be able to do it.. Here's a few potential issues:

First off, a preparation of dmt needs to be analysed just to get an idea about its profile. Then do a time-coursed oxidation (as typically described - dissolve dmt in methanol, add equal volume of H2O2, wait). Time-course means that you set up a reaction and then you sample it at t=0 hours, then 30 min, 1 hour, 2 hours, 3 hours, overnight, after 3 days (or similar). Intuition is important here....

The thing is, between me and the GC-MS, there are a few days, or up to a week of wait.. The other thing is that Im not sure if I can introduce a hydrogen peroxide with methanol in the GC-MS, or if I have to evaporate it all and then dissolve whatever is left in methanol, I can ask. The other thing is, with the temperature of the GC-MS, if there's any unreacted hydrogen peroxide, it might affect the results (like further oxidize in a way that at room temperature wouldnt?). I could test with TLC though, but im neither sure how the h2o2 affects in a plate. The first plate in this thread was a mixture of the dissolved dmt in methanol and peroxide, while the one I posted later in this thread was with the whole thing evaporated and then dissolved only in methanol. The GC-MSed one was also with the whole thing evaporated and then redissolved.

I can ask about the enzyme in the pharmacies...

One thing I think would be ideal to test in mid-long term is to have same batch of DMT, and have it stored well in the freezer or fridge, part of it store it improperly at room temperature (make test after some months), and part of it oxidize and test. Then compare results.

You mentioned there would be a way to tell appart how much of the n-oxide was actually converted back to the parent compound in the GC-MS but you didnt explain how... Or did you meant there ISNT a way?

The only way I can think of is wait for LC-MS when that is available... Any other ideas?

Dozuki, NIST guessing is at times incorrect, so you have to see first of all what probability it gives (for bufotenine and peak 8 for example it was around 15%? ) . Also things to check for are: Mass Peak (it doesnt match up with bufotenine...), other very distinct fragments that are varying. Also, I analysed some Anadenanthera seeds and they came out quite later in retention time like at around 7.1 instead of 6.35 or something. And while retention time can vary, that Anadenanthera also had a bit of dmt which came out at 4.48, which is pretty similar to the DMT in this sample, and therefore we can expect that the bufotenine wouldnt be sooo different either.

 

[Infundibulum]

^^
Well, I know that you'll probably need to wait for few days for the samples to be analysed:d you won't have to couple the h2o2 oxidation with immediate ms analysis...The idea is that you stop the reaction by neutralising (or destroying) h2o2 after, say 1 2 or 3 hours after incubation, then you can dry the sample and store till the time comes for it to be injected to the gcms. Ideally there should be some timepoints stored to be analysed at your own leisure.

You mentioned there would be a way to tell appart how much of the n-oxide was actually converted back to the parent compound in the GC-MS but you didnt explain how... Or did you meant there ISNT a way?

By taking ratios; the ratio of n-oxide vs dmt should be constant for complete conversion of dmt to n-oxide. If e.g. there are 3 dmts to 1 n-oxide, and assuming that upon ejection there were no dmt species, only n-oxide species, then it is clear that 75% of the n-oxide is shown as the parental compound. if there's unreacted dmt upon ejection then the ratio should be, e.g. 5 dmts to 1 oxide. In a just 1 second conversion you should get minimal oxide, so there'll be truly minimal if any shown, but as the reaction proceeds the ratio will be stabilised (=complete conversion).

Note that whether dmt or n-oxide gets degraded to other crap does not matter one bit because we use ratios.

 

[endlessness]

If the enzyme can be found only, no? So ideally I could test some control dmt, some control dmt with the enzyme, and a couple of different n-oxide samples. But this is supposing this enzyme works well without interfering too much with results of course. (and if I can insert such a mixture of things in the GC-MS without problems...)

As for your explanation, Im sorry youre gonna have to explain that again. You just said "assuming that upon ejection (I guess you mean injection) there were no dmt species", but how can we know that in the first place? We could only tell if it made a difference in ratio between some try and another but we still cannot know how much actual n-oxide was there. Also, one possibility I just imagined is the possibility that the rate/ratio of conversion to the parent compound might change depending on the amounts there are of the substances. Lets say if there's ony small amounts, proportionately more of it will change back to parent compound with heat, but maybe if there's a lot, then only smaller amount changes? This would even further screw up our test results if it does happen like that (and I dont know enough about this analysis to know if its true or not..)

Another question I have: What possible difference can using h2o2 10% be from 30% ? Appart frm maybe faster reactions, could the qualitative nature of reactions change too (i.e. different byproducts ) ?