Anadenanthera colubrina subjected to flash chromatography

[69ron]

SWIM extracted some very low quality Anadenanthera colubrina seeds using 99% IPA in a Soxhlet for 18 hours. This was concentrated down and 2 parts water was added. The pH was adjusted to pH 3 with tartaric acid. This was defatted 8 times with DCM. Then the pH adjusted to pH 9.5 and extracted 6 times with DCM. This evaporated down to a sticky goo which caused lots of nausea when vaporized and was extremely weak. This was then dissolved in a tiny amount of methanol, then diluted 6 times with MEK, which caused lots of precipitates to form (these precipitates are all inactive and are discarded). The remaining MEK was concentrated down to dryness, and then it was dissolved in a tiny amount of a 50:50 mix of ethyl acetate and methanol.

The extract amounted to 0.2% of the original seeds used. That’s extremely small for Anadenanthera. Usually it contains 20 times this amount! What horrible seeds.

The alkaloid mix was then subjected to flash chromatography.

There were 8 fractions eluted, with increasingly polar solvents.

Fraction 1 was eluted with pure ethyl acetate. Something like DMT N-Oxide eluted. This fluoresced light blue in UV light. When dried it crystallized as amber yellowish crystals resembling DMT. This amounted to 13% of the material that eluted. This was tested for activity. At 7 mg vaporized it produced a slight DMT N-Oxide effect along with some head tension.

Fraction 2 was 75% ethyl acetate and 25% methanol. The next thing that eluted was some amber material that fluoresced blue in UV light. It dried to a single solid yellow amber mass with no crystals present. This amounted to 27% of the total eluted material. This has not been tested yet.

Fraction 3 was 50% ethyl acetate and 50% methanol. The next thing that eluted was bufotenine mixed with amber material. This dried and formed bufotenine crystals surrounded by solid amber material. This amounted to 35% of the material eluted. This was the largest amount eluted from the column.

Fraction 4 was 25% ethyl acetate and 75% methanol, the next thing that eluted was bufotenine mixed with amber material. This dried and formed bufotenine crystals surrounded by solid amber material. This amounted to 7% of the material eluted.

Fraction 5 was methanol, the next thing that eluted was some amber material. This dried and formed a few crystals surrounded by solid amber material. This amounted to 9% of the material eluted. This was not tested.

Fraction 6 was 75 % methanol in water. This dried to amber crystals. This was 1% of the eluted material.

Fraction 7 was water. This dried to a grayish crusty material. This was 1% of the eluted material.

Fraction 8 was vinegar. This dried to an amber brown material. It smelled like burnt sugar. This was 7% of the eluted material.

The column remained filled with dark brown material that could not be eluted in reasonable time. This was 40% of the original started material.



So to sum it up, from the original seeds we obtained 0.2% alkaloids soluble in MEK. From that 0.2% only 55% eluted from the column for a total of 0.11% of the original seed material. From that, only about 30% was bufotenine. So it contained approximately 0.033% bufotenine and about 0.0077% of a DMT N-Oxide like alkaloid.

Talk about weak seeds! These seeds were worthless!

While some of these might look wet in the pictures, they are all actually completely dry. Fraction 2 especially looks wet. But it’s bone dry.

Does anyone have any idea what the solid amber mass is in fraction 2?

 

[69ron]

SWIM tested fraction 3 for activity at 7 mg vaporized. It immediately produced very slight nausea for about 30 seconds and tingling sensations in the head. The slight nausea was completely gone in 1 minute. The tingling started to expand to the rest of the body becoming very pleasant, as expected. Visual effects started in about 2 minutes and peaked after about 15 minutes. The entire experience lasted about 90 minutes. Up until the peak there were lots of auditory hallucinations. From the peak onwards the experience was extremely euphoric. The very mild side effects lasted less than 1 minute while the rest of the experience was completely side effect free.

Fraction 3 was obviously pretty clean bufotenine.

Unfortunately, separation using flash chromatography didn’t get rid of the amber coloring. All fractions had the amber coloring to various degrees with fraction 4 being the darkest amber.

The amber material eluted best in fraction 4 which was 25 % ethyl acetate and 75 % methanol.

Does anyone have any idea what this amber coloring is?

Any idea how to remove it from fraction 3? He tried activated carbon but it ate up all the bufotenine.

 

[69ron]

SWIM Tested fraction 2. It's actually bufotenine, and quite pure at that. There are no crystals present and I’m not sure why. Vaporizing as little as 7 mg was enough to produce mild 3D visual effects that hovered in mid air. There were lots of auditory hallucinations. The effects were very clean, without side effects (except for the initial 30 seconds with slight nausea and tingling before the main effects start).

I wish SWIM knew how to get rid of the amber colored material. As is, it’s very clean in effects, but just amber. For practical use it's fine and doesn't need any further purification.

 

[69ron]

SWIM used a very tiny column for this test. It was a standard medicine dropper about 3 inches long and about 1/4 of an inch thick. He plugged it with cotton and added 2 inches of flash chromatography silica that was topped with sand. He used vacuum instead of pressure (its much easier) to elute the solvent mixes. The amount of extract he used was 100 mg dissolved in 0.5 ml of ethyl acetate and 0.5 ml of methanol. The column used was really smaller than it should be fur that amount. But it worked.

The solvent volumes were 4 ml for each fraction.

So a very cheap way to purify bufotenine from an Anadenathera alkaloid mix is to use flash chromatography, and elute the DMT N-Oxide (or whatever it actually is, I’m not sure) with ethyl acetate, and then the bufotenine with a 3:1 mix with ethyl acetate and methanol.

By doing this simple procedure, you can get rid of the sticky texture of an Anadenathera alkaloid extract and get rid of nearly all the side effects.

Don’t be put off by the flash column chromatography. It’s very easy to perform. Even a child could do it. You can use a standard medicine dropper as your column. You don’t need fancy equipment. You will need a source of vacuum or pressure to speed up the process. You will also need flash chromatography silica gel, a little sand, the solvents, some cotton, and any kind of column (a medicine dropper is great for small quantities).

Pure bufotenine is far nicer than the alkaloid mix you usually get by extracting from Anadenathera using an A/B tech. The side effects experience from the alkaloid mix can be pretty intense. The batch SWIM used for this test was extremely weak and produced lots of side effects (body tension, head tension, long lasting nausea, and other unpleasant bodily sensations).

If you’re interested at all in extremely pure bufotenine, this is the way to go. SWIM thinks bufotenine (5-HO-DMT) is far better than DMT as a vaporized experience, but orally, DMT is better. Vaporized bufotenine lasts about 90-120 minutes (depending on dosage), it causes almost no “mind fuck” at all, and it causes both visual and auditory hallucinations. It has very mild side effects which only last about 30 seconds (very slight nausea and tingling in the head).

 

[69ron]

What’s good about this test was that finally a simple method was found to get rid of the side effect causing compounds in Anadenanthera. They are all very polar and easy to remove because they can’t be eluted from the silica gel even with methanol! So you simply pass your 1:1 mix of methanol and ethyl acetate dissolved alkaloid mix through silica gel, and all of the good stuff elutes and all the bad stuff stays stuck on the silica gel! You don’t even need to run a proper column, you just use the silica as a filter medium! It’s really quite cool!

This might also work using Whatman 1PS filter paper, which should effectively remove all the polar compounds in the same way they stick to the silica. SWIM will try this. He’ll dissolve the alkaloids in 1:1 of methanol and ethyl acetate, and then pass them through Whatman 1PS filter paper. This paper has silica gel on it, and so the polar alkaloids should stick to the paper and not pass through, just as they do in a column! SWIM is quite excited about this possibility.

 

[Entropymancer]

Very nice work there Ron! I wonder if there's any analytical chemists interested in Anadenanthera who might be interested in identifying these seperate fractions (particularly the ones causing side-effects). Still, it's very cool to have a clearer picture of what's coming out of this acetone/citrate extraction.

Great work.

Speculation based solely on the color: might the brown coloration (fraction #2) be a beta-carboline?

 

[69ron]

It could very well be, but vaporizing fraction #3 and then #2 the following day both gave the effects of nearly pure bufotenine. Which was a surprise for SWIM. Fraction #1 felt like DMT N-Oxide with no bufotenine effects felt at all.

The following compounds have been found in Anadenanthera (according to several different sources)

XLogP Compound
?.? 2-methyl-6-methoxy-1,2,3,tetrahydro-beta-carboline
?.? 1,2-dimethyl-6-methoxy-1,2,3,4-tetrahydro-beta-carboline
1.0 Serotonin
1.3 Bufotenine N-oxide
1.4 N-methyl-serotonin (Unknown activity)
1.6 Bufotenine
1.7 2-methyltryptoline (2-methyl-1,2,3,4-tetrahydro-beta-carboline)
1.7 DMT N-oxide
1.7 5-MeO-NMT (Unknown activity)
1.8 N-methyl-tryptamine (NMT) (Unknown activity)
1.9 5-MeO-DMT (Active at 2-10 mg)
2.0 DMT

The XLogP information for two of the three beta carbolines found in Anadenanthera is unknown. So I have no idea where they would be eluted, or if they stayed on the column. But one of them, 2-methyltryptoline, might elute along with bufotenine. It has a similar XLogP, but is slightly less polar.

SWIM’s tests show that pure ethyl acetate could not elute bufotenine, but did elute a DMT N-Oxide like compound that crystallized. So it’s likely that pure ethyl acetate could only elute everything from XLogP 1.7 and up. That would include the beta-carboline 2-methyltryptoline.

SWIM’s tests show that ethyl acetate with 25% methanol is able to elute nearly pure bufotenine (after eluting with pure ethyl acetate) along with a brown or amber material that might be some 2-methyltryptoline coming through. Whatever the amber stuff is, it seems to have little activity in the 7 mg dosage range tested. So it would appear that ethyl acetate with 25% methanol is able to elute everything from XLogP 1.6 and up. If the amber or brown material is one or more of the beta carbolines above, then they are very weak compared to bufotenine.

According to other papers, serotonin elutes with dilute hydrochloric acid. So it should not elute with methanol, or if it does it should take tons of solvent to elute it.

N-methyl-serotonin is fluorescent. I can’t find much information about it. It’s much less polar than serotonin and bufotenine N-Oxide. It’s possible that it is eluted with ethyl acetate with 75% methanol.

The main reason for this work is to find out what’s causing the side effects, and to remove them. From this test it’s clear that the side effects are caused by compounds that are more polar than bufotenine and can therefore be easily removed. But which one of the more polar compounds are responsible for the side effects? Serotonin? Bufotenine N-oxide? N-methyl-serotonin? Or one or more of the beta-carbolines? Or a mix of them?

 

[flyboy]

Yeah Ron i've been working with a recently got batch of columbrina and they were very weak compared to those from the same supplier 6 months ago. I thought it was me but i compared smoking one seed from each batch and the difference was huge.

 

[69ron]

I've heard the potency varies quite a bit by location and season for the same plant.

There's apparently a season when they are most potent. I would like to know what time of year that is.

The mature seeds are said to be the most potent. So whatever time of year the seeds are mature, that’s the time to buy them.

SWIM has had seeds that were extremely potent and nearly side effect free. And others that only produced side effects and nothing else, all from the same vendor at different times of year. SWIM hasn’t been keeping a log of that though.

 

[kemist]

WELL DONE 69RON! :shock:

I wonder if there's any analytical chemists interested in Anadenanthera who might be interested in identifying these seperate fractions (particularly the ones causing side-effects).

ILPT is a chemical analyst. Sadly he is not as wealthy as 69ron`s swim is and he currently have not access to the lab. That`s a effin shite that is. Yopo is soooooo interesting.


WERE THIS SHITTY SEEDS FROM THIS VENDA 100% Guarantee on ethically harvested popular ethnobotanicals.... ????

 

[69ron]

They were from BBB. BBB has been good before. I think all vendors vary in their quality. I've heard the seeds vary a lot in potency by strain, season and location.

SWIM will do more work on this in the near future. Hopefully a simpler way can be found.

 

[kemist]

They were from BBB. BBB has been good before. I think all vendors vary in their quality. I've heard the seeds vary a lot in potency by strain, season and location.

Thank you very much