Argon gas is readily obtainable - inert atmosphere work may be of value in conjunction with something as labile as psilocin.

Agreed. Looking at the literature papers again, the drying is always done under inner atmospheres, air drying could be the cause of weak bioassay's in many experiments so far.

That's why a vacuum chamber should work, same principle as when you purge the solvents out of your oil to make wax or shatter, no oxygen to mess with the product while it's being purged in a vacuum

I'm now fairly certain that simple air drying should be avoided. Also, avoiding light is important.

Attaching a paper that discusses the subject. Interestingly, the found that N2 drying was superior to vacuum drying. However, they do not spell out their vacuum drying technique. One question is, if the vacuum chamber atmosphere is purged first with N2 or CO2, will vacuum be my just as good as drying over N2?

One interest thing is that they found vitamin C protected the product during vacuum dry and storage (see plots below). Another interesting thing is that they used ethyl acetate (available in the US hardware stores as MEK substitute) and ammonia to elute psilocin. We can try this on the cation resin which may give a purer product. Why? Because ethyl acetate is a protein crashing solvent, so it should leave proteins behind.

More stuff to consider. Work is ongoing

Quick update.

10g of mushrooms and 3/4 teaspoon of Vit C where steeped in 70C water for 1h to try to convert psilocybin into psilocinH+ and move it into the water. This tea was filtered (final pH 4.35) and shaken with Na cation exchange resin in a Mason jar for 10 minutes. The tea was then filtered out and saved (plan to bioasy this, should not be active if both the phosphatase enzymes and the cation exchange resin worked so that actives ended up as psilocinH+ binded to the resin).

The beads were washed with water until water ran clear (normal and UV light). Then the beads were extracted (aka eluted) with baking soda water (~pH 8.5) which became tawny and fluorescent under UV. After this elution these beads (calling them mother resin beads) were set aside for other tests

Followed downwardsfromzero's advice and bioassayed 1/5th of this basified water (neutralized it with vitamin C first to make a delicious effervescent drink), unfortunately it was not active.

So where are the actives? I took small samples of the mother resin and tried different tests. One was elutimg with ammonia and ethyl acetate (MEK substitute at the hardware store). The acetate became cloudy. The water was also tawny, and then started changing color to green, indicating that something new was being eluted.

So maybe the baking soda water was not strong enough to elute psilocin?

As mentioned before, will test the original tea that was treated with the resin to see if any actives remain there.

Ideas welcome, more tests to follow, including boiling the water at the end to denatured proteins.

Interesting, I put some of the mother ion from the previous post in alkaline water again and also in | High Pobability of Braindamage by Creepy non tested Drugs (forced by scammer 69ron) | (no color change after shaking) and then ran it in the microwave for one minute, it boiled for 20s or so. The water solution came out cloudy, so something happened.

Does the temperature and/or microwave help elution?

I wonder if at higher temperature it is easier to pull psilocin out off the resin at mild pH (if indeed it is trapped in there). Water solubility changes a lot for psilocin from reading the info out there, so it's plausible I guess. That would be ideal since a bunch of non active twany stuff was eluted already at room temp (from the bioassay in the previous post).

Edit: Attaching some literature on the subject. Though I don't quite understand it, it looks like temperature can change how things elute.

That colour change from tawny to green suggests to me oxidation is occurring - tawny + blue = green?

Any progress on inert atmospheres?

Ah, I hadn't thought of the color combo, makes sense. That supports the possibility that baking soda in water at room temp may not be strong enough to elute psilocin out of the resin I'm using (if binded to it).

I've looked into innert atmospheres, they are a bit tricky to set up. Vacuum evap is easy though. It can also also be put in a small freezer. So, for a couple hundred bucks freeze drying should be possible. I got a list of hardware ready to order if needed. If we get a positive bioassay coming out of the ion resin and we can't crystallize it, I will pull the trigger on the freeze drying setup to see what happens.

Alright, I drank 1/10 of of the 10g mushroom filtered tea that had been steeped in 500ml of water with 3/4 tsp of Vit C at 70C for 1h (final pH 4.3) and then treated with cation resin. The tea had been kept in the fridge (wrapped in aluminum foil in a mason jar) for a few days.

Result: Some nervous initial energy, but no significant effects. I fell asleep quickly which is not possible for me at all with mushrooms. The tea had all the funky taste of shrooms though

I think this indicates the psilocybin did convert into psilocinH+ during the acidic 70C 1h water steep and was subsequently absorbed by the ion exchange cation resin.

Based on the results from trying to recover (elute) the psilocin, it seems that room temp baking soda water does not elute it - but it does elute something that does not seem active. Maybe just water soluble proteins with -OH groups.

Several more tests have been done and based on color changes (towards a greenish blue) other systems acould be eluting psilocin (for example ethyl acetate and ammonia, or microwaved hot baking soda water).

Out thhese elution options tested, one that I'm going to focus on more is baking soda + 75% everclear. A promising green fluorescence clear liquid was eluted by this system from the mother beads (which had already previously been washed until baking soda water ran clear).

I have a sample of this 75% ethanol elute that was neutralized with VitC quickly after elution | High Pobability of Braindamage by Creepy non tested Drugs (forced by scammer 69ron) | was also added before neutralizing it. I'm hoping the goods are in it and plan to bioasy this.

If bioassay of this elute is positive, will try to dry it in air to see | High Pobability of Braindamage by Creepy non tested Drugs (forced by scammer 69ron) | can protect psilocin. If activity is lost during air dry will try to crash a hypothetical | High Pobability of Braindamage by Creepy non tested Drugs (forced by scammer 69ron) | complex (with acetone). If that fails, will invest a couple hundred bucks in the equipment to lyophilize (freeze dry) the (hopefully active) neutralized elute + | High Pobability of Braindamage by Creepy non tested Drugs (forced by scammer 69ron) |.

Edit:Added a couple articles discussing | High Pobability of Braindamage by Creepy non tested Drugs (forced by scammer 69ron) | complexation of curcumin (which also has an -OH group that degrades in Alkaline conditions). To make a complex they did basify and then quickly neutralized curcumin in the presence of | High Pobability of Braindamage by Creepy non tested Drugs (forced by scammer 69ron) |. Above something similar was don't, but the | High Pobability of Braindamage by Creepy non tested Drugs (forced by scammer 69ron) | was added after the ion resin pull, instead of before the drug is put in the Alkaline solution. Next time I'll modify the order, so that | High Pobability of Braindamage by Creepy non tested Drugs (forced by scammer 69ron) | can complex at t=0.

Result: whatever eluted with alcohol and baking soda was not active.

Among the many possible issues I think I may be having trouble eluting the beads. Bellow is a picture of the beads in a baking soda solution after a few days. Looks like something that is still stuck to the beads is degrading.

Gonna try to elute again with washing soda.

Another possible issue is that the psilocin is binded to a protein and that protein is stuck hard to the ion resin. Maybe denaturing proteins with heat at the end ot the tea steeping step could help (boil at 100C for a few minutes)?

Anyway, will keep on trying but ideas welcome.

I did a test on the beads that are turning greenish blue after refusing to elute anything into room temp alkali solutions:

Boiled them in alkali water. They released a bunch of stuff. The beads stayed discolored, but much less intensity. I filtered the elute, acidified it and put it on fresh beads. Surprisingly, it smelled funky, with a shroomy odor. Then, I filtered this out, rinse the beads and extracted in alkali water at room temp. They released a bunch of stuff into solution.

So by boiling the beads that had stopped eluting into a base at room temp, some stuff was released. When this stuff was acidified and put through a set of fresh cation beads, at least two fractions could be recovered: one that did not bind to the beads in acid conditions and was stinly, and another one that did bind and could be released into alkali solution at room temp (before boiling this was not happening).

I think this is consistent with some kind of change during the boil that makes the extract more friendly to ion exchange elution? Seems consistent with protein denaturalization through heat?

I don't know, maybe I'm wrong. One next test as mentioned before is to boil the next tea after the enzymatic conversion and see how that behaves.

OK, so after boiling a new tea extract I'm still not getting great elution with baking soda (confirmed no significant activity from eleute) or washing soda.

However, I did a test with a little bit of ammonia. A lot of stuff eluted based on color changes (????).

This is confusing to me since it does not seem only pH driven. Why would the ammonia elute so much more (assuming color and UV change means elution here)?

I'm confused by this, but it is what I see happening. Any help to understand is appreciated.

I'll need to find a safe way to bioassay the ammonia elute, perhaps evaping until the smell goes away and then neutralizing it. Or I could try to elute with dilute NaOH and skip ammonia (could the carbonate ion be getting in the way?). Will be doing more testing in the near future.

Answering my own question, it could be that the pH is changing during the ion exchange resin pull? I've been setting up the pH before it goes into solution. This could be an issue for sodium carbonates because if Na+ replaced what is being eluted, since some of the biological molecules should have the form XH+, that could liberate H+ and the liberated H+ would neutralize the elution. Ammonia could be a little more resilient to this the way I was doing things.

For example (R- represents the negatively charged cation resin functional group)

NaHCO3 + R-XH+ -> H2CO3 + X + R-Na+, this generates carbonic acid which neutralizes the elute and stops elution.

So, this is simply a case of me being inexperienced with ion resins. The answer is simple, monitor the pH during the ion resin pull. 🤦‍♂️

I ran some elution tests on a "mother" resin. This time a 4% NaOH solution was used to adjust the pH during the "batch" elution process.

First used water. Looks pH was 8.3, the 4% NaOH moved it up to 9.4 with 3 drops, the pH increase was continuous as far as I could tell and stuff was being eluted based on UV and visible light changes.

Next I used everclear (75% alcohol). This was interesting. The pH would shoot up after each drop of NaOH and then drop over the next few minutes. I think this is consistent with eluting something in the form

R-XH+ + NaOH -> R-Na+ + X + H2O

Three drops were used during the everclear elution, this is what the pH did (⌚ =~ 5 minutes)

8.5 -> 💧 -> 11.6 -> ⌚ -> 8.7 -> 💧 -> 11.7 -> ⌚ -> 8.9 -> 💧 -> 11.9 -> ⌚ -> 9.4

This elute was pulled from the resin and acidified to pH6.5 with a dash of vitaminC. | High Pobability of Braindamage by Creepy non tested Drugs (forced by scammer 69ron) | was also present to try to protect from oxydation.

Will be testing this process further, doing bioassays and reporting back.

After further testing, adding 4% NaOH drops elutes a lot a more stuff than the weaker baking and washing sodas. Typically when doing this, pH shoots up to 11 or so, then comes down within minutes and liquid becomes more fluorescent. I repeated this process until the pH stabilized at 9.4 after dropping (about 5 NaOH additions).

However, the resulting liquid is not active after neutralizing with vitamin C and doing a bioassay. Maybe the pH spikes could be hurting the psilocin, or the psilocin has not yet been eluted (I'm assuming Psilocin was originally bound to the resin since the original tea treated with resin is not active).

Reading more about this, the lab folks use buffered solutions with increasing ionic strength to help elute drugs. Sometimes they add proteins that help elute other proteins.

So to elute psilocin in mild conditions these additives may do the trick. Stuff like ammonium acetate (which can be sublimated off) and/or an arginine (a basic amino acid that should have a positive charge and may compete with psilocin binding to the resin).

Etid: Attaching some info on how to select pH and buffers. Also attaching a publication from 2016 discussing how arginine helps with elution. Also, in this thread someone claimed arginine can be precipitated by glutamine, with is surprising but can be tested.

I tested ammonium acetate on the beads that had been treated with NaOH. More stuff eluted out.

So we can say that we are not eluting everything in the beads yet. This may explain the poor bioassay results so far. Focus now is on making sure everything is eluted and then doing more biassays.

Next tests will be using arginine. Unlike ammonium acetate one can safely bioassay this eluting "helper". Plan to use an arginine base + organic acid to adjust the pH and have a buffered eluting solution that is safe to bioassay. For example arganine and vinegar together, or arganine and vitamin C.

Below are the charges vs. pH for arginine (1) and psilocin (2). Arginine will be more positive and could be a good agent to displace psilocin. Alcohol may help elute psilocin too since it would increase psiclocin solubility into the eulte and decrease arginine solubility pushing it onto the resin (I believe, this is just speculation on my part).

I'm getting the protein supplements from the health store and will do more tests in the future.

After reading about this more, turns out that an ion exchange method was published for psilocin/psilocybin.

They used what appears to be a sulfonic based cation resin, similar to what we are working with now but with HPLS. The mushrooms where extracted with acidified methanol and that was loaded into resin with a column geometry. The column was eluted under acidic (pH 3) buffer conditions with increasing ionic strength (250mM) using water and up to 20% ethanol in one of the papers.

So the precedent exists with HPLS (attaching two published works that talk about it and adding the picture below). Something similar may be possible to do here at the nexus using a simple gravity column I hope. We could use ammonium acetate as the ionic elution buffer under slightly acidic conditions and evaporate it off. HPBBCD to protect the vulnerable psilocin while increasing its solubility into the elute may be useful. Finally, arginine could help elute the actives if needed, but may become part of the final product.

I'm also attaching a very nice mushroom review paper that I enjoyed and wished I had found sooner.

Note that I'm backing off work on batch elution since it is proving difficult. Column elution makes sense since the drugs can be pushed out even if some level of affinity exists between the ion resin and the drug.

Some new learnings, results, and thoughts on ongoing work:

- One issue that can arise with pH adjustments is that solubility goes down at the pI, making elution less likely.
- It is important to use buffers with stable pH and known ionic strengths to get consistent results.
- A way to elute could be bellow the pI (still in ionic form and soluble) while increasing the ionic strength. This is good because it avoids alkali conditions that could degrade the actives.
- Typically, charged molecules stick to ion resins when ionic strengths are < 100mM and being to elute above that. The ionic strength at which elution happens depends on the molecule of interest and even the nature of the buffer molecules.
- Too high ionic strength can lower solubility and turn off elution into the liquid (salting out). This salting put effect depends on the buffer molecules too (e.g. ammonium sulphate being particularly strong for salting out effects).
- Adding some ethanol (say 20%) during the elution could be useful (keep solubility high and protect psilocin).
- When working with psilocin the phenolic ring is sensitive to degradation from air and light. It needs protection and handling with care. Maybe | High Pobability of Braindamage by Creepy non tested Drugs (forced by scammer 69ron) | can help as mentioned before. Freeze vacuum dry many be needed if | High Pobability of Braindamage by Creepy non tested Drugs (forced by scammer 69ron) | does not stabilize the molecule before drying.
-Step column elution and batch elution are claimed to be equivalent in the literature. That puts the very simple batch elution back on the table.
- The elution conditions are very important. Need to have enough ionic strength to detach psilocin from the resin, but not too much ionic strength to salt it out. Also need an acidic mild pH to protect it from alkali oxidation and too keep it in ionic soluble form. Keep low lighting conditions. Quite a few things to consider, but seems doable.
-A color reagent is very helpful to figure our what is going on. I had vanillin in the pantry so made a vanillin/HCl/ethanol reagent. This was applied to different column fractions and it does look like different stuff is eluting at different times. Got orange, deep red, deep blue, and no reaction colors (see image below). This is very promising

So after a lot of failures (or after succeeding at finding what does not work ) here is the protocol that is shaping up to try next.

1) Extract mushrooms into vitamin C water at 70C for 1h (5g of VitC per 10g of mushrooms). Final pH should be close to 4 after extraction. Adjust to pH6.5 (with baking powder for example). Add water if needed to final extract to lower ionic strength below 100mM (guessing we need a final volume of 1L of water per 5g of almost neutralized VitC)
2) Condition resin with 50mM pH6.5 VitC/baking soda buffer, verify pH is stable after going through resin. This can be done with the batch method (Mason jar and resin mixed and then filtered to separate). Since we are using an Na+ ion resin already, pH should be very stable, but it is good to verify. If one is using an H+ resin this step would be very important and very necessary I think.
3) Save a small extract sample and load the rest onto conditioned resin (Mason jar batch method).
4) Mix extract and resin. Sample extract with reagent and compare to the sample reference extract. Abscense or change of reaction indicates indoles are on the resin (I think). Mix until reagent result is stable/unchanging. If resin attachment concerns exist try diluting the extract to lower ionic strength and do more reagent tests.
5) Rinse resin with fresh conditioning solution until clear.
6) Elute at pH6.5 and 20% ethanol by slowly increasing the ionic strength (in 50mM steps?). Check for indoles with reagent on a small sample of each elute.
7) Bioassay different elutes. Keep in mind that more than 5g of VitC may not be ideal, 3g or more may cause discomfort. Keep track of the molarity and volume of what is available for bioassay to know how much Vitamin C is being ingested.

It this works, we could know the elution condition for psilocin. | High Pobability of Braindamage by Creepy non tested Drugs (forced by scammer 69ron) | may alter the elution too. After that and if successful we could move on to volatile buffers like ammonium acetate for elution which can be evaped off to get a relatively pure product. If | High Pobability of Braindamage by Creepy non tested Drugs (forced by scammer 69ron) | does not protect psilocin during evaporation freeze vacuum drying may be needed. If | High Pobability of Braindamage by Creepy non tested Drugs (forced by scammer 69ron) | works it could open up new ROIs.

I'm sure I'm still missing learnings and will make more mistakes, so input welcome.