.

Interesting. I like how you bring proper science into forums that are otherwise typically not scientific.

thanks
just passing along ideas I got from working in the biotech industry.
they may be employed in other areas of interest

grasses come to mind, and possibly, psilocybes


the beauty of this method is, with a column and strong cation-exchange resin,
one could get relatively pure DMT freebase using any A/B method..just filter the acidic (pH 4 - 5) MHRB solution, add it to the packed column, add your prepared base (~ pH 13.6). rinse the column with MEK substitute (ethyl acetate).

Anyone have experience with this? The "batch" method where resin beads are simply mixed with the extract is interesting since no column is needed and a simple mason jar may be sufficient. This seems very accessible. Also, it was neat to see the phosphate group having a good affinity to an anion exchange resin, maybe that translates to psylocybin?

How about something like this:

- Methanol extract of dry ground mushrooms.
- Mix extract with an anion exchange resin in a mason jar and give time to react (hope is that abeads absorbe -1 charge psilocybin at biological pH?)
- Rinse the beads in fresh methanol. (Edit: using a rinse pH~5.1 may be better, see next post)
- Prepare a cold vinegar/methanol (or HCl/methanol) solution with pH~4.1 and add it to the beads in a mason jar, psilocybin should move from the beads into solution? One question here is that if the resin has OH- as a counter ion, it may be hard to keep the pH at 4 and more acid may be needed (?). A Cl- counter ion based resin may work better (?). Keep cold to try to avoid converion to psiclocin which is unstable.

Then dry the acidic methanol and see what we get.

Anyone have thoughs or tried something like this already? Also, I could not find any discussion about H+ or OH- counter ion release affecting pH in the material Benz posted, does anyone have more info on that? There are mixed resin beds for water that use these counter ions that are cheap and used in aquariums...

There is a very nice free database with protein isoelectric points.

For example, the protein pIs for white button mushrooms are shown below (as an example). Not surprisingly, there's a lot

During the resin bead rinsing step, a pH of 5.1 (one unit above psilocybin's pI) may be better to remove as much junk as possible.

Notice that some proteins are still expected to make it through with a the anion resin catch/release strategy (those with pI near pH~4.1). Moar work would be needed to cleanup those, but it would be very interesting to see how the dry extract looks after the anion resin treatment.

One could also convert the psilocybin to psilocin by heating up the acid extract. Then increase the pH back to 5.1 and catch the pesky surviving proteins in anion resin while the now positive +1 psilocin makes it through. That should be very pure, and could dry to a salt if the unstable psiclocin does not break down.

Ok, so I went ahead and ordered some cheap anion resin that is used for hone aquariums. It is used for water purification, so the counter ion should be OH-. We should expect that pH will be increased when the resin starts to work and exchanges OH- for other negative ions. So, I think I'll try to condition the resin with salt water to avoid this by replaceling OH- with Cl- (the salt water should become basic). Then, the hope is that the negatively charged phosphate group replaces the Cl- when the resin is mixed with the mushroom extract.

So something like this,

- Resin conditioning: Mix OH- anion resing with salt water until pH stops increasing. Rinse resin with fresh water to remove excess salt and NaOH.
- Resin-psilocynin binding: Mix conditioned resin with methanol mushroom extract in a mason jar and adjust pH to 5.1 by adding HCl/water
- Resin clean-up: Rinse resin with fresh methanol/water/HCl solution at pH = 5.1 until wash dries clean
- Elution: Extract rinsed resin with cold methanol/HCl/water solution at pH = 4.1
- Recovery: Dry elution over a fan in a cool environment, solids should be psylocybin + any proteins with pI <~ 4.5 ish. There may be ways to purify this further.

Will see what happens. One assumption here is that the charge state of Psilocybin vs pH in water/HCl will be the same as in Methanol/Water/HCl (as read by a cheap pH meter). Does anyone know if this is wrong or of any adjustments are needed? May need some trial and error resin binding/rinses/elutions as HCl is adjusted.

Maybe the proper way to do this is to use anion Cl- based resins, but I cannot find them cheap and easy to buy.

Edit: According to this, the pH should be measured/adjusted in the water, then add the water to the methanol.

Attaching a practical guide for using ion exchange resins. Plan to start by following what they reccomend, except that instead of a column, a simple mason jar will be used. Their tea methanol extract treatment to get polyphenols looks similar to what could be done on a mushroom methanol extract to get psilocybin (may need to be gentle with pH/temperature so psilocybin does not break down).

Looks like the general procedure is similar to the initial thoughts above:

(1) condition the anion(cation) resin, (2) bind drug to resin with a high(low) pH, (3) rinse resin, (4) release drug from resin with an low(high) pH.

I'll be starting mushroom experiments, any guidance or ideas welcome. Thanks!

To guess/estimate psilocybin to anion resin binding vs pH, the pKa was looked at. The sum of the species in a negative charge state (that is, the % of psilocybin in anion form) is assumed to bind to the resin. This gives a first order plot of what may be expected when adjusting the pH.

The resulting plot is below. At pH > 8, ~95% of the psilocybin is expected to be in anion form (bound), and at pH <5.5, ~95% is expected to be neutral or positive (unbound). Earlier I mentioned that the biological pH (6-7) should be good enough to bind the psilocybin, but now it looks like that was an incorrect assumption.

To be as gentle as possible with the pH conditions, while also having a chance at being effective, the pH will we adjusted around these values when trying the anion resin assisted psilocybin extraction. Temperature will also be kept low (~fridge temps).

I think this roughly makes sense. Let me know if something is/looks wrong - want to try to start experiments in a reasonable place. Thanks!

Semi-random question - I have some ion-exchange resin, probably from a water softener of unknown providence. How might I go about ascertaining its properties? Will it typically be anion resin, which if I understand what you're saying above, is a cationic resin that binds anions?

An anion resin binds anions I believe.

I think water softener is usually a cation resin that binds Ca2+ to it. It exchanges Ca2+ for H+ typically (sometimes Na+ though which is not considered to cause hard water). If you mix a water sample with calcium cloride together with an H+ cation resin, the pH should go down I think (Ca is absorbed producing HCl).

There are also deionazing resins that have both anion and cation resins mixed in. These are the most common ones available comercially. They typically exchange both the + and - ions of a salt for H+ and OH- (water). If you mix in a water sample with CaCl2 with this kind of resin, the pH should stay flat and the salt taste should go away. They could work for pH based zwitterion extractions I guess (?).

The commercial anion resins are less common and are usually used in a two-stage process in conjunction with a cation resin in aquariums. Some hobbyist like to separate their anion and cation filters for some reason. They claim more control and superior performance, buy I don't understand why that would be. To recoginze an OH- based anion resin mixing some CaCl2 salt in will increase the pH (Cl will be absorebed and Ca(OH)2 will go into solution).

I'm learning about this and I could be wrong though, so take this info with a grain of salt (no pun intended).

Some resings also change colors. I've started to experiment with the anion resin I have. It is blue to begin with. Once 5 %vinegar is added, the color changes over time to translucent amber (it took about 10 minutes with vigorous shaking), indicating that OH- has been replaced by acetate. The issue I have is that after rinsing the acetate conditioned resin multiple times with fresh water the pH is staying low. I was hoping/thinking it would go back to neutral once the excess vinegar was rinsed out. This is confusing me at this time. On the plus side, the resin is very easy to handle with a reusable coffee filter.

Maybe the conditioned resin call still bind to some small ammount of OH- in a dynamic equilibrium (where some acetic ions move into the water and some OH- go into the resin). Taking those OH- out of solution would increase the H+ concentration, making the conditioned resin in water slightly acidic despite repeated washes. There is another effect raising the pH which is released acetate ions combining with H+ to form non disociated acetic acid. In the end the balance of all these reactions could result in a slightly acidic solution. After each wash with water, a little bit of acetate ions move into the fresh water to repeat the process resulting in consistently getting low pH.

Also, the fresh un-conditioned resin saturated with OH- is slightly basic when fresh deionized water water is added according to my calibrated pH meters. In this case the resin may be releasing some OH- in exchange for salt anions, looks like a couple part per million of NaCl salt could change the pH by 1 unit.

Interesting stuff.

Started testing the aquarium anion resin on one type of mushroom extract today.

Used 339mg of cleaned up dried methanol extract (cleaned by acetone co-precipitation, and natphta washes - first picture). It represents of the order of 20g of dry mushrooms (rough estimata, a lot split experiments where done on the parent batch). Since I haveve not bioassayed this sarting material yet, I'm not sure if the actives are in it, buy I suspect they should be.

Sorry I'm not more precise, this is just work in progress. However, several interesting things happened I want to share. Here are the steps followed:

1) Fresh anion resin was rinsed with distilled water until the pH was in the 8.5 range about 5 rinses were needed. THe pH started at 10 right out the bag and the resin had an ammonia-like odor that went away with the rinses.

2) The extract was dissolved in ~50ml water in a mason jar (which became tan in normal light and fluorescent under UV).

3) This water was mixed with two teaspoons of rinsed resin. The pH shot up to 11 and the taness and fluorescencence dissapered from the water after a couple minutes. Presumably this is from the resin exchanging mushroom extract anions for OH-.

4) The resin was rinsed with distilled water until the pH was ~ 8.5. This took about 4 rinses. The water did not become tan or fluorescencent at any time.

5) Distilled white 5% vinegar was added to the water resin mix at pH 8.5 (picture before adding the vinegar nis the second one below). The pH would drop down to ~3.5 quickly, but uppon mixing would go up to ~ 9.5 (from acetate being exchanged for OH-).

6) After adding about 25ml of vinegar, the beads strated to change color from blue to amber. The water solution became tan and fluorescent (third picture) with a stable pH of 3.5 (presumably from mushroom extract material such as proteins and psilocybin being released).

7) Resin was filtered and an additinal water rinse/filter collected which had a pH of 4.2 and was slightly tan/fluorescencent (a 3rd rinse was done but not used since it had lost all fluorescence). The water from the first two resin filtrations was combined and put under a fan.

Will post the result after the dry. Probably will not be crystals since a lot of proteins could make it through this crude mason jar process. I wanted to control the pH better to minimize the number of proteins coming through, but that was not possible with a resin loaded with OH- ions.

However, it is very interesting how the resin grabbed and then released the tan/fluorescent compounds in the mushroom extract. The water rinses between the grab/release should clean up sugars and other soluble compounds nicely. Anions that don't change charge with pH and have more affinity to the resin than acetic acid will also be cleaned up.

Unfortunately, the resulting anion resin water from the previous post dried to a goo/tar (image below).

I think it is still very interesting how the anion resin was able to trap and then release the tan color and fluorescencence in the water. Because of this I'll be doing more tests.

Things that may help improve the extract cleanliness (based on tips attached earlier in the thread):

- Use anion resin conditioned with acetone (instead of -OH). This will make adjusting the pH simpler. Also, it will avoid using large amounts of vinegar before the pH changes during the resin pull. Too much vinegar in solution may displace all/most of the anions in the resin, bit we only want to displace psilocybin.
- Before the binding resin step, pull at low pH (<5.5) with anion resin as a cleaning step.
- After the resin binding step, use alcohol (isopropyl) to rinse the resin.
- When pulling the resin use IPA + vinegar + water. Minimize vinegar isto get a pH of 5.5.
- May need to pack the resin in a filter column instead of simply moxing in a mason jar.
- What else?

So for the next test looking at something like this:

- Hippie hot water extract on plain chopped up mushrooms
- Filter and cool tea quickly (ice?)
- Optional? adjust extract to pH<5.5 with drops of vinegar and wash with conditioned anion resin. Remove this resin.
- Adjust tea pH to 8.5 with drop(s) of ammonia
- Add anion resin (conditioned with acetate), keep pH >= 8.5
- Filter out resin and rinse it with IPA
- Extract resin with IPA + pH 5.5 water / vinegar

Interested in any other options/ideas. Thank you.

Ok, so I did this starting with 54g of fresh mushrooms (5.4g dry estimate). Cooled tea with ice and did the optional "defat".

The hippie tea does change when adding the anion resin. Below is a picture of the crude tea (1st picture) and a slightly more clarified tea after the "defat" pull with anion resin (second picture).

Thing is not much is left after evaporating the pH8.5 pull.

So, I went back to the leftover tea a day later (it looked slightly milky at this point) and added fresh -OH resin. The pH quickly shot up from 8.5 to ~11+, which is an indication that anions in are being exchanged for -OH from the resin. After a few minutes the tea at this pH turned a beautiful amazing blue (3rd picture).

I recovered this last resin, rinsed it with IPA, and "pulled" it with vinegar. The water/vinegar became slightly cloudy/tan (4th picture), so it looks like something came through. This pull is being dried and I'll report back.

It looks like the acetic acid conditioned anion resin did not do a good job at picking up psilocybin. Maybe a higher pH or an -OH resin may do the trick. Even if the anion resin is able to take out the psilocybin from solution, in this example enough (or maybe all) was left behind to degrade into (presumably) blue quinone oxydation products.

This residue dried to a hard resin. Upon testing it was innactive.

One possible interpretation is that psilocybin was not attched to the anion resin before the final acid pull. Instead it dephosphorilated and oxydized to a blue quinone which remained in solution.

Prehaps using lower temperature, avoiding the IPA rinse, avoiding the initial pH4 step, and/or a column geometry may help. However, I'm not going to pursue this path right now.

It is interesting though that the ion exchange resin picked up other gunk.

Not going to give up yet. I'm going to get mixed bed and cation resins. Will try something like this:

1) Hippie mushroom tea (filtered and made cold)
2) Add mixed bed resin, mix, and filter (keep water)
3) Add cation resin, adjust pH to 4 with HCl, mix and filter (keep water)
4) Add cation resin, adjust pH to ~1.5, mix and filter (keep resin). Psilocybin may not survive this harsh pH even at low temp (but not sure, anyone know?).
5) Rinse resin with IPA - keep this separate and evap.
6) Add minimal amount of water to the resin from the previous step. If the pH is below 4, adjust it to 4 by adding mixed bed (or anion) resin slowly. Mix and filter (keep water).
7) Evap

Another option is to do a 1h 70c temperature cycle after 3), and then continue at step 4) using pH ~ 5 since we may now be dealing with psilocin. To release the psiclocin from the resin the pH would need to be moved to ~9 though, which may destroy it if is kept there for too long (work fast and at low temperatures).

To increase the pH in step 6) a source of anion resin is used. Idea is that the Cl- ions will be exchanged with OH-. If instead ammonia is used the resin will be resistant to the pH changes as NH4+ will be replaced by H+ until the cation resin is exahusted. Since the resins themselves adjust the pH this gives another possible way to proceed:

1) Hippie mushroom tea (filtered and cold)
2) Add cation resin until pH~4 (will this happen?), mix and filter (keeping water)
3) Add cation resin until pH~1.5 (will this happen?), mix and filter (keeping water)
4) Rinse resin with IPA, keep filtered IPA separate and evap.
5) Add water to resin and if needed anion resin until pH ~ 4.
6) Filter water and evap.

Then, there is this extreme short method;

1) To a cold filtered mushroom tea add excess cation resin, pH will drop significantly (shooting for pH < 2). Filter out water and rinse resin with 99% IPA. Add fresh water and small amounts of anion resin (pH will rise) until pH is ~ 4. Filter water and evaporate it.

Anyhow, will be trying something like these options next. I'm really just guessing here and any input is welcome.

Update: I tried the low pH strategy (1.5) to pick up psilocybin with a cation resin but was not successful.

Mindlusion had some good input in the live psychedlic research cybernetic support center (also called the nexus chat).

The resin can act as catalists for hydroliysis (psilocybin -> psilocin). This could explain some of the issues seem in previous experiments. To avoid this, focus will be on isolating psilocin during the next experiments.

Here is a possible approach. I also have a TDS meter (Total Disolved Solids, which is really a conductivity meter) to see if it can help understand what is happening.

1) Make cooled filtered mushroom tea
2) Add cation resin slowly until pH drops ~ 4. Add mixed resin after that (if pH rises add more cation resin so pH stays at 4). Once tea stops changing color (or TDS meter reading is stable?), remove resins. Hoping this is a "defat" step and psilocybin has remained stable since and has not attached to the resins at this pH. Any natural psilocin would be lost since it would attach to the resin in theory.
3) Heat-cycle acidified tea (70C) for 1 hour. Hope is that psylocybin hydrolizes to psiclocin+ and H2PO4- (at pH 4 the phosphate- ion is a great leaving group). Check TDS, does it go up indicating hydroliysis has happened?
4) Add mixed bed resin to absorbe both psilocin+ and phosphate-, exchanging them for H+ and OH- (water) keeping the pH stable. Does TDS drop to the the reading before hydroliysis indicating this is happening?
5) Recover resin
6) Cover resin with 91% IPA. Add ammonia slowly. Idea is for NH4+ to displace the ions on the resin: first any left over H+ (during this period pH should be stable) and then psiclocin+ at which point pH can rise. Psilocin+ can depronate and act as a butter to the pH increase (a change in the rate of pH/TDS increase may be seen if this depronation happens).
7) Remove resin and evaporate the IPA and see what happens.

Interested in any other ideas or if you see something that I'm missing, misunderstanding, or misassuming feedback would be greatly usefull. Thank you.

Ok, I think we can say that the ion exchange resin can absorve psilocinH+ (along with other ions and protenated proteins).

120g of fresh mshrooms were extracted with dilute vinegar (pH~4) for 1h (to convert psilocybin to psilocin). This extract was split into two. Jars, to the right jar ion exchange resin was added (mixed bed, H/OH based). The pH was surprisingly stable. After about 10 minutes with intermittent shaking, the jar with the resin stopped fluorescing (see first image, jar with resin is on the right). The loss of fluorescencence indicates that the resin absorbed something. A TDS (total dissolved solids) meter was also used and the tea with the resin dropped from 600 ppm to ~50ppm.

The resin was filtered out (liquid moved to another jar), rinsed with fresh vinegar, rinsed with fresh IPA, and set aside (more on what happened to it tomorrow).

The jar without the resin pull and the jar with the liquid that had been pulled by the resin were left in the fridge for several days. The jar that had not had the resin pull turned a blue/green color while the jar with the resin pull did not. I think this is evidence that the resin did indeed pull the psiclocin out of the vinegar extract (second picture, liquid resulting from the resin pull on the right).

This resin was extracted with ammonia water. It was difficult to adjust the pH, I think this is because of the presence of both anion and cation resins loaded with H+ and OH- ions. Eventually the pH was adjusted to 9ish and the resin released fluorescent material into the water.

This water was evaporated with the aid of a fan. No color changes indicating psilocin degradation were observed. Both fluorescent and other residue could be seen in the dish. 99%,IPA and limonene did not pick it up, it seem to washed out some material. 75% ethanol picked l up the fluorescence immediately and very effectively. This resulted in a clear tawny liquid which seems active at very low dose, but repeats at higher dose are needed to confirm the low dose effects were not placebo. Image of the liquid is below. It dries to a managebe residue. If funaric acid is added it dries to a more crystaline like material.

Next, will move to cation resin loaded with Na+ to make the pH adjustment straightforward. This process is not practical in my opinion because of how the H+/OH- mixed bed resin resists pH adjustment.

Ideas and suggestions from monkey mind's other than mine welcome. Thank you.

I can't offer any info but I always look forward to your posts and the work you do, I cannot wait for a proper writeup/tek on how you do it. Thanks for keeping us all updated appreciate it.

Thanks sbc1.

Another day another test. This time, based on the learnings so far, moved to Na+ based cation resin. These are widely available to remove water hardness (trade Ca2+ for Na+) and are very cheap.

There may be an interesting result here.

Here is what was done:

-177g of fresh mushrooms vinegar tea, kept warm for an hour in an attempt to convert all psilocybin to psilocinH+.
- Mixed in Na+ cation resin, hoping to capture psilocinH+ with the resin (along with other cations)
- Recovered resin and washed it with fresh water a few times and once with fresh 75% everclear.
- Pulled resin with the everclear and a touch of ammonia (very little) the everclear becomes tawny and fluorescent indicating we are pulling (aka eluting) something from the resin.
- Ph was at 9.6 for my particular pH meter. Since a Na+ resin is used, the ammonia may have been exchanchanged to NaOH, so instead of evaporating, the pH was brought down to 4.2 by adding tartaric acid. This means that Na tartrate and/or Ammonium tartrate could be in solution (along with hopefully Psilocin Tartrate).
- The acidified pull was dried.
- Result is 3.0g of crystalline brown material (we've learned that this is expected when drying relatively clean mushroom extract and adding a crystalline organic acid). Going by weight difference, about 600mg of mushroom material is present (psilocin should only be 170mg, so at the most, the mushroom material pulled is roughtly 25% pure if the psilocin cam through this process and survived). Proteins that can pronate and depronate like psilocin in can be picked up with this process also I think (see post #3, of concern would be proteins with pI~ 9 for which there is a secondary density hot spot) . Pictures below.

This result needs to be bioassayed. If active will try to clean it up with washes, etc. I think dry acetone can pick up the excess tartaric acid and leave the salts behind (hopefully we don't revert to a resin after that wash)