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Capn Blue Beard
The Majestic Beard



Registered: 03/14/13
Posts: 755
Last seen: 8 years, 5 months
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Acquire the Psilocybin & Psilocin Content
#18135330 - 04/19/13 12:57 PM (11 years, 1 month ago) |
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I want to get into selective cloning for potency, but I need a way to determine the actives before I go about cloning 'not so potent' fruits.
Any help or links would be greatly appreciated.
-------------------- My Level 5 Trip Report Having a Troubled Grow? Read This! Capn Blue Beards Brothel - Old Grow Log "I honestly dont grow drugs. I just try to provide them the perfect environment, they have to grow themselves."- MoGrow
** I have been gone for 10 months, any and all information I supply could be old, wrong and unintentionally fictional. I apologize for this in advance and recommend you wait for more information before settling on my advice.**
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StygianKnight
A Mushroom

Registered: 03/12/12
Posts: 2,717
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Re: Acquire the Psilocybin & Psilocin Content [Re: Capn Blue Beard]
#18136329 - 04/19/13 04:33 PM (11 years, 1 month ago) |
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Feed them to big scientific machines. Feed them to people and get their experiences. Either way, someone's gotta eat it.
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fastfred
Old Hand



Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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Re: Acquire the Psilocybin & Psilocin Content [Re: Capn Blue Beard]
#18136354 - 04/19/13 04:40 PM (11 years, 1 month ago) |
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Semi-quantitative TLC is your best bet.
You'll need some TLC plates, a few solvents, and some p-DMAB or other colorimetric reagent.
There is an older article floating around about using the p-DMAB (and/or other reagents) and a spectrometer. Unless, or even if, you have a spec laying around I suggest TLC. There is plenty of literature out there on the subject.
If you can't find what you're looking for post back and I'll dig something up for you.
Actually, I do have something for ya!

That should give you some starting values and solvent systems to try. I believe p-DMAB is more sensitive and specific than most other reagents out there.
Basically, you will take a known weight, extract and/or dilute to a proper concentration, spot on your TLC plates, place the end in the solvent to run the plate, develop the plate by spraying with the reagent, wait a fixed time, then score the spots by size.
Once you get going, TLC is about the cheapest test you could ever come up with. If you're cheap you can wash the plates with some solvent to run off the spots, dry them, and reuse the plates at least a few times. Lots of info out there about making your own plates and running them, it's all chem 101 or 213 level techniques.
If you really wanted to get serious you can work out your own standards or determine the best dry weight -> extraction solvent volume. You can easily score the spots against each other, or you can use a standard, or you can use the dilution method. The dilution method is where you find the minimum concentration where you can detect a spot on the plate. You can probably find literature values for this, which will allow you to get a fairly accurate absolute concentration value.
There's also some software programs out there where you can scan the plates into the computer and it will analyze the the size, color intensity, and density across the spot.
-FF
-------------------- It drinks the alcohol and abstains from the weed or else it gets the hose again. -Chemy The difference between the substances doesn't matter. This is a war on consciousness, on our right to the very essence of what we are. With no control over that, we have no need to speak of freedom or a free society. -fireseed "If we are going to have a war on marijuana, the least we can do is pull the sick and the dying off the battlefield." -Neal Levine (MPP) I find the whole "my drug should be legal but yours should be illegal" mindset disgusting and hypocritical. It's what George Bush and company do when they drink a cocktail and debate the best way to imprison marijuana users. -Diploid
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thenilsmeister
Shroomery addict


Registered: 04/23/12
Posts: 262
Last seen: 3 years, 1 month
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Re: Acquire the Psilocybin & Psilocin Content [Re: fastfred]
#18147533 - 04/21/13 11:47 PM (11 years, 29 days ago) |
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Damn thats cool
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mycofever
Part-Time



Registered: 10/13/12
Posts: 605
Loc: USA
Last seen: 5 years, 1 month
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Re: Acquire the Psilocybin & Psilocin Content [Re: thenilsmeister]
#18151910 - 04/22/13 07:17 PM (11 years, 29 days ago) |
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fastFred you are just full of knowledge.
-------------------- Patience will help you keep your sanity.It will insure your success if you are patient in all aspects of mushroom growing.When you rush you are prone to make mistakes and all of your efforts are wasted.
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Kizzle
Misanthrope


Registered: 08/30/11
Posts: 9,866
Last seen: 2 months, 9 days
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Re: Acquire the Psilocybin & Psilocin Content [Re: mycofever]
#18152763 - 04/22/13 09:39 PM (11 years, 29 days ago) |
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I think any method you use you're going to want to clone the mushroom before it's tested which mean cloning some duds in the process.
Edit: In retrospect I suppose it might save you the time of actually cultivating them. Getting a decent clone to use just by trial and error isn't that hard but it would be nice to have a less subjective method to use.
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Edited by Kizzle (04/22/13 09:53 PM)
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trade_om
An Old Omelette

Registered: 10/19/03
Posts: 172
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Re: Acquire the Psilocybin & Psilocin Content [Re: Kizzle]
#18171285 - 04/26/13 10:55 AM (11 years, 25 days ago) |
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TLC is good for qualitative analysis. Quantitative analysis becomes a bit more involved. To get real data and results you would have to plot a concentration curve to "calibrate". Also, quantitative without digital assistance is next to impossible, unless you are comparing very different concentrations. Digitally enhanced TLC (search this term on google) also has problems such as the algorithm used by most household/office scanners and cameras to average and balance the colors. But its doable if you can spot several known concentrations and scan/photograph them. If the camera/scanner is no good, you will not get a linear concentration curve.
The RF table FF posted is a good reference. Another good one would be methanol/chloroform (or methylene chloride) 1:9 ratio. Psilocybin rf = 0.00 Psilocin rf = ~0.8
-------------------- Trading omelettes is fun..... :P
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KnotHardly
Stranger


Registered: 06/24/09
Posts: 35
Loc: Wouldn't You Like To Know
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Re: Acquire the Psilocybin & Psilocin Content [Re: trade_om]
#18171654 - 04/26/13 12:32 PM (11 years, 25 days ago) |
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Here's how the United Nation recommends quantitative analysis of psilocybin/psilocin, and mescaline as well. Looks relatively simple if one had access to proper lab equipment.
Manual For Use By National Narcotics Laboratories
-------------------- "I collect mold, spores, and fungus." ~E. Spengler
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fastfred
Old Hand



Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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Re: Acquire the Psilocybin & Psilocin Content [Re: KnotHardly]
#18172682 - 04/26/13 04:01 PM (11 years, 25 days ago) |
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Quote:
TLC is good for qualitative analysis. Quantitative analysis becomes a bit more involved. To get real data and results you would have to plot a concentration curve to "calibrate".
Qualitative and quantitative results just depend on how you apply the technique. I alluded to some of the techniques above.
Quote:
Also, quantitative without digital assistance is next to impossible, unless you are comparing very different concentrations.
Fortunately, changing the concentration is well within our power. I suggest the minimal observable spot method would be pretty good for our uses.
Using the literature, we can find quite a few references on the minimal observable spot concentration. Since we don't have an easy way to prepare good standards, using the literature values will have to be good enough.
I don't have them on hand at the moment, but assume for the discussion that the minimal spot is 20ng/mL. Simply take your sample and prepare a series of dilutions. Spot out those dilutions and find the one that gives the minimal observable spot. Now back-calculate that using the lit. value of 20ng/mL and you have your sample concentration. From there you can create your own standards using dilutions from that original sample and use them to score all your other samples, create curves for your digital analysis, etc..
While that method probably isn't good enough for publication... it will give you a pretty good idea of your absolute concentration of the original sample.
No matter what method you use, you're never going to get that precise of a measurement of what was in the initial material. Even with GC/MS you still have to deal with extraction efficiency, reactions during extraction, etc.. This is always going to be the major source of error, so I don't think the downstream accuracy is really all that important.
You also can't GC psilocybin due to it being dephosphorylated during the vaporization, so if you want cin/cybin ratios or a better estimate of the total actives you're pretty much stuck with TLC.
Since we're most likely talking about relative concentrations anyways, TLC is a pretty darn good method. With proper extraction and dilutions it should be really easy to compare two samples and figure out the relative potency.
I should also point out that the solvents, plates, developers, etc. are easy to get and not all that expensive. We're talking about doing these tests using quite small volumes, so the cost is quite cheap.
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The RF table FF posted is a good reference. Another good one would be methanol/chloroform (or methylene chloride) 1:9 ratio. Psilocybin rf = 0.00 Psilocin rf = ~0.8
Thanks for the reference, but that doesn't seem like a particularly good system since the cybin doesn't seem to move from the start. Do you have a cite? Perhaps they converted all their cybin to cin.
Thanks again, for the lab manual cite. Here is the relevant info. Starts on page 33.
Quote:
[Retyped and organized by FF] Solvent A: n-Butanol 20, Acetic acid 10, Water 10 Solvent B: Methanol 100, Concentrated ammonia solution 1.5
Rf in solvents A and B, respectively. Psilocybin -- 34, 5 Psilocin Rf - 59, 39 Baeocystin -- 59, 39
They suggest the detection limit is 20ng for psilocybin and 10ng for psilocin using a p-Dimethylaminocinnamaldehyde developer. They claim that Ehrlich's reagent is less sensitive.
Sounds like this would make a fun project. We've talked about this for quite a few years now. Searching my posts for "TLC" will probably bring up some useful results.
-FF
-------------------- It drinks the alcohol and abstains from the weed or else it gets the hose again. -Chemy The difference between the substances doesn't matter. This is a war on consciousness, on our right to the very essence of what we are. With no control over that, we have no need to speak of freedom or a free society. -fireseed "If we are going to have a war on marijuana, the least we can do is pull the sick and the dying off the battlefield." -Neal Levine (MPP) I find the whole "my drug should be legal but yours should be illegal" mindset disgusting and hypocritical. It's what George Bush and company do when they drink a cocktail and debate the best way to imprison marijuana users. -Diploid
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trade_om
An Old Omelette

Registered: 10/19/03
Posts: 172
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Re: Acquire the Psilocybin & Psilocin Content [Re: fastfred]
#18175275 - 04/27/13 12:51 AM (11 years, 24 days ago) |
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Quote:
Qualitative and quantitative results just depend on how you apply the technique. I alluded to some of the techniques above.
I dont mean to be anal, but from a chemist stand point, our eyes are definitely not good detectors.
Quote:
Fortunately, changing the concentration is well within our power. I suggest the minimal observable spot method would be pretty good for our uses.
Using the literature, we can find quite a few references on the minimal observable spot concentration. Since we don't have an easy way to prepare good standards, using the literature values will have to be good enough.
comparing say 20ng/ml with say 25ng/ml, the difference will be un-noticeable to the naked eye!
Quote:
You also can't GC psilocybin due to it being dephosphorylated during the vaporization, so if you want cin/cybin ratios or a better estimate of the total actives you're pretty much stuck with TLC.
HPLC FTW. easily done, can find a few standard methods for cin/cybin
Quote:
Thanks for the reference, but that doesn't seem like a particularly good system since the cybin doesn't seem to move from the start. Do you have a cite? Perhaps they converted all their cybin to cin.
This is what I use. I like that the cybin does not move at all, since it is so easy to distinguish between the 2. no confusion, no what ifs. cybin stays in the bottom. cin goes to the top.
Quote:
They suggest the detection limit is 20ng for psilocybin and 10ng for psilocin using a p-Dimethylaminocinnamaldehyde developer. They claim that Ehrlich's reagent is less sensitive.
Ive only ever used Ehrlich's for indoles and it has satisfied me.
-------------------- Trading omelettes is fun..... :P
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fastfred
Old Hand



Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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Re: Acquire the Psilocybin & Psilocin Content [Re: trade_om]
#18179865 - 04/28/13 02:58 AM (11 years, 23 days ago) |
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> I dont mean to be anal, but from a chemist stand point, our eyes are definitely not good detectors.
It's easy to get trapped in that mode of thinking. But the human eye is quite good at +/- tests and comparisons.
Thinking that expensive equipment can actually do better is giving it way too much credit. You just need to learn how to use your eyes to their best advantage.
> comparing say 20ng/ml with say 25ng/ml, the difference will be un-noticeable to the naked eye!
It's a simple spot or no spot test. If you can't see the spot it's less than 20ng, if you can it's more. With a series of dilutions you can determine 20ng as precisely as you want.
> HPLC FTW. easily done, can find a few standard methods for cin/cybin
That's great if you've got a pump, column, and detector handy. Otherwise it's not so useful. I still think extraction efficiency and degradation/conversion during extraction are going to be the dominant factors, so I question weather expensive quanitation methods would even be worth it.
> I like that the cybin does not move at all, since it is so easy to distinguish between the 2.
But it's useless for quanitation and bad practice even for qualitative determination. Without a Rf it's just sitting in the start puddle with any other junk in your extraction that didn't move.
-FF
-------------------- It drinks the alcohol and abstains from the weed or else it gets the hose again. -Chemy The difference between the substances doesn't matter. This is a war on consciousness, on our right to the very essence of what we are. With no control over that, we have no need to speak of freedom or a free society. -fireseed "If we are going to have a war on marijuana, the least we can do is pull the sick and the dying off the battlefield." -Neal Levine (MPP) I find the whole "my drug should be legal but yours should be illegal" mindset disgusting and hypocritical. It's what George Bush and company do when they drink a cocktail and debate the best way to imprison marijuana users. -Diploid
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trade_om
An Old Omelette

Registered: 10/19/03
Posts: 172
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Re: Acquire the Psilocybin & Psilocin Content [Re: fastfred]
#18180044 - 04/28/13 04:56 AM (11 years, 23 days ago) |
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The OP asked for a method to mesaure the actives content, not a method to check if the actives are there!
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It's easy to get trapped in that mode of thinking. But the human eye is quite good at +/- tests and comparisons.
I was under the impression this is meant to be a thread for measuring amount of alkaloids, not to verify that the alkaloids are there.
Quote:
That's great if you've got a pump, column, and detector handy. Otherwise it's not so useful. I still think extraction efficiency and degradation/conversion during extraction are going to be the dominant factors, so I question weather expensive quanitation methods would even be worth it.
long methanol soak under inert atmosphere in the dark. extracts everything, and the inert and dark conditions minimize any losses to oxidation/photo-oxidation.
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But it's useless for quanitation and bad practice even for qualitative determination. Without a Rf it's just sitting in the start puddle with any other junk in your extraction that didn't move.
. True that baeocystin will not run as well, but non indolic crap which stays at bottom does not stain with ehrlich's. So for quantitation it does not matter if you have a spot at rf=0. In fact there are many published papers which utilize similar TLC methods for a variety of materials.
-------------------- Trading omelettes is fun..... :P
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nooneman


Registered: 04/24/09
Posts: 14,700
Loc: Utah
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Re: Acquire the Psilocybin & Psilocin Content [Re: Capn Blue Beard]
#18180074 - 04/28/13 05:27 AM (11 years, 23 days ago) |
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Most people don't go quite as far as all that. OP sounds like he just wants to know how to end up with a quality isolate.
Generally, you start with a bunch (100+ preferably) of isolates on agar, and fruit all of the isolates keeping track of which is which. Then you sample the end product to determine potency subjectively, and throw away the isolates that resulted in crap potency and/or crap flushes. Keep doing that until you end up with an isolate that has good potency and good flushes.
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trade_om
An Old Omelette

Registered: 10/19/03
Posts: 172
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Re: Acquire the Psilocybin & Psilocin Content [Re: nooneman]
#18180133 - 04/28/13 06:16 AM (11 years, 23 days ago) |
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nooneman : tolerance builds rather quickly. If the tester does not space the self administration far enough, subjective results will be skewed. other than that bioassay is the best bet for the non chemist or at least the non chemistry-inclined-person.
-------------------- Trading omelettes is fun..... :P
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fastfred
Old Hand



Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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Re: Acquire the Psilocybin & Psilocin Content [Re: trade_om]
#18182446 - 04/28/13 05:49 PM (11 years, 23 days ago) |
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Quote:
I was under the impression this is meant to be a thread for measuring amount of alkaloids, not to verify that the alkaloids are there.
Hence the discussion of quantitative TLC. There are plenty of reports in the literature from mycologists who would have had access to more involved analytical methods.
TLC is a good choice because it's very cheap, very fast, and more than accurate for our needs.
Quote:
long methanol soak under inert atmosphere in the dark. extracts everything, and the inert and dark conditions minimize any losses to oxidation/photo-oxidation.
Kind hard to do a 3x extract under inert atmosphere in the dark, same problem with concentrating the extracts.
What you're suggesting is just too expensive, way over the top, and pointless to our needs. With the TLC you can pretty easily construct a fairly accurate standard and get decent quantitative results and excellent relative results. Anything fancier just isn't needed.
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So for quantitation it does not matter if you have a spot at rf=0.
I don't agree with that statement. You could have any other number of unknowns involved in your matrix. Since you can't know the unknowns involved, if you don't get separation then you don't have valid results. The whole point of TLC is to separate out your components and analyze their spots. If you don't separate them all you've done is analyze your extract after a wash.
-FF
-------------------- It drinks the alcohol and abstains from the weed or else it gets the hose again. -Chemy The difference between the substances doesn't matter. This is a war on consciousness, on our right to the very essence of what we are. With no control over that, we have no need to speak of freedom or a free society. -fireseed "If we are going to have a war on marijuana, the least we can do is pull the sick and the dying off the battlefield." -Neal Levine (MPP) I find the whole "my drug should be legal but yours should be illegal" mindset disgusting and hypocritical. It's what George Bush and company do when they drink a cocktail and debate the best way to imprison marijuana users. -Diploid
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