The Measure of the Mushroom
Taken from PM&E Volume Five
(OCR'd by GluckSpilz)
- SUBJECTIVE AND OBJECTIVE TESTING
- OTHER POTENTIAL CHROMOPHORES
- THE CORRECT WAVELENGTH TO MEASURE THE DMAB CHROMOPHORE
- LIGHT SPEEDS THE TEST REACTION
- DEFINING THE EXTRACTION SOLVENT
- SAMPLE WEIGHT
- OTHER INFLUENCES ON THE TEST
- INTERPRETATION OF THE DMAB TEST
- THE COLORIMETRIC TEST
- TESTING SOLUTIONS PREPARATION
- THE PARA-DIMETHYLBENZALDEHYDE COLORIMETRIC TEST
- THE PARA-DIMETHYLBENZALDEHYDE COLORIMETRIC TEST
The Mushroom Entheogen explores the relationships between hard mycological chemistry and visionary experiences related to psilocybin mushroom use. In PM & E vol. 1 we were presented with optimum harvesting/storage techniques. A study of the bluing reaction with ways to inhibit its onset was presented. In PM & E vol. 2 the relationships between mushroom pretreatment agents and various forms of dehydration were presented, with emphasis on optimum psychoactivity. In PM & E vol. 4 instructions are given for constructing a vacuum dehydration system. HPTLC (high performance thin layer chromatography) comparisons were noted upon samples.
We continue this series with an overview of psilocybin potency testing - both in the laboratory and through implicit meditation and physiological / psychological observation. PM & E is exceptionally proud to bring this installment of The Mushroom Entheogen to our readers. So why do you need to test your psychedelic mushrooms for their potency?
There are two good reasons: either to see the affect of some experimental procedure on the final concentration of the active tryptamines(i.e. psilocybin and psilocin) in the mushrooms (which is pretty much what these articles are about) or more important, to know the subjective intensity of the dosage you may plan to take. You may remember the anecdote from the previous article about my friend who mistakenly took too much mushroom powder. He came very close to needing some medical help, because he thought he was losing his mind.
Neither of us had any idea that we had made a measurement error with our dose of mushrooms and he had taken twice the amount of what would have been a large dose. I ended with about half of a large dose. I was fine but he panicked and the knowledge that I had taken what I thought was the same dose made it even worse, because as far as I was concerned he was being totally irrational.
This extreme example of overdose is more likely to be the rarity. What is more common and frustrating is "under" dosing. If you are like me, a mushroom trip is a special event for which I need to plan the time. With family and job responsibilities I can no longer take a day off on the weekend anytime I feel like it. Too many times I have planned for a day of tripping only to end up with a mild buzz and a loaded feeling, not that altered state of awareness and consciousness which is characteristic of the full mushroom trip. I needed a mushroom testing procedure. Knowing what the active tryptamine concentration is before taking the mushrooms can prevent the possible problem of over or under dosing.
One aim of my research, besides reducing the toxicity of the mushrooms, is to maximize the psilocybin content of the cultivated mushrooms and to stabilize the quantity biosynthesized from flush to flush of a particular strain of P. cubensis by controlling environmental and nutritional factors. In my own research I found that as I experimentally changed these growth-affecting factors, my particular strain's concentration, as measured by the test procedure described at the end of this article, increased by a factor of four or five.
In their research, Bigwood and Beung echo this same variation in the concentration of psilocybin in the controlled cultivation of P. cubensis. But because of their large variation in what they felt was a rigidly controlled growth environment, I am inclined to conclude that they were not controlling all the possible factors which control the growth and biosynthesis of psilocybin. They found that in their own cultivation, concentrations varied by a factor of four and, even worse, specimens from other sources varied as much as ten fold.(4)
An upcoming article, using the results of the mushroom sample testing will show how, by careful control of the mushroom nutritional and environmental growth factors, one can minimize this large flush-to-flush tryptamine(the major molecular grouping in psilocybin/psilocin and other related compounds) concentration variation.
Because of even less environmental and nutritional control, this sample-to-sample variation is further exacerbated if you collect samples from the wild. Besides strain differences(i.e. genetic differences), microenvironmental and growth substrate nutritional differences contribute to large variations between specimens, even collected close together. Christiansen, et al found from their studies of the psilocybin concentration of many different samples of P. semilanceata in Norway, that the content varied by a factor slightly greater than ten.(7)
If ten-fold variations exist between mushrooms of the same species, imagine the potential for variation between different psychedelic genera. Mushrooms which contain the hallucinogenic tryptamines include the genera Concybe, Panaeolus, Psilocybe, and Stropharia. (12) If you are collecting any of these varieties for psychedelic purposes, you may wish to consider a test of their relative strength before taking them. If you plan to take the mushrooms fresh, then with a little experience with one of the field tests described later you will be able to estimate their relative concentration. You can tell not only from the final intensity of color of the reaction but also from the speed with which the sample develops a color.
A final point on the need for a test: if you happen to be someone who buys psychedelic mushrooms, you may want to know just what you are getting for your money. Ideally, if it were legal to sell, a mushroom dealer should be aware of the relative strength of his different batches of mushrooms and should sell the dried mushrooms not by weight but by what is necessary for a moderate-dose trip.
SUBJECTIVE AND OBJECTIVE TESTING
"Okay," you say, "So maybe it would be helpful to be able to test the mushrooms I buy or grow, but I am not a chemist and I want something simple. There are two basic types of testing: subjective and objective. Subjective testing of mushrooms is descriptive. It is easy and cheap and requires only attention to one's own mind, but it does take time.
Objective testing, on the other hand, is quantitative. It is simple, usually quick, repeatable, but can in some procedures, require complex and expensive equipment. Although I have identified two forms of testing, we need both to know the psychedelic effectiveness of an unknown batch of mushrooms or to communicate what our batch of mushrooms will be like to someone else.
The problem with subjective testing is standardizing the method. Because of the vagaries of the mind, one needs to control the set and setting under which one performs his subjective testing. By controlling these two factors, although very difficult at that, one can establish a common reaction(e.g. degree of energy, quantity of hallucinations, their colors and shapes, the ease of feeling at-one with external objects or concepts, etc.) to a standardized dose. This reaction can be the gauge which one uses to compare all other subjective tests. This subjective response to a standardized dose will help one to know how much to take later.
The problem with objective testing is that no matter what value of concentration(usually expressed in mg of psilocybin/ gr. of mushroom) one finally arrives at from his test, it does not tell him what the subjective experience will be like. To know what the subjective experience is, one still needs to take the psychedelic. By doing this a few times for various concentration levels, one can extrapolate a subjective intensity value from an unfamiliar objective test value, thus dispensing with the need for subjective testing.
The advantage of having an objective test concentration value is that it can communicate what the personal experience will be like to anyone who has taken the time to compare several batches of mushrooms subjectively after finding an objective test concentration value for each batch. By comparing their experiences with a few corresponding numerical values, one can infer from a new objective test value the intensity of the personal experience when taking an unknown batch of mushrooms.
The subjective effects may vary considerably from one individual to another but it is the intensity and duration which will change linearly with the increase in the objective test value.
Subjective tests need to be done only a few times (and recorded for future comparisons) when comparing different concentration levels of mushroom strengths, after which you can rely solely on the objective test for evaluating new samples of mushrooms. Whereas, if one opts to use the subjective test only, then one will need to test each batch by actually taking a small, standardized dose. Then from this tedious evaluation one can determine the amount needed for whatever level of tripping one wishes to reach later.
Besides the problem of taking a sample dose for each and every batch of mushrooms, the subjective test has another difficulty when used alone. Its results can be difficult to cornmunicate to someone else because the phenomenal experience may vary radically from one individual to another. For instance, someone may describe a reaction to a five gram mushroom trip as giving him a feeling of strong sexual energy with a keen awareness of his physical self.
Whereas, you may take the very same mushroom powder and even expect and perhaps look forward to a similar reaction, only to experience a very introspective trip in which the last thing on your mind is sex. The objective test value allows a means of communicating the intensity of the mushroom trip without describing the experience it evoked. By itself subjective testing has its problems, but when supplemented with objective, quantitative testing, it can become a predictive tool for us to use. And also, by itself, objective testing conveys no real meaning to us about the subjective nature of the trip. For the reason that the objective test tells us no personally valid information, one can conclude that the foundation for any quantitative test is our own subjective testing.
I have used the following procedures and guidelines in my own subjective testing. Use your own guidelines, but the primary rule is to watch the effects of the psychedelic on your body and mind. It helps to know your mind well before evaluating the changes caused by a psychedelic. The best way to understand your own mind is to regularly practice some form of mental meditation techniques in which the emphasis is on alert consciousness in an ever-increasingly calm mind.
The Subjective Test
- One can argue about the effects of set and setting on the psychedelic experience, but no matter the outcome of the argument one generally does have more physical effects, greater duration and depth of the trip with ever-increasing doses of psychedelics. Observe these differences in the trip's length and depth for different amounts of mushroom, preferably from the same mixture of mushroom powder (all properly stored so that the interval between tests does not degrade the quality of the mushroom).
- Observe your mind with little or no other sensory inputs during the trip. The best way to do this is to close your eyes or be in complete darkness, plug your ears or be in an absolutely quiet environment and lie or sit completely still. After sitting or lying like this for a few minutes, notice the intensity of the colors in the mind's eye or projected in the dark. Observe the sharpness of the edges and forms. Is the nature of the forms benign or malevolent? Do you experience dreamy hallucina- tions or patterns? Is your mind clear or dreamy or sleepy?
- Observe the nature of your perceptions in an eyes-open mode in a well-lighted environment. Notice the rippling effect around objects. Do you have colored patterns or hallucinations projected on the environment. This is sure indicator that you have taken a strong dose.
- Listen to sounds or music and feel their effect on your emotions and observe how they change the hallucinations or patterns.
- Observe the quality of your consciousness. Are you cloudy, sleepy, moody, willful, clear, alert? Note all physical side effects, such as nausea, headaches, muscle aches, stomach cramping, aching joints and other uncomfortable symptoms. Many aches and pains can be a result of psychosomatic manifestations during a psychedelic trip. But many times impurities in the mushrooms can initiate the side effects, too. All the above are indications of the quality of the trip and consequently the mushroom quality. Generally, as I perfected my growing and storage techniques, the above physical symptoms diminished.
- Watch how you respond physically. How is your coordination, such as your ability to walk and talk?
- I have noticed that left brain functions--those usually associated with concrete, analytical thought processes--become harder to perform with increasing doses of psychedelics. Can you perform simple math tasks? Do you have trouble expressing ideas in speech? Can you give directions to a familiar location to someone?
- How fast does the trip come on? How long is the "rush?" How long before you start to come down from the psychedelic portion of the trip? I find that the drug effects last no longer than six to eight hours, but as I increase the dosage or strength of the mushrooms, the trip comes on faster, the rush lasts somewhat longer and the psychedelic portion gradually increases from as little as thirty minutes to as much as five or six hours. By reassessing your trip as it progresses using some of the evaluation criteria as described in the above points, you can observe the course of the trip accurately and predict its length and intensity.
- When establishing a subjective baseline for your trips, it is important to standardize the set and setting of your trip so that you minimize the results of such variables on the trip. Try to take it in the same type of environment, preferably at the same time of day. Do not take an evaluative trip if you are in a negative mood. My friend recommends jogging, or other aerobic exercises, to help elevate and stabilize one's moods.
Having evaluated your mushroom samples subjectively, you are well on your way to being able to plan for an entheogenic, or ecstatic trip because you know how much to take for the sought-after experience. The emphasis of these articles is purposely limited to the use of the mushrooms for the more introverted and spiritually expanding psychedelic experience. True, many of the preparation, growing techniques and even some of the suggestions for directing the energy of the experience(a later article) can apply to trips which focus on interpersonal relationships or even for those who just want to have fun for an afternoon, but you will not need to work as hard for these non-entheogenic experiences. My background with psychedelics, and primarily with the "magic mushrooms," has shown me that the highest quality mushroom experience and states of consciousness come with effort and planning between trips and a tremendous burst of yearning during the actual trip.
Apparently a change in consciousness takes effort and time. The more intense the concentration of effort and desire for such a change, the faster is the change in consciousness. The next section may be superfluous to your needs if you are not drawn to such objective evaluation of your mushrooms. But even if you do not elect to do any chemical testing of your mushrooms the discussion might help you to understand the meaning of the values which I will describe in upcoming articles on environmental and nutritional influences on the growth and psychedelic tryptamine production in P. cubensis. So I encourage you to read at least the general theory and interpretation of the test's results. The actual test procedure is at the end of the article for those who wish to do their own chemical evaluations.
General theory and various detection and measuring schemes.
Objective test results can give a numerical value which tells how much, and for some tests, what is in a mushroom. One can find a relative concentration value or an absolute concentration value. If one has access to the pure psychedelic tryptamines then one can derive the absolute concentration by measuring an accurately weighed amount of the pure sample and then comparing that test result with the value obtained for the unknown sample. If one does not have a pure sample it does not matter because the numerical test results will indicate that one sample has more of the measured tryptamine than another and to what degree it has a greater concentration.
These numerical or objective test results greatly help communicate the relative strength of different batches of mushrooms to any one else who may have a different subjective interpretation than yourself. Each individual chemical behaves differently from all other chemicals because of its unique structure. Based on this uniqueness of each chemical, objective tests are possible. One type of objective testing relies on the unique absorption of specific wavelengths of light absorbed by each unique chemical.
In other words, each chemical has a different color, although the "color" is usually not in the visible spectrum. Another aspect of each chemical which is often used in designing a test procedure is that it will interact with or react with other chemicals completely differently.
See Figure I.
Tests can be developed which are also based on the similarity of various compounds with the understanding that a related portion of the molecule will react similarly. For instance, there are several active molecular components in the entheogenic mushrooms but the most important component includes the general family of molecules, called tryptamines. All these tryptamines have in common the indole ring in their molecular makeup. Tests have been developed which show whether this indole ring (as part of the larger tryptamine molecule) is present in a solution and to what degree.
The several different types of tests which are available to the scientist include spectrophotometry, colorimetry and chromatography . Spectrophotometry measures the degree with which the molecule under investigation absorbs light at specific wave lengths. But because most organic molecules absorb best in the infrared or ultraviolet spectrum and these instruments are expensive for the average hobbyist, I have not pursued these techniques.
Chromatography is a technique which separates mixtures of compounds by passing the mixture while in solution through a specially prepared media(the "sorbent") of highly refined sand, called, "silica gel." Silica gel is the most common sorbent used, but other less common sorbents can be used. The mixture of various molecules interact differently with the molecules on the surface of the silica gel as they pass and thus slow their movement to a greater or lesser degree.
After migrating through the silica gel for a distance, the mixture of compounds segregate into separate bands of pure compounds. Chromatography generally falls in two modes, depending on the apparatus used with the silica gel to separate the sample--thin layer chromatography(TLC) and column chromatography of which high pressure liquid chromatography(HPLC) is another technique subgroup commonly used in the lab.
As the name indicates, in TLC a thin layer of silica gel is applied to a glass or plastic plate then the sample is streaked or spotted near the bottom of the plate. The plate is then put into a glass tank in which a small amount of a particular solvent mixture, determined by experimentation, has been poured. The silica gel, being porous, allows capillary action to draw up the solvent which pulls the sample, too. As the sample moves each pure chemical in the mixture moves at a different rate thus causing a separation into bands of the pure component molecules of the sample mixture. Different techniques are available to make each pure chemical band visible.
See Figure 2.
For a given sorbent and solvent a particular compound(e.g. the psychedelic tryptamines) will always move up the TLC plate the same relative distance when compared to the distance that the solvent was allowed to creep up the plate. For example on one occasion a researcher let the solvent develop on the TLC plate for one hour and the solvent moved up the plate 10 cm. After using either a chemical dye to detect the chromategraphed spots or a UV lamp, he found that the spot he was most interested in moved 5 cm up the plate or half way between the starting point and the solvent front. On another occasion he only let the plate in for 45 minutes and the solvent moved only 8 cm. Because he knows the compound has an Rf(an abbreviation referring to relative migration distance up a TLC plate of a pure compound) of 0.5, then under identical conditions for the same chemical, the spot of interest will move half way up the plate or 4 cm. And so it does in practice.
The experimental literature will usually have these relative distance values for most compounds, which are always less than 1.0 (An Rf of 1.0 would indicate that the compound moved with the solvent all the way up the plate; therefore its relative distance when compared to the distance that the solvent moved is 1.0.) After a TLC separation it is easy to see if a particular compound is present by looking for a band which occurs at the correct distance up the plate. The intensity of the color of the band will indicate the concentration of the compound, or one can actually scrape the band off the plate and measure the nearly pure compound by some of the other techniques available.
HPLC(or High Pressure Liquid Chromatography) is a form of column chromatography. The sample is applied at the top of high pressure capable column with sorbent in it, then the solvent is pumped through the column at high pressures (usually 1000 psi or greater). At the other end of the column a spectrophotometer monitors the solvent for an absorbance which indicates an organic compound is coming off the column. The experimenter can view the output from the spectre photometer via a graph output or a video screen.
The area under the peaks which represent each different molecule are proportional to the concentration. With samples of the pure tryptamines, one can calculate the absolute concentration of the compounds investigated. An HPLC solvent is pumped through the column until most of the sample has been washed off. Instead of distance traveled through the silica gel as in TLC, the time it took to wash the compound off the column before it was detected by the spectrophotometer is used to determine what the molecule is.
If you read the technical literature you will see HPLC mentioned often. Its advantage is far greater sensitivity and ability to resolve many more compounds which may be present in an unknown sample. Its disadvantage is cost. The average HPLC set up may cost $10,000. Whereas one can buy pretty much all he needs to perform TLC for about $100 to $200.
Which brings us to the last general detection technique and the one of choice for most of my research--colorimetry. Colorimetry is similar to spectrophotometry in that a solution of the sample absorbs specific wavelengths of light and the wavelength and the degree of absorbance can tell much about the sample. But in colorimetry the light absorbed and the consequent color of the solution measured falls within the visible spectrum. Also, because the absorption peaks cover a much broader range of wavelengths than in the ultraviolet (UV) or infrared (IR) regions, the spectrophotometer used can be much less sensitive and can use coarser methods of breaking up the light spectrum to irradiate the sample. The instrument used for colorimetry is called not a "spectrophotometer," but a "colorimeter" and can use filters rather than the much more expensive diffraction grating monochrometer used in spectrophotometers.
The trick with colorimetry is making the sample molecule which normally does not absorb in the visible spectrum(i.e. 400 to 700 nanometers, which is the wavelength of light from the deepest reds to the faintest violets) visibly colored. Chemists have found that there are molecules which by themselves are uncolored but when combined with certain other molecules will form a color. These chemicals are called "chromophores" and the presence of this color indicates that the molecule under test exists in the solution and the intensity of the color tells one how much of the compound is in the solution.
The problem with colorimetry is that most chromophores do not combine specifically with a unique molecule but with a portion of the molecule under test. For example, in the test which I used in my research, the chromophore reacts with the indole ring of the psychedelic tryptamines to form a blue or purplish color. Indole rings are not specific to psychedelic tryptamines. There are many molecules other than the psychedelic tryptamines which have indole rings.
And to further complicate things, the chromophore will react with other nitrogen containing centers, although without the usual blue or violet color. The net result can be a hodgepodge of color which overlaps to a greater or lesser degree with the specific wavelength, or color, which the colorimeter is viewing. The colorimeter is dumb; it does not know the difference between an absorbance at 570 nanometers which is caused by urea or psilocybin or a little of both. Generally, the non-active compounds have absorbances far enough away in the light spectrum so that they do not interfere with the psychedelic tryptamine readings, but this is not always the case. I will discuss this interference and how it relates to the interpretation of the test's results in a later section.
Spectrophotometric theory tells us that the measured absorbance of a compound is directly related to its concentration in solution. In other words as absorbance increases so does the concentration. If we test one mushroom for active tryptamines and find that it has an absorbance of 0.600 and then test another and find that it has an absorbance of 0.900, we can say that the latter one has a greater concentration of tryptamines than the first. Sure, we do not know the absolute concentration in mg/gram of the psilocybin/psilocin, but who cares? We can as easily relate to 0.600 A as we could relate to 2 mg/gram in our own subjective experience.
What is in a mushroom? What are we measuring?
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