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The Mushroom Entheogen - The Measure of the Mushroom

by

C.B. Gold

Taken from PM&E Volume Five

(OCR'd by GluckSpilz)


Table of Contents:
Summary
Subjective and Objective Testing
 The Subjective Test
 Objective Testing
  General theory and various detection and measuring schemes
  p-DMAB Test Paper
  The theory behind the reference colorimetric test
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
 Equipment
 Chamicals
 Supplies
  Colorimeters
  Balance
  Large borosilicate test tubes
  Matched cuvettes
  Test Tube Holder
  Chemicals
  Plastic sheet
Testing Solutions Preparation
 Color development reagent
 Acetic Acid Extraction Solution
The para-Dimethylbenzaldehyde Colorimetric Test
Conclusion
Bibliography

Summary

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 communicate 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 hallucinations 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.

It is best to take the mushrooms on an empty stomach during an evaluation because different foods will affect the nature of the trip. Also, having food in the stomach will slow the absorption of the psilocybin and give the impression that you had less. If you are correlating objective test concentration information with subjective intensities, make sure you always use the same amount of mushroom. I found that one gram was enough to test. It usually was not enough to put me on a full-blown trip, but was plenty to observe all the psychedelic manifestations, particularly if I closed my eyes and plugged my ears and practiced meditation techniques which I know.

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.

Objective Testing

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.

Figure 1 - Shows indole-tryptamine derivatives. Similarities between naturally occurring bodily compounds and hallucinogenic compounds allows for the psychedelic effect.

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.

Figure 2 - Shows a rendition of the TLC (Thin Layer Chromatography) processing. This method of visual analysis allows chemical present within the mushroom to be compared next to one another.

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 spectrophotometer 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.

Because of the potential for ambiguity in the test results through the chromophore's multiple color reactions, it may be appropriate to review the literature to see what constituents of P. cubensis others have found in their research. Some of these other natural compounds will react with the test and add to the test value even though they are not active tryptamines. And others are active tryptamines which because of their somewhat different psychophysiological activity can modify one's trip significantly for better or worse. We need to know what these are, too.

As far as active tryptamines in the mushroom, the two with the greatest concentration in P. cubensis are, of course, psilocybin and psilocin.13 p.109 The "tryptamine derivatives" are called such because of their similarity to serotonin. This class has an INDOLE group and a DIMETHYLAMINE group. The tryptamine derivatives include the brain transmitter substance, serotonin, the essential amino acid, tryptophan, the fast acting but short lasting psychedelic, DMT, and of course psilocin and psilocybin. "Active tryptamines" refers to the various psychedelic tryptamines, including psilocybin, psilocin and all their analogs. See Figure 1 for examples of the various tryptamines.

Repke points out that any psilocin detected in mushroom samples may in fact be an artifact caused by hydrolytic cleavage of the phosphate group off the psilocybin molecule in the handling and sample preparation.14 In fact, other analogs can be easily formed by the various enzyme systems and the presence of oxygen. I tried to make this point in the first article when I emphasized the importance of low temperature, vacuum drying when preparing the mushrooms for storage or the care needed in preparing the mushrooms for ingestion, for it is these analogs and breakdown products which are most likely the cause of the headaches, mental cloudiness and achiness which are not normal side effects of synthetic psilocybin.

Most literature references which I read noted that little psilocin was present in mushrooms. They may not have been able to find any psilocin with the psilocybin, because it is an artifact or perhaps because of the ease with which psilocin is oxidized. In the work of Bigwood and Beug, however, they found that after the second flush the psilocin levels are significantly high. In fact, they range from about ten to thirty percent of the total active tryptamine concentration (concentration of psilocybin and psilocin in this paper).4

Apparently, baeocystin, which is another psychedelic analog to psilocybin and psilocin, plays a major role in the natural biosynthesis of the psilocin and psilocybin in the mushroom and it is present in small but significant levels in P. cubensis. Based on the various samples tested in the cited literature, the range extends from 0.001% to 0.02% baeocystin of the mushroom's dry weight. Repke and the others found that baeocystin was never found in mushrooms which did not already have psilocybin present also. To put this in perspective, psilocybin usually makes up about one percent of the dry mushroom weight. (Baeocystin forms a pink to purple to blue color reaction in the presence of Ehrlich's reagent, which is similar to the test reagent which I describe at the end of this article.)14

The researchers, Beung and Bigwood found through their TLC work that they could isolate 12 distinct spots (i.e. different compounds) on the silica gel plate. Besides psilocybin, psilocin and baeocystin, their tentative conclusion is that the other spots represent N-methyl and tryptamine analogs of psilocin and psilocybin.3 Another tryptamine found in mushrooms is tryptophan.9 This will react with the test reagents with a similar color as psilocin and will consequently add a small amount of absorbance to any test result, giving a slightly false high reading.

The test which I used to quantify the amount of psilocybin and psilocin in the P. cubensis mushrooms reacts with other nitrogen containing compounds, although it is most sensitive to indole containing compounds when read at the prescribed wavelength.10 The common amino acid, glycine, is one such nitrogen containing compound found in abundance in the mushroom.18 Another nitrogen based compound which has been found in the mushroom is urea. The yellow color change of the test indicates the presence of one of these ubiquitous compounds. Luckily, yellow adds only a small amount of absorbance to the value of the active tryptamines when read at 570 nm, the test wavelength.3

Casale, in reviewing the literature, mentions that besides the compounds already mentioned, ergosterol, ergosteral peroxide and a,a-trehalose have also been found in the methanol extracts of the Psilocybe mushrooms.5 I could find no other mention in the literature about other tryptamine analogs, toxins, enzymes or hormones which may be present in P. cubensis and thus affect one's subjective experience. Agurell, Blomkvist and Catalfomo1 identified a lengthy list of possible tryptophan metabolites which might show up in the psilocybe mushrooms: 6-hydroxytryptophan; kynurenine; tryptophan; kynurenic acid; xanthurenic acid; psilocin; tryptamine; methyltryptamine; dimethyltryptamine; 3-hydroxyanthranilic acid; anthranilic acid; N-acetyltryptophan; and indoleacetic acid. Agurell and Nilsson2 demonstrated in their paper a tentative biosynthetic route for psilocybin for which any of the intermediates could exist in the mushroom, too. The synthetic pathway proceeds from tryptophan to tryptamine to N-methyltryptamine to N,N-dimethyltryptamine to psilocin to psilocybin.

Any and probably all precursors to psilocybin can be found at one time or another in P. cubensis. Some may be bound to proteins or enzymes which may tie them up for any chemical reaction or assimilation in the body. The consequence of the presence of all these other non-active tryptamine or nitrogen containing compounds which react with the test reagent is to artificially raise the absorbance or apparent concentration. In testing, the change in the absorbance does not necessarily mean a change in the active tryptamine (i.e. psilocybin/ psilocin) concentration at all. Some quick and easy tests for field or the non-technically inclined.

A simple test described in High Times to determine whether one has inadvertently purchased LSD laced mushrooms is to mash the mushroom in some methanol and let it sit overnight. Decant the methanol the next day and hold the extract up to a black light. If the liquid glows blue then you have LSD containing mushrooms, which, as far as I know, do not exist.17 p. 252 Norland describes a few colorimetric tests which can be used to identify mushrooms which contain tryptamine derivatives.13 p.116 You may find them more useful than the longer and more complicated test procedure at the end of this article. You do not require a colorimeter for test results and if you can live with an eyeball color comparison and your memory, you can at least estimate the concentration differences between mushroom flushes.

  1. A simple test for indole-containing compounds and tryptamines is to crush a small piece of mushroom into 1/2 ounce of vodka or ethyl alcohol ("denatured alcohol" or the hardware store "shellac thinner" is fine) and mix. Add 3-4 drops of hydrochloric acid (or the hardware store variety called, "muriatic acid") then drop a pine tree shaving into the solution which will turn "cheny red" in the presence of indoles.
  2. Another test for indoles uses a small crushed piece of mushroom in 1/2 oz. of either methanol or ethanol (or Vodka). If you are interested in testing for psilocybin use methanol; if psilocin use ethanol or vodka. The difference in solubility between the two active tryptamines account for the difference in the solvents used. Mix well then filter. Let evaporate overnight or use a steam bath or a hair dryer to dry. Spot the residue on filter paper and let dry. Spray or drop on the following developer. In order for the test to work effectively the developer must be made fresh. To make the developer, add one drop of 37% formaldehyde to 15 drops of concentrated sulfuric acid. Psilocin should turn green to black where as psilocybin should turn yellow to green-yellow; green is normal. Orange-brown indicates amphetamines or LSD.
  3. Ehrlich's reagent is a name of a mixture which is used to detect indole compounds which have been separated on a TLC plate. After spraying the test solution on the plate, a colored spot will form where such an indole-like compound lies. The reagent is made from pimethylaminobenzaIdehyde (5%) (abbreviated DMAB) in concentrated hydrochloric acid (HCl).9 Another variation of the Ehrlich's reagent is 2% DMAB in HCl-ethanol (1:1). This reagent gave the following color changes: psilocybin turned reddish-purple then faded to violet whereas psilocin yielded a strong blue color which faded to violet.18
  4. If you are interested in pursuing a TLC testing procedure, see Leung, Smith and Paul for the various solvent systems which can be used to separate out the constituents of the mushroom. Also, you will be able to use the information about the expected relative distances (Rf values) which psilocin and psilocybin will travel up the plate for each solvent system. This information plus a test reagent such as the Ehrlich's will help to establish if psilocybin or psilocin or both are present in the mushrooms you test.9

For my own TLC use, I found that the solvent system which Beung and Bigwood used in their research worked best. They found that they could obtain the greatest resolution of the most spots by using a solvent mixture of n-butanol-acetic acid-water (12:3:5) with silica gelplates.3 In my use of this solvent system, T found that the solvent ratios mix well. Some of the other literature suggests solvent mixtures which are not homogeneous after shaking, but instead quickly separate out into two layers, making these other solvent mixtures difficult to use in TLC.

In my own separations, I used a UV light and the fluorescent version of the TLC plates for detection. I did not have access to a fine mist sprayer which is required if using the Ehrlich's reagent. I could only distinguish six spots in contrast to the twelve which Beung and Bigwood found when they used both detection schemes (i.e. Ehrlich's reagent and UV lamp). In another test procedure I made a test paper which can detect the presence of indole compounds by using p-DMAB (para-dimethoxybenzaIdehyde) as the detection reagent or chromophore. Although the paper is not sensitive to low levels of indoles, I found it useful for quick checks of mushroom extracts for the presence of psilocybin/psilocin.

p-DMAB Test Paper

  1. Add 1.0 gram of p-DMAB to 28 mil of ethanol (denatured). Stir until dissolved.
  2. Add water to the above mix until 60 mil total volume achieved.
  3. Soak some filter paper in the solution and let dry completely. One can use a hair dryer to speed the drying but do not use too hot of an air flow. The heat will destroy the p-DMAB and consequently the test paper's usefulness. Store the paper in a tight jar in the refrigerator.
  4. To use the paper add a drop of the extracted sample to the paper and let dry. Hold the test paper over the mouth of a bottle of concentrated hydrochloric acid. The fumes will develop the purplish/blue color quickly. If the fumes do not develop the color try adding a drop of hydrochloric acid on the paper next to the sample spot. As the hydrochloric acid diffuses into the filter-test paper and comes close to the sample spot, the color, purple or blue will form if the sample is positive for indole compounds.

The theory behind the reference colorimetric test

In the preceding sections I have outlined the general parameters of colorimetric testing and in the last section listed the procedures for several test which are easy to set up and read. The test which I have selected for measuring the active tryptamine content in the various experimental samples from the last four years, is based on a color reaction with paradimethylaminobenzaldehyde (DMAB). DMAB is the common reagent ingredient in several other tests mentioned above including the Ehrlich's test.

To review the general colorimetric test again, the indole part of the tryptamines reacts with DMAB in a solution conducive to driving the reaction to completion thus forming a colored complex which can be visualized or read with a colorimeter or spectrophotometer. The intensity of the color (i.e. absorbance) is proportional to the concentration of the tryptamine content of the solution. The use of this test is common in the literature in slightly different formats. The test which I developed for measuring the indole-like compounds, psilocybin and psilocin, was originally used in a slightly modified form to measure ergot alkaloids.16 Lysergic acid and ergotamine tartrate are ergot derivatives - both precursors to LSD and other pharmaceuticals and can be tested using DMAB.

Besides the more sensitive but more complex HPLC (High Pressure Liquid Chromatography) testing procedures, various researchers have used other means of quantifying psilocybin and psilocin. Leung and Paul used a quantitative TLC (Thin Layer Chromatography) method in which the least amount of chemically pure psilocybin to cause a reaction with Ehrlich's reagent (a 5% solution of p-DMAB in hydrochloric acid) was compared to the least amount of a test sample from extracted mushroom tissue needed to induce a color change. One assumes that the psilocybin concentration is equal in both cases and then computes the percent concentration of the psilocybin in the mushroom by knowing the amount of mushroom which was extracted.11

Other Potential Chromophores

The test does not have to be restricted to DMAB as a chromophore. Although in the following testing procedure and in all the technical literature, para-dimethylaminobenzaldehyde has been used as a color developing agent, another chemical, para-dimethylaminocinnamaldehyde may be used. This particular developing agent will yield a much more intense color than the DMAB and will consequently be more sensitive.

This additional sensitivity may be necessary if you are using a more crude colorimeter with a broad bandpass filter (i.e. more than 30 NM). Instead of the accepted reading wavelength of 570 nm used for the DMAB test, you should use 625 nm for para-dimethylaminocinnamaldehyde.15 But in most applications this increased sensitivity will cause too dark a reaction color to develop and thus be hard to read on the colorimeter scale.

Still another reagent used similarly to the DMAB reagent (Erlich's reagent) is the Pauley reagent. This reagent uses diazotised sulphanilic acid. It is more specific than DMAB in that it does not react with psilocybin or urea, but only with psilocin, giving a deep red-orange color.18 (I do not have details on which wavelength to measure this color reaction.)

The Correct Wavelength to Measure the DMAB Chromophore

A few researchers used DMAB in their colorimetric test and generally the wavelength they used to read the indole containing compounds has been 570 nm. The spectrophotometric peak of both psilocybin and psilocin after reacting with DMAB is sufficiently broad so that one can use another wavelength close to 570 nm without affecting the sensitivity of the test. This could be important if you use a filter colorimeter and the available filters do not include the specific wavelength of 570 nm.

In figure 3 I have constructed a spectral absorbance graph for a positive DMAB test for some mushroom powder. I took transmittance readings every 10 nm from 400 nm to 610 nm and then converted the transmittance values to absorbance, which I later plotted. The best wavelength to read a colorimetric test is usually at one of the absorbance peaks. As you can see, the test could be read at more than one wavelength based on this criteria. Another possible absorbance maxima besides the approximate 580 nm peak is on the 550 nm peak. It is probable that these two peaks represent psilocin and psilocybin. The large peak around 410 nm may be a secondary peak for psilocybin, which is usually seen as purple - a combination of red and blue.

When analyzing the graph remember that this represents spectral absorbance not transmittance. Therefore for the color blue one would look for an absorbance peak in the red area of the spectrum (i.e. near 600 nm).

Figure 3 - Shows the spectrum of p-DMAB test reactions. This test allows color reactions, such show amounts of specific chemicals in comparison.

Light Speeds the Test Reaction

It was from the paper of Agurell, Blomkvist and Catalfomo which I discovered the principle outline of the test which T present here.1 In that paper they suggested the use of a UV light which will speed the reaction with the DMAB chromophore. I have substituted that step by letting the reaction develop under fluorescent lights for a longer period of time. Even at that, the reaction continues to develop for 24 hours after initiation. The researchers mentioned also produced a calibration curve which could prove useful. But these researchers not only extracted the psilocybin but also purified it to some extent before testing their samples. You may be able to get a "ball park" absolute concentration in mg/gram of mushroom by extrapolating from this curve and by purifying your mushroom extract. Consult the reference for more details on purification if you are interested. (A future article will outline a purification procedure.)

The DMAB reaction requires at least thirty minutes to reach a plateau of color development under fluorescent light. Note figure 4 which shows this. Because the reaction actually continues for up to 24 hours, you will need to accurately time the development period and then standardize this time for all tests. I use 30 minutes because the greatest color changes have occurred by this time and I prefer not waiting too long for the results. Another good reason for using a shorter development time period is that psilocin and other related tryptamines which are present do gradually degrade, thus altering the value of concentration for the active tryptamines if allowed to sit in solution while the test is developing.

Figure 4 - Shows the time absorption scale surounding colorimetric testing.

Defining the Extraction Solvent

The researchers, Agurell, et al., tested the extracted and somewhat purified psilocybin. They did not test for psilocin. To avoid a tedious and lengthy sample preparation, I wanted to extract, then read the mushroom sample without purification. Ideally, I wanted to use an aqueous solution to avoid organic solvents and to test for both psilocybin and psilocin during the same test. But psilocin has difficulty dissolving in water, and psilocybin is easily dissolved in water. Since psilocin is especially unstable in alkaline solution, I felt that an acidified aqueous solution would be the best to use as a solvent.19 Many TLC tests confirmed that the acetic acid-water solution which I finally decided on did, in fact, extract both the psilocybin and psilocin.

I use an acetic acid-water extraction solution to help extract the psilocin and psilocybin more completely and also, to lower the pH so that the active tryptamines will be more stable. Without the acetic acid the solution will quickly react with atmospheric oxygen in the presence of endogenous enzymes to form a strong blue product and in the process destroy some of the psilocybin/psilocin. Also, the color blue itself will interfere with the test results, since the reaction yields a blue or purple color for tryptamines. Specifically, psilocin yields a brown-deep blue and psilocybin a yellow-green and purple color. In contrast, LSD will react with DMAB to form a blue-purple color.17

Apparently, others have also found that a dilute acetic acid solution is an excellent solvent for both psilocin and psilocybin. Not only does the solution completely extract both tryptamines but the solution extracts other interfering substances to a lesser degree. Casale also notes that if one heats the extraction solution of dilute acetic acid to 70 degrees centigrade for ten minutes, then the psilocybin is completely converted by dephosphorylation to psilocin.5

I have found on my own that heating the acetic acid solution eliminated whatever bluing reaction was occurring in the enzyme denaturing environment of the low pH extraction solution. That psilocybin is converted to psilocin is a plus, too. It means that the color reaction will form a more pure color and is therefore easier to interpret the test results.

Besides measuring the color of the developed reagent when it has stabilized somewhat, it is also important to measure a sample of the mushroom extraction as soon as possible. The dilute acetic acid slows the degradation of the psilocin-like tryptamines but does not totally inhibit this degradation. The longer you wait to perform the test on your sample, the lower the value will be. I found the reduction to be approximately 10% after 20 hours. Interestingly, the greater the concentration of active tryptamines as measured on a fresh sample, the greater the effect of time in reducing the apparent concentration.

Sample Weight

I arrived at the sample weight of 0.5 gram powdered mushroom by running a test on four sample masses: 0.2 grams, 0.5 grams, 1.0 gram and 1.5 gram. For the extraction volume of 20 milliliters, the 0.5 gram sample works best. The larger mushroom samples tend to float on top of the extraction solution in the large test tube and have to be constantly stirred so that the powder remains in the extraction solution. The smaller amounts of mushroom powder become increasingly harder to weigh accurately and precisely. Also, the smaller samples have less of a color in the developed reaction making it harder to read the spectrophotometer.

Other Influences on the Test

An interesting but unexplained influence on the color test came from a slight, but apparently significant, change in my standard procedure. If for some reason I used solvents in the preparation of the mushroom material and did not extract the mushrooms, but then totally evaporated the solvent leaving the original mushroom powder ostensibly unchanged, the test color shifted to a more pink color and consequently changed the absorbance from 570 nm. I noted this color shift primarily when I used methanol. Perhaps methanol reacts with something in the mushroom and this product in turn reacts with the DMAB. The point is that the test results cannot be compared to other test results for which you have modified the test procedures. Common sense may tell you that a particular modification may not matter, but in fact, the modification may change the results dramatically.

A more dramatic example of trying to compare apples and oranges as far as the colorimetric test for psilocybin/psilocin happens when I have tried to pre-purify a mushroom powder sample by extraction. My extractions were attempts to clean up the mushroom powder of non-active tryptamines. The colorimetric test becomes a more pure color but because other chemical entities have been removed which also react with DMAB, the overall absorbance drops, giving one the immediate impression that not as much psilocybin/psilocin exist in the mushroom powder sample when, in fact, just as much psilocybin/psilocin is present.

Interpretation of the DMAB Test

The DMAB test reacts with other than psilocin/psilocybin. The DMAB colorimetric test is not a perfect test. The numerical results can be somewhat misleading when used to indicate the concentration of psilocybin/psilocin, the two most common psilocybian analogs in P. cubensis. In the above section on "what is in the mushroom and what are we measuring?", I made the point that the test is not specific to just these two tryptamines. The test reacts with nitrogen-containing indoles, of which the tryptamines are a larger molecule which incorporates the indole group. The test is most sensitive to these indole-containing compounds but still can react with other indoles besides tryptamines.

DMAB reacts to form different colors with other indole containing compounds or other reactive nitrogen-containing molecules. For instance, psilocin typically is blue and psilocybin is purple or purple-green. And tryptophan which is chemically similar to both develops a deep blue color which is different enough from both to make it difficult to use as a standard as I had hoped.

The evidence one can obtain from the different color reactions for different indole compounds can help to evaluate the test results. Note the color mixture after the test has developed. Record the various colors. How pure a color are they? The more pure the color the greater the purity of the tryptamine present in the mushroom.

In my own TLC work I have consistently seen four separate bands which react with DMAB:

ZoneRfColor with DMAB
10.137Dark Violet
20.275Pure Violet
30.550Grey Blue
40.965Pink Orange

Each of the above "zones" represent a different chemical compound in the mushrooms which I tested.

By knowing that the DMAB test reacts with other indole compounds besides psilocin/psilocybin, one can conclude that the numerical results of the test represent the summed absorbances (i.e. concentrations) of the various tryptamines or other reactive compounds present in the mushroom, not just the absorbance of psilocybin or psilocin.

One needs to keep in mind that the DMAB test can lead to ambiguous results when trying to make conclusions about environmental or nutritional influences on the biosynthesis of psilocybin/psilocin. What may be occurring is a change in the mixture of the various tryptamines in the mushroom along with an increasing or decreasing or static test result. The ultimate confirmation would be the subjective test, because a change in the tryptamine concentration will change the nature of the psychedelic trip.

One can mistakenly interpret the test results in other ways. In addition to the extra absorbance because DMAB reacts with other compounds in the mushroom extraction besides the ones we are most interested, the general enzyme class of phenol oxidases which are widely distributed in different species of mushrooms can use DMAB as a substrate and can thus reduce the absorbance of the test artificially. By reducing the concentration of DMAB, these enzymes effect a falsely low value for the tryptamines. This is another reason I use a low pH extraction solution (i.e. acetic acid in water) and heat the extraction mixture. Both of these procedural details should reduce the likelihood of losing DMAB through enzymatic activity.8,p. 108

As noted in the article on harvesting and storage, some ions will interfere with the color development of the DMAB. In particular, my experience shows that the bisulfate ion inhibits the reaction. Sodium bisulfite can be an alternative to vitamin C as an antioxidant and enzyme inhibitor for mushroom storage.

Quantifying the contribution of non-psilocybin/psilocin factors which react to DMAB. When I began subjectively testing the effect of nutritional factors on the growth of my variety of P. cubensis, I noticed that the increase in absorbance of the DMAB test, which relates to an increase in concentration, did not seem to be proportional to the large increase in the subjective effects of the mushroom powder. A small apparent increase in the concentration (absorbance value) of the DMAB test doubled the subjective effects of the mushroom. My earliest concentration values for the mushrooms tested were 0.6 A (absorbance units). When I subjectively tested mushrooms which had increased to 0.8 A, I was surprised to have a trip which seemed twice as strong.

After careful thought, I concluded that the initial concentration value was composed of two or more colored constituents which added to the total value and that the active tryptamines which made the trip possible were a comparatively small percentage of this total color intensity.

To obtain evidence to confirm or deny this hypothesis, I used the process of paper chromatography to separate out three different mushroom powder samples measuring approximately 0.60 A, 0.80 A and 0.85 A by the DMAB test. After separating the mushroom extracts, I cut out the zone which corresponded to psilocybin, using its known Rf value for paper chromatography and a 2% solution of DMAB in ethanol and hydrochloric acid (1:1) to conclusively identify the width of the zone by its color reaction. After cutting out the psilocybin zone, I extracted it in 5% glacial acetic acid at room temperature overnight. I retested this extraction and compared the results with the extraction and test of the remainder of the cut up paper chromatogram which held the other factors in the mushroom extraction.

After correcting for variations in the sample volumes streaked on the paper and in spite of the poor separation achieved on the paper as compared to silica gel TLC, the results clearly showed that the other unknown but DMAB reacting factors in the mushroom increased their concentration with the increasing absorbance at a slower rate than psilocybin. In other words, the concentration of psilocybin increased with the increase in absorbance, which follows colorimetric theory, but as the absorbance increased, the psilocybin concentration contributed a greater percentage to the total color intensity of the various constituents which react with DMAB. This is in direct conflict with colorimetric theory which states that the concentration of a solution with an absorbance of 1.0 is twice that of a solution with an absorbance of 0.5. But colorimetric theory is for a solution of a single absorbing molecule and as stated previously, there could be as many as twelve different DMAB reactive compounds in the mushroom, each increasing its concentration at a different rate as stimulated by nutritional or environmental changes.

So at this point one can only say that as the absorbance of the test increases, the concentration of the active tryptamines increases - but we still do not know by how much. To try and answer this, I put together a graph at the end of my four years of research based on my subjective experiences of the strength of a trip as compared to the absorbance of the DMAB test. My log shows that I had at least several trips at several different concentration levels: at 0.5 to 0.6 absorbance; at 0.7 to 0.9 absorbance; at 0.9 to 1.1 absorbance and at greater than 1.1 absorbance. By using the intensity, duration and phenomenal experiences of the 0.5 absorbance experience as my reference, I compared each plateau with the previous one and then converted the increase into units of the base reference experience.

For example, I know from written records of my experiences that the second plateau (0.75 A) feels twice as strong as the first (0.5 A) and the third level is 1.5 times as strong as the second, and so on. The following graph (Figure 5) shows these results.

Figure 5 - Shows the psychedelic's trance strength compared to the colorimetric tests.

There are several points that we can glean from the graph:

  1. The increase in the active tryptamines is in fact linear for this range and this variety of P. cubensis. Apparently for every increase in the mushroom test value of 0.2 to 0.3 A the experience intensity increases by the equivalent of the 0.5 A mushroom experience.
  2. The paper chromatography extraction experiment discussed earlier obtained a value of the other-than-psilocybin factors' absorbance as 0.35 A for the 0.6 A mushroom powder. The test was inconclusive on this matter because paper chromatography is such a rough separation procedure, but it suggests that the value for the absorbance for the other-than-psilocybin factors increases only slightly as the absorbance increases. Interestingly, if one extrapolates the subjective experience graph to the "0" experience point, the absorbance obtained corresponds to the point after which one will begin to experience the psilocybin. This extrapolated absorbance approximates 0.3 A which supports the paper chromatography results that showed that the first 0.3 A of a mushroom test value is color from non-active tryptamines and other DMAB reactive compounds. The value also means that all the samples had at least 0.3 A of non-active, but DMAB reactive, junk in the mushroom. Of course some of the active tryptamines, which can intensify the trip, may not necessarily be a pleasant addition to the trip either.
  3. Although my work on increasing the concentration of active tryptamines through environmental and nutritional manipulation was successful, my secondary goal of reducing the concentration of the junk in the mushroom was not particularly successful Apparently, the level of non-active, but DMAB reactive substances, stayed about the same. It is impossible for me to know without more extensive testing using HPLC whether as the concentration increased, the mixture and relative concentration of the various active tryptamines, such as psilocybin, psilocin, baeocystin and their analogs, changed in the mushroom powder.

In conclusion, then, when reading the upcoming article(s) on growth factors for P. cubensis, keep in mind that as the test values go up the subjective experience increases in intensity, too. But, an increase in the objective test value cannot be used to predict an equivalent percentage increase in the subjective experience. In fact my experience has shown that for a modest increase in the test absorbance, I increased the subjective amount of active tryptamines two or even three times.

The Colorimetric Test

(Items marked with an "*" have expanded notes and explanations following the equipment and supplies listing.)

Equipment

  • Colorimeter/Spectrophotometer*
  • Hot Plate
  • Quart Pan
  • Balance capable of weighing at least one gram and
  • accurate to 0.1 gr*
  • Electric Coffee grinder

Chamicals*

  • 25 grams p(para)-Dimethylamino benzaldehyde
  • 1 pt concentrated. Sulfuric Acid
  • 1 pt Glacial Acetic Acid
  • 125 gr Ferric Chloride
  • Deionized water

Supplies

  • 1 ea. 250 mil plastic graduated cylinder
  • 1 ea. borosilicate 250 mil beaker
  • 2 ea. 125 mil amber narrow mouthed bottle with caps
  • 1 ea. 1000 mil bottle with cap
  • 12 ea 20 X 150mm borosilicate glass culture tubes*
  • 6 ea matched cuvettes for the colorimeter*
  • Polyester cosmetic balls (available at most supermarkets)
  • 6 ea. Small (60mm) plastic funnels
  • 1 ea. Multiple funnel rack
  • 1 Box Filter paper: 9 cm Coarse (like Whatman 4) and 9 cm Medium (1 or 2)
  • 1 ea. Plastic test tube rack (to hold about 10 of the 20 mm tubes)
  • 1 ea. Plastic test tube rack (Nalge 5900-0007)$
  • 1 ea. Test tube cleaning brush
  • 1 ea. Volumetric pipette 20 mil
  • 1 ea. Pipette 10 mil in 1/10 mil
  • 1 ea. Pipette 1 mil in 1/100 mil
  • 1 ea. Pipetting Aid (either Bel-Art F-37898 or Nalge 3780- 0100)
  • 1 ea. glass stirring rod
  • 1 ea. stainless spatula, double blade-rounded and squared
  • 1 ea. 500 mil plastic wash bottle
  • 1 ea. bright color nail polish or airplane glue
  • 1 ea. small polypropylene plastic sheet about 4" square*
  • 1 ea. thermometer, 0 to 110 degrees Centigrade

*NOTES:

Colorimeters

I acquired a used Bausch & Lomb Spectronic 20. I understand that not everyone is in the position to so easily buy such equipment. Try the yellow pages for used laboratory equipment dealers. If not specifically listed, try laboratory suppliers. Usually, they have a service department and may have used spectrophotometers or colorimeters. Some brand names to look for are older models of the Bausch & Lomb Spectronic 20, the Coleman Jr. and the Turner. These companies sold many of these lower cost spectrophotometers for many years and you might be able to buy one comparatively inexpensively, say 100 to 200 dollars.

Another option is to buy a new colorimeter but without all the expensive features that the above units have. For the application of reading at a fixed wavelength and a broad color peak, such as in this test, a low cost colorimeter is all that is necessary. All it needs is the proper filter (i.e. as close to 570 nm as possible), a cuvette or test tube holder and a scale in per cent transmittance.

There are three companies that I know of which can offer a low cost colorimeter:

  • Chemtrix (P.O. box 1359, Hillsboro OR 97123; 800-821-1358) has a colorimeter (20A) for $269;
  • Hach (P.O.Box 389 Loveland, Colorado 80539; 800-525-5940) has a single parameter DR100 colorimeter for about $200 (contact Hach about supplying a transmittance scale and the proper filter)
  • Hellige (877 Stewart Ave., Garden City, N.Y. 11530; 516-222-0302) has a meter readout photometer for about $125.

Balance

The Ohaus triple-beam balance is a recognizable standard school lab scale. Presently these cost about $80 to $90 from almost any lab dealer or even hobby shops or some specialty hardware stores. But for this test and most work with mushrooms, you will not need the capacity, or ability to weigh large as well as small masses, offered by this triple-beam. I do not have company names but I have seen in High Times advertisements for small, Inexpensive scales which can weigh accurately up to 30 grams.

Large borosilicate test tubes

Of the twelve test tubes set aside six. These will be used "as-is" with out matchine. The other six need to be volumetrically marked for the standard extraction volume of 20 milliliters. To do this, simply fill up the 20 mil volumetric pipette with water then add it to the test tube to be marked while standing in the test tube holder. Then mark the bottom of the meniscus with an indelible marking felt pen. Do the same with the other five test tubes.

Matched cuvettes

Cuvettes should be one centimeter ID, but because of variations in the extrusion Process for the tubes the ID differs slightly. This irregularity in the internal diameter and the occasional streaks or variations in the thickness in the glass walls of the tubes will result in cuvette-to-cuvette differences in the amount of light from the colorimeter filter passing through the cuvette and solution. This variation can be as much as five percent transmittance. To increase test-to-test precision, some of the manufacturers of the colorimeters mentioned above offer matched cuvettes as an option. Matched cuvettes have been selected so that the amount of light which is absorbed by the walls of the cuvette is essentially the same from cuvette to cuvette. For the price it is usually easier to buy them. If you have to prepare matched cuvettes follow this procedure:

  1. 13 mm OD culture tubes will have a nominal ID of 10 mm. Buy at least a couple of dozen.
  2. Chose several and fill them with water. Insert them into your colorimeter and set the wavelength to 570 nm or as near as your filter will allow.
  3. Set the meter to an arbitrary mid-meter position. Then slowly rotate the cuvette. Notice the variation as you rotate the cuvette the full 360 degrees. Choose the culture tube with the least variation.
  4. Continue with another set of a few cuvettes and keep selecting the least variable cuvette until you have six to ten cuvettes.
  5. Now match the cuvettes by making an arbitrary mark with nail polish or airplane enamel on the open lip of a cuvette such that this mark can be used to align the cuvette in its holder. Note the meter readout.
  6. Take another pre-selected cuvette and rotate it until it matches the transmittance of the first marked cuvette. Mark it with nail polish or paint, too.
  7. Continue until all the cuvettes are matched and marked. When using the cuvettes to measure the transmittance of the DMAB test reaction, be sure to always align the mark with the pre-determined alignment position in the cuvette holder. Usually the holder itself has a mark or ridge to reproduce the correct cuvette position.
  8. It is possible that the initial cuvette which you arbitrarily marked may not match any or only a few other cuvettes. Just start over but use another cuvette as your initial reference cuvette.

Test Tube Holder

The particular holder I use is an open style which is one tube deep and seven across. I use this holder for heating the extraction solution in a one quart pan. To make it fit in the pan, I cut the holder in two sections of three test tube capacity each. When using this holder with the test tubes make sure you balance the weight of the tubes in the three slots. The holder tends to float. For instance if you put a single test tube in the holder's end, the holder will float up dumping the tube into the heated water and thus losing your mushroom sample. To avoid losing your sample, when extracting a single sample put the tube in the center of the holder, or if using two test tubes, put them on either end of the holder.

Chemicals

I have used technical grade chemicals throughout all my testing. Technical grade is somewhat less pure than reagent grade and usually less expensive. The sulfuric acid could be reagent grade which would be somewhat clearer than the slightly yellow-brown technicalgrade. Sulfuric acid is used in the color development solution and is read on the colorimeter, so clarity and lack of particles floating around would help readability and reproducability. I have had no problems with the less expensive technical grade by always using a sample blank to compare all mushroom test samples against it. A sample blank is used in colorimetry to set the "zero" point. It is usually the test reagent with a distilled water sample rather than the material or extraction to be tested. If the test reagent is colored in the spectral region of the test before any reaction, then by using a sample blank one can offset the colorimeter by this degree of color so that the test result is not artificially high.

Plastic sheet

All that is necessary is a piece of plastic to cover your thumb so that when you shake the culture tube with the test reaction mixture the sulfuric acid will not burn your skin. A piece a couple inches square should suffice. Most plastics are unaffected by sulfuric acid.

Testing Solutions Preparation

Color development reagent

This mixture is the actual solution which when in the presence of an indole or indole-like compound will develop a color from a near colorless solution.

  1. Weigh out 0.2 gr of p-dimethylaminobenzaIdehyde (DMAB) and put it into the 250 mil beaker.
  2. Make a 20% aqueous ferric chloride solution by weighing out 20 gr of the ferric chloride into 100 mil of distilled or Deionized (DI) water in the 250 mil graduated cylinder. Wear gloves. Ferric chloride can be corrosive. Make sure it all goes into solution. If any particles remain insoluble, filter the solution. Store the finished solution in a 125 mil bottle and label. Rinse out the graduated cylinder.
  3. Using the 1 ml pipette and the pipette-aid (never suck up corrosive or toxic chemicals in a pipette!) draw up 0.3 mil of the stock ferric chloride solution (#2,above). Add this to the 250 mil beaker with the DMAB already in it.
  4. Add 35 mil of distilled or Deionized water to the 250 mil beaker and stir the water to dissolve the DMAB as much as possible.
  5. Add 65 mil of Sulfuric Acid from the graduated cylinder to the beaker with the DMAB. Be careful with sulfuric acid. Wear gloves, an apron and preferably a face shield. When adding the acid, set the beaker on the table. Do not hold the beaker. An exothermic reaction will take place when you add the acid to the water present in the beaker and it will be too hot to hold. Gently stir the mixture. The heat of the acid-water mixture will drive the remaining DMAB into solution.
  6. Let the mixture cool down and pour this stock solution into a 125 mil bottle and label it. Do not put hot liquids into the bottles; they may break.

Store the DMAB solution in a dark, cool location, preferably in the freezer section of your refrigerator. If you cannot store it somewhere cold, the solution will gradually darken and thus be unusable for colorimetry. I have successfully stored my stock solution in the freezer for over a year and it has not darkened noticeably. At room temperature you will probably need to discard the solution after a few months.

Acetic Acid Extraction Solution

This stock solution is used to extract the mushroom of its active tryptamines. Then a sample of this extract is tested using the above DMAB color development solution.

  1. Measure out 50 mil of glacial acetic acid in the 250 mil graduated cylinder. Fill the cylinder to the 250 mil mark with DI or distilled water and pour it into the 1000 mil bottle. Rinse the cylinder with 3 X 250 mil amounts of DI water into to the 1000 mil bottle to make a total of 1000 mil stock solution of 5% Acetic Acid.
  2. Distilled vinegar (i.e. "white vinegar") can be substituted for 5% acetic acid. Distilled vinegar is approximately 5% acetic acid.

The para-Dimethylbenzaldehyde Colorimetric Test

Figure 6 - Shows the basic procedure for colorimetric (p-DMAB) testing.
  1. Set up the table or bench on which you will do your test with all the equipment in the above illustration spread out. Turn on the colorimeter or spectrophotometer to begin warming it up and adjust or set the proper wavelength (570 nm or close to this). Most have light sources which need to be on for 20 to 30 minutes to stabilize the spectral output. The older models also have slow-to-warm-up electronics.

  2. Turn on the heating plate and begin to heat the water filled quart pan. Do not let the water boil. The boiling action will tip over the test tubes with the samples spilling out into the boiling water.

  3. If you have not done so, use a coffee mill to grind up the dried mushrooms which you wish to test. Weigh out 0.5 grams of the ground mushroom. It's best when using a scale to use a piece of paper on which you weigh the mushroom. Find out how much this paper weighs before adding any mushroom powder then add this weight to the required half gram sample. This total weight in grams is what you should set the scale for if you have a triple beam-type scale. With the weighed mushroom powder on the weighing paper, cup the paper to form a trough and pour the sample into an empty, large (20 mm X 150 mm) unmarked test tube.

  4. Add 20 mil of 5% Glacial Acetic Acid to the sample. Stir the sample with a glass stir rod making sure all the mushroom powder is wetted with the acetic acid solution. Put the test tube in the plastic holder and immerse the holder in the preheated near-boiling water. Note the time or start a timer. Heat this suspension for 30 minutes. This time is not critical. If you should heat it for longer than thirty minutes it will not make much difference. Too long will oxidize the psilocin in the solution and cause a false low reading of the sample. Also, the acetic acid solution does evaporate.

    Every ten minutes or so stir the suspension with the stirring rod. The mushroom powder tends to float on the acetic acid solution which cannot optimally extract the mushroom powder when it is not suspended. Also, the top of the powder can be exposed to air and will oxidize to a dark blue. Again, this can lead to a false low reading.

    The immersion of the glacial acetic acid solution in near boiling water with the mushroom powder during the extraction phase of the test is important to deactivate any enzymes which can cause bluing, and thus tryptamine loss, in the mushrooms.

    Without heat and with time the mushroom extract solution will slowly turn blue-green until it becomes dark blue after several days.

    As noted in the above section on theory, use a thermometer to make sure that the extraction solution reaches 70 degrees Centigrade for at least ten minutes. This temperature converts all the psilocybin to psilocin which makes for more pure test color.

  5. While the mushroom powder is extracting, set up the next step: the filtering and sample development. For each sample, use one small filtering funnel. In the funnel put a polyester ball. Using the wash bottle, wet the cosmetic ball then press any excess water out of the cosmetic ball with your finger. Discard this water if it went into the collection test tube.

  6. After 30 minutes or so, remove the test tube holder from the water bath. Turn off the heat plate (or stove). Just before the initial filtering, stir the suspension one more time. Pour the test tube contents over the top of the polyester cosmetic ball in the small filtering funnel which drains into a large (20 X 150 mm) test tube on which you marked the 20 mil level. Do not worry if you cannot get every last drop of the suspension out of the test tube.

    Let the filtrate pass through the small filter completely by gravity. Sometimes pressing the cosmetic ball will succeed in driving more water extract off the polyester fibers. This step is important because if you don't get all of the original, undiluted extract in the final test sample, your concentration value will be falsely low. I have occasionally forgotten to do this squeezing step and have had readings which were off by 25%.

    Although you could get more consistent results by just using filter paper instead of the cosmetic ball, by filtering the thinly mucilaginous mushroom suspension with the cosmetic ball you can save literally hours of waiting for all the suspension to get through the paper filter.

  7. Now use the wash bottle filled with distilled (or deionized) water and rinse the sides of the test tube used to extract the mushroom sample with a few milliliters of water. Note that the cosmetic ball filtered extract does not reach the 20 mil mark on the test tube. This loss of fluid came primarily from evaporation when you were heating the mushroom suspension.

    For reasons of standardization all our test solutions need to have a total volume of 20 mi. Shake the unmarked test tube which you just added some rinse water to and pour an estimated amount of the rinse water which will fill up the test tube to the 20 mil mark over the cosmetic ball filter. This rinse will remove most of the residual extract left on the polyester ball and will dilute the initial filtrate up to the 20 mil mark. Watch the filtering carefully and remove the filter to the sink once the level has reached the 20 mil mark.

    The largest errors can occur in the filtering and dilution steps. If in the process of washing the filtrate and bringing up the filtered volume to the 20 milliliter mark, you add too much water to the top of the funnel filter you will dilute some of the extracted sample which will not then pass through the filter.

    Since you have reached the 20 ml mark you are forced to discard the water which may contain some of your sample. The results may then be artificially low. Solution: add smaller amounts of rinse water with the squeegee bottle, so that you will not leave any of the liquid mushroom extraction on the filter that will then not be part of the test.

    If in the process of rinsing the sample on the cosmetic ball and adjusting the 20 mil volume, you should over-fill the test tube beyond the 20 mil mark, just correct the final absorbance by multiplying the absorbance by a fraction consisting of the volume which was filtered (over 20 milliliters) as the numerator and 20 as the denominator.

  8. This rough filtered extract is your test sample but needs to be filtered again so that it is clear and will not add absorbance to the colorimeter reading because of its cloudiness. Pour this extract through another small plastic funnel with a folded piece of filter paper in it. Fold the filter paper on itself three times (i.e. into eighths).

    Most extracts will have some colloidal suspension in them, making them look slightly cloudy. Even filtering these with a fine filter will not remove all the turbidity. For most filtering you will need to remove only coarse particles and the Whatman #4 filter paper will suffice. For heavier suspensions use the finer, but slower, Whatman #2 filter paper. The idea here is to obtain a reasonably clear sample which has the least amount of turbidity. Turbidity, if excessive, will give false high colorimetric readings.

    For the receiving container for this filtrate, use another large test tube as the receiver. After a few milliliters of fluid has passed through, you can stop the process if you wish. I usually like at least enough prepared sample for a couple of tests (each tests requires 0.5 or 1.0 ml of sample), just in case I wish to repeat a reading.

  9. The preceding two-step filtering process should take 10 to 15 minutes. While the filtering is proceeding, using the pipette-aid and the 10 mil pipette, draw up 2 mil of the DMAB reagent and dispense it into a matched cuvettes/small test tube. Continue dispensing the DMAB reagent into a matched cuvette for each sample or test you may wish to run. Now you are ready to develop the color and read the final absorbance. If you store the DMAB test solution in the freezer, allow it to return to room temperature before using it in a test.

  10. Using the pipette-aid and the 1 mil graduated pipette, draw up a 1 ml sample of the mushroom extract to be tested. If you anticipate an absorbance reading above 0.700 A or so, then draw up only 1/2 mil of sample, but before doing so add a 1/2 mil of Deionized or distilled water to the small reaction test tube/matched cuvette.

    The reason for this dilution is that for colorimeters with a transmittance scale, the most readable portion of the scale lies above 20% transmittance. Lower transmittance than this corresponds to higher absorbance and greater possibility of reading errors and poor precision. I checked to make sure that both ways of testing equaled the same value after making the necessary multiplication of the diluted sample by two.

    The total combined test volume must be 3 mil for both the sample and the DMAB reagent. The aqueous sample will not immediately mix with the much denser DMAB reagent but will rest on top until mixed.

  11. Make sure your timer is ready. Hold your thumb over the reaction tube with the plastic sheet between the thumb and the test tube and turn the tube upside-down then back again. Do not shake because the shaking will add bubbles which can remain on the walls of the test tube and interfere with the colorimetric reading later. Either start the timer or note the time immediately. After exactly 30 minutes, read the transmittance on the colorimeter (or absorbance if your instrument has a direct liner conversion to absorbance from transmittance).

    Sometimes the reaction mixture in spite of filtering the sample will become turbid. If so, dilute with 3 ml of DI water, read and divide the absorbance result by two. This dilution will usually water down the turbidity enough to be able to read the test.

    It is important to test the mushroom extraction solution soon, otherwise the bluing and other oxidation reactions will breakdown the active tryptamines and cause you to have a false low value for the test's absorbance. By testing the mush- room extract solution at regular intervals, I found that no significant reduction of the active tryptamine concentration occurred until after six to eight hours.

    I have always conducted this test under two 40 watt fluorescent lamps. In light of the research paper which used UV to accelerate the DMAB color reaction6, fluorescent lamps may have more of their spectral output in the near UV than incandescent lights or sunlight coming through a glass window. The absence of such lighting in your test area may slow the reaction down and thus cause you to have lower readings after the 30 minutes than mine. I have not confirmed this, but be aware of this possibility.

  12. At the end of 30 minutes, read the colorimeter scale and note the value for later reference. When reading the meter (unless the meter is digital) look directly down on the needle to the scale. If you should read the needle at an angle, your reading will not be as accurate, nor reproducible. As the value of the transmittance becomes smaller, errors in reading make a greater difference in the final absorbance (or concentration) value. Some meters have a mirror below the scale. By looking down on the needle such that you cannot see the needle's reflection, then you know that you are looking directly down on the needle and meter. Try to interpolate or "guesstimate" the value if the needle should fall between two divisions and show this as part of your recorded value (e.g. 14.6%) If you have a colorimeter or spectrophotometer with a direct absorbance output, your test value will be directly proportional to concentration of the tryptamines present in the mushroom sample. If, on the other hand, your colorimeter has a transmittance scale (i.e. 0-100%) and only a log absorbance scale (i.e. the distance between integers gets smaller as the numbers get larger), you will need to convert your transmittance to absorbance. The formula is: Absorbance Log (1/.01 X Transmittance in %).

    The easiest way to calculate this is to buy an inexpensive calculator with a "log" and "1/x" or reciprocal functions. Then calculate by entering the transmittance as a number between one and one hundred, divide by 100, push the "1/X" key then push the "Log" key. The result will be the absorbance which is proportional to concentration.

Conclusion

It is my hope that the above helps to explain the test values which will be used in the upcoming articles. The next article on the environmental and nutritional influences on the growth and biosynthesis of psilocybin in P. cubensis will make use of this test extensively. The last section of this article, of course, is for those people inclined to do their own testing. I encourage anyone with any amount of chemistry background to try this test. The test values will help immensely in planning your own entheogenic experiences with the "magic mushroom."

Bibliography

  1. Agurell, S., Blomkvist,S. and Catalfomo, P., "Biosynthesis of Psilocybin in Submerged Culture of Psilocybin cubensis: Part I. Incorporation of labeled tryptophan and tryptamine," Acta Pharm. Suecica, Vol. 3 (1966), 37-44.
  2. Agurell, S., Lars, J. and and Nilsson, C., "Biosynthesis of Psilocybin: Part II. Incorporation of Labelled Tryptamine Derivatives, Acta Chemica Scandinavica, Vol. 22, No. 4 (1968), 1210-1218.
  3. Beung, M.W. and Bigwood, J., "Quantitative Analysis of Psilocybin and Psilocin in P. Baeocystis by HPLC and by Thin-Layer Chromatography, Journal of Chromatography, Vol. 207 (1981), 379-385.
  4. Bigwood, Jeremy and Beug, Michael W., and others, editors. "Variation of Psilocybin and Psilocin Levels with Repeated Flushes (Harvests) of Mature Sporocarps of Psilocybe Cubensis (Earle) Singer," Journal of Ethnopharmacology, Vol. 5 (1982), 287-291.
  5. Casale, John F., "An Aqueous-Organic Extraction Method for the Isolation and Identification of Psilocin from Hallucinogenic Mushrooms," Journal of Forensic Sciences, Vol. 30, No. 1(Jan., 1985), 247-250.
  6. Catalfomo, P. and Tyler, V. E., Jr., "The Production of Psilocybin in Submerged Culture by Psilocybe cubensis, Lloydia, Vol. 27, No.l (March 1964), 53-63.
  7. Christiansen, A.L. and Rasmussen, K.E., and others. "The Content of Psilocybin in Norvegian Psilocybe semilanceata," Planta Medica: Journal of Medicinal Plant Research, Vol. 42 (1981), 229-235.
  8. Haard, Richard and Karen. Poisonous & Hallucinogenic Mushrooms, 2nd Edition. Cloudburst Press, 1977.
  9. Leung, A. Y. and Smith, A. H., and others. "Production of Psilocybin in Psilocybe Baeocystis Saprophytic Culture, Journal of Pharmaceutical Sciences, Vol. 54, No. 11 (November 1965), 1576-1579.
  10. Leung, A.Y. and Paul, A. G., "Baeocystin and Norbaeocystin: New Analogs of Psilocybin from Psilocybe baeocystis," Journal of Pharmaceutical Sciences, Vol. 57, No. 10 (October 1968), 1667-1671.
  11. Leung, A.Y. and Paul, A.G., "The Relationship of Carbon and Nitrogen Nutrition of Psilocybe baecocystis to the Production of Psilocybin and its Analogs," Lloydia, Vol. 32, No. 1(March, 1969), 66-71.
  12. Lewis, Waiter H., Medical Botany: Plants affecting Man's Health. Chapter 18, Hallucinogens. Wiley-Interscience, John Wiley & Sons, 1977.
  13. Norland, Richard. What's in a Mushroom. Pear Tree Publications, 1976.
  14. Repke, David B. and Leslie, Dale Thomas, and others. "Baeocystin in Psilocybe, Conocybe and Panaeolus," Lloydia, Vol. 40, No.6 (Nov-Dec 1977), 566-578.
  15. Sarvicki,editor. Photometric Organic Analysis, Vol 31. Wiley-Interscience, 1970.
  16. Snell & Snell,editor. CoIorzetric Methods of Analysis, 3rd Edition, Volume IV. Van Nostrand, 1954.
  17. Stafford, Peter. Psychedelics Encyclopedia. From Chapter 4, "Mushrooms". Berkeley, California: And/Or Press, 1977.
  18. Weeks, Amold R. and Singer, Rolf, and others. "A New Psilocybian Species of Coplandia," Journal of Natural Products, Vol. 42, No. 5 (1979), 469-474.
  19. Windholz, Martha, editor. The Merck Index. Ninth Edition, Entries #7711 and #7712. Rahway,N.J.: Merck & Co., Inc., 1976.



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