The Mushroom Entheogen, The Measure of the Mushroom by C.B. Gold

Explores the relationships between hard mycological chemistry and visionary experiences - From Pyschedelic Monograms and Essays #5

Summary:


The Mushroom Entheogen explores the relationships between hard mycological chemistry and visionary experiences related to psilocy- bin mushroom use. In PM & E vol. 1 we were presented with opti- mum 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 con- structing 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 test- ing - both in the laboratory and through implicit meditation and phys- iological/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 charac- teristic 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 culti- vated mushrooms and to stabilize the quantity biosynthesized from flush to flush of a particular strain of P. cubensis by con- trolling 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 varia- tion in the concentration of psilocybin in the controlled cultiva- tion 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, concen- trations 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 mush- room nutritional and environmental growth factors, one can minimize this large flush-to-flush tryptamine(the major molec- ular grouping in psilocybin/psilocin and other related com- pounds) concentration variation.

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 sim- pie, 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 psy- chedelic 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 sub- jective 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 con- cepts, 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 concentra- tion 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 sev- eral batches of mushrooms subjectively after finding an objec- tive 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 indi- vidual to another but it is the intensity and duration which will change linearly with the increase in the objective test value. The 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 dif- ficulty when used alone. Its results can be difficult to cornmu- nicate 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 reac- tion, 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.

The Subjective Test.

1. One can argue about the effects of set and setting on the psychedelic experience, but no matter the outcome of the argu- ment 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).

2. 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?

3. 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.

4. Listen to sounds or music and feel their effect on your emotions and observe how they change the hallucinations or patterns.

5. 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 mani- festations during a psychedelic trip. But many times impuri- ties in the mushrooms can initiate the side effects, too. All the above are indications of the quality of the trip and conse- quently the mushroom quality. Generally, as I perfected my growing and storage techniques, the above physical symptoms diminished.

6. Watch how you respond physically. How is your coordi- nation, such as your ability to walk and talk?

7. 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 express- ing ideas in speech? Can you give directions to a familiar location to someone?

8. 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.

9. 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 aero- bic 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 mani- festations, 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 pur- posely limited to the use of the mushrooms for the more intro- verted 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 con- sciousness come with effort and planning between trips and a tremendous burst of yearning during the actual trip.

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 concen- tration value. If one has access to the pure psychedelic trypta- mines 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 commu- nicate the relative strength of different batches of mushrooms to any one else who may have a different subjective interpreta- tion 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 spe- cific 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 chemi- cals completely differently.

See Figure I.

Tests can be developed which are also based on the similar- ity 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 tryp- tamines 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 chroma- tography . 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 mix- ture of various molecules interact differently with the mole- cules 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 mix- ture of compounds segregate into separate bands of pure com- pounds. 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 chromatogra- phy(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 sol- vent 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 exam- pie 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 chromate- graphed 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 sol- vent 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 com- pound, or one can actually scrape the band off the plate and measure the nearly pure compound by some of the other tech- niques 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 (usu- ally 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 col- umn. 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 concen- tration 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 col- umn 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 men- tioned 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 con- sequent 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 "color- imeter" and can use filters rather than the much more expen- sive 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 them- selves are uncolored but when combined with certain other molecules will form a color. These chemicals are called "chro- mophores" 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 psyche- delic 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 view- ing. 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 dis- cuss 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 absor- bance 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?

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 psycho-physiological 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, psiloc- ybin 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 psyche- delic 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 cleav- age of the phosphate group off the psilocybin molecule in the handling and sample preparation.(l4) 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, vac- uum 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 psilo- cin 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 bio- synthesis 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 psilocy- bin 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 rea- gent 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 absor- bance 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.(l0) The common amino acid, glycine, is one such nitrogen containing compound found in abundance in the mushroom.(l8) 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 expe- rience. Agurell, Blomkvist and Catalfomo (1) identified a lengthy list of possible tryptophan metabolites which might show up in the psilocybe mushrooms: 6hydroxytryptophan; kynurenine; tryptophan; kynurenic acid; xanthurenic acid; psil- ocin; tryptamine; methyltryptamine; dimethyltryptamine; 3- hydroxyanthranilic acid; anthranilic acid; N-acetyltryptophan; and indoleacetic acid. Agurell and Nilsson (2) 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 tryp- tamine 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 con- taining mushrooms, which, as far as I know, do not exist.(l7 p. 252)

Norland describes a few colorimetric tests which can be used to identify mushrooms which contain tryptamine deriva- tives.(l3 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 mem- ory, you can at least estimate the concentration differences between mushroom flushes.

1) A simple test for indole-containing compounds and tryp- tamines 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, "muri- atic 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 over- night or use a steam bath or a hair dryer to dry. Spot the resi- due on filter paper and let dry. Spray or drop on the following developer. In order for the test to work effectively the devel- oper 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 p-dimethylaminobenzaIdehyde(5%) (abbreviated DMAB) in concentrated hydrochloric acid (HC1). (9) Another variation of the Ehrlich's reagent is 2% DMAB in HC1-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.(l8)

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 mush- room. Also, you will be able to use the information about the expected relative distances (Rf values) which psilocin and psi- locybin 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 sys- tem, T found that the solvent ratios mix well. Some of the other literature suggests solvent mixtures which are not home- geneous 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(dena- tured). 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 com- pletely. 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 posi- tive for indole compounds.

The theory behind the reference colorimetric test.

In the preceding sections I have outlined the general parame- ters of colorimetric testing and in the last section listed the pro- cedures 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 para- dimethylaminobenzaldehyde (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 origi- nally used in a slightly modified form to measure ergot alka- loids.(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 psi- locybin 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 chro- mophore. Although in the following testing procedure and in all the technical literature, para- dimethylaminobenzaldehyde has been used as a color developing agent, another chemical, paradimethylaminocinnamaldehyde may be used. This partic- ular 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 applica- tions 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 dia- zotised 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.(l8) (I do not have details on which wavelength to measure this color reaction.)

THE CORRECT WAVELENGTH TO MEASURE THE

A few researchers used DMAB in their colorimetric test and generally the wavelength they used to read the indole contain- ing 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 10nm 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 spec- tral 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).
See Figure 3.

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.(l) In that paper they suggested the use of a UV light which will speed the reaction with the DMAB chrome- phore. 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 out- line 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 con- tinues 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.

See Figure 4.

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 purifica- tion. 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 psilo- cin is especially unstable in alkaline solution, I felt that an acidified aqueous solution would be the best to use as a sol- vent.(l9) 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 inter- fere with the test results, since the reaction yields a blue or pur- ple 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.(l7)

Apparently, others have also found that a dilute acetic acid solution is an excellent solvent for both psilocin and psilocy- bin. Not only does the solution completely extract both trypta- mines but the solution extracts other interfering substances to a lesser degree. Casale also notes that if one heats the extrac- tion 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 solu- tion. 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.

SAMPLE WEIGHT

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 some- thing 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 proce- dures. Common sense may tell you that a particular modifica- tion may not matter, but in fact, the modification may change the results dramatically

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 sec- tion on "what is in the mushroom and what are we measur- ing?", 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 mole- cules. 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.

Zone / Rf / Color with DMAB

1 / 0.137 / Dark Violet

2 / 0.275 / Pure Violet

3 / 0.550 / Grey Blue

4 / 0.965 / Pink 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 absor- bances(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 oxi- dases 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 concen- tration of DMAB, these enzymes effect a falsely low value for the tryptamines. This is another reason I use a low pH extrac- tion 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.

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 pro- portional to the large increase in the subjective effects of the mushroom powder. A small apparent increase in the concen- tration(absorbance value) of the DMAB test doubled the sub- jective 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 concentra- tion 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 per- centage 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 separat- ing the mushroom extracts, I cut out the zone which corre- sponded 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 tempera- ture 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 concen- tration contributed a greater percentage to the total color inten- sity 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 colori- metric theory is for a solution of a single absorbing molecule and as stated previously, there could be as many as twelve dif- ferent 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 con- verted the increase into units of the base reference experience. For example, I know from written records of my experiences that the second plateau(0.'ir 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.
See figure 5.
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 experi- ence intensity increases by the equivalent of the 0.5 A mush- room experience.

2) The paper chromatography extraction experiment dis- cussed 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 chrom- atography 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 cor- responds 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 com- pounds. The value also means that all the samples had at least 0.3 A of non-active, but DMAB reactive, junk in the mush- room. 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 particu- larly 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 mix- ture and relative concentration of the various active trypta- mines, such as psilocybin, psilocin, baeocystin and their analogs, changed in the mushroom powder. In conclusion, then, when reading the upcoming article(s) on

THE COLORIMETRIC TEST

EQUIPMENT AND SUPPLIES

EQUIPMENT

Colorimeter/Spectrophotometer*

Hot Plate

Quart Pan

Balance capable of weighing at least one gram and

accurate to 0.1 gr*

Electric Coffee grinder

CHEMICALS*

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

12ea 20 X 150mm borosilicate glass culture tubes*

6ea 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 under- stand that not everyone is in the position to so easily buy such equipment. Try the yellow pages for used laboratory equip ment dealers. If not specifically listed, try laboratory suppli- ers. 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 appli- cation of reading at a fixed wavelength and a broad color peak, such as in this test, a low cost colorimeter is all that is neces- sary. 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:

1) Chemtrix(P.O. box 1359, Hillsboro OR 97123; 800-821- 1358) has a colorimeter (20A) for $269;

2) 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)

Balance

Large borosilicate test tubes.

Matched cuvettes.

Cuvettes should be one centimeter ID, but because of varia- tions in the extrusion Process for the tubes the ID differs slightly. This irregularity in the internal diameter and the occa- sional 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 per- cent 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.

Test Tube Holder.

Chemicals.

Plastic sheet.

TESTING SOLUTIONS PREPARATION:

Color development reagent.

This mixture is the actual solution which when in the pres- ence 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 weigh- ing 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 pipetteaid(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.

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.

THE para-DIMETHYLBENZALDEHYDE

COLORIMETRIC TEST

See figure 6.

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 warm- ing it up and adjust or set the proper wavelength(570nm 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 mush- room powder on the weighing paper, cup the paper to form a



trough and pour the sample into an empty, large (20mmX150mm) 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 pre- heated 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(20X150mm) 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 occasional-ly 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 deion- ized) 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 esti- mated 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 20ml mark you are forced to dis- card 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.

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 corre- sponds 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 neces- sary 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 col- orimetric 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 sam- ple will become turbid. If so, dilute with 3ml 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 sig- nificant reduction of the active tryptamine concentration occurred until after six to eight hours.

I have always conducted this test under two 40 watt fluores- cent lamps. In light of the research paper which used UV to accelerate the DMAB color reaction(6), fluorescent lamps may have more of their spectral output in the near UV than incan- descent lights or sunlight coming through a glass window. The absence of such lighting in your test area may slow the reac- tion 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 read- ing 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 "guess-timate" 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 transmit- tance 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 absor- bance. 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 "l/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 encour- age 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
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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.
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.... To be continued.

This document Copyright Peter Meyer