The Measure of the Mushroom
C.B. Gold
Taken from PM&E Volume Five
(OCR'd by GluckSpilz
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.(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, 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.(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 experience. 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; psilocin; 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 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 thepresence 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.(l7 p. 252) Norland describes a few colorimetric tests which can be used to identify mushrooms which contain tryptamine derivatives.(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 memory, you can at least estimate the concentration differences between mushroom flushes.
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
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.(l8) (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 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 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).
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 chromephore. 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.
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 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.(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 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.(l7)
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 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.
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 comparedto 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.
[ NEXT SECTION ]
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.(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, 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.(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 experience. 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; psilocin; 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 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 thepresence 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.(l7 p. 252) Norland describes a few colorimetric tests which can be used to identify mushrooms which contain tryptamine derivatives.(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 memory, you can at least estimate the concentration differences between mushroom flushes.
- 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.
- 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.
- 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 (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)
- 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)
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
- Add 1.0 gram of p-DMAB to 28 mil of ethanol(denatured). Stir until dissolved.
- Add water to the above mix until 60 mil total volume achieved.
- 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.
- 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.(l8) (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 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 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).
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 chromephore. 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.
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 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.(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 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.(l7)
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 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.
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 comparedto 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.
[ NEXT SECTION ]