STATEMENT OF MR. LEVINE,

CHEMIST, BUREAU OF NARCOTICS.

MR. LEVINE: The Beam test seems to be the most widely used chemical test for the identification of Marihuana, and was first introduced by Dr. W. Beam, of the Wellcome Tropical Research Laboratory, of Khartoum. The alkaline test in 1911 and the acid Beam test in 1915 have come to be accepted as specific for Marihuana, although a lot of samples have
failed to respond. Workers in Europe and north Africa have used it and tried it on a large
number of plant materials. In general it is agreed that no other material responds
characteristically to the test.
It has been attacked by some chemists, notably Trolle and Rende, who said they obtained the Beam test on a mixture of drugs consisting of ginger, coriander, licorice, nutmeg, and several other things. Other workers, notably Fahamy and Keiy in Egypt, and Papavassiliou and Liberato, in Greece, applied the test to these materials, both individually and in the mixture, and failed to get a positive response to any of them.
As I stated before, a large number of authentic samples of Marihuana failed to respond to the Beam test, or gave a very slightly dirty purple color, which might be mistaken for just a
dirty color. So a number of modifications of the Beam test have been introduced to try to improve the results obtained in the test.
One of the steps taken to improve the test was the use of adsorbent charcoal to remove chlorophyll from the solution of the resin. This was reported independently by Fahamy and Keiy, Bouquet, and by the Bureau of Narcotics Laboratory.
One of the tests developed in the Bureau of Narcotics Laboratory is the ethyl acetate test. Ethyl acetate is used as a solvent, because it is a good solvent for the resin, it has a low boiling point, hence is easily evaporated and it can be treated with activated charcoal, which removes most of the chlorophyll, and other substances, which would interfere with color development, but takes out very little of the material responsible for the Beam test.
In carrying out the test, a sample of Marihuana is extracted with a portion of ethyl acetate. The solvent is treated for a few seconds with darco or norite and filtered.
The filtrate is divided between two porcelain dishes and evaporated on a steam bath before a fan. To one of the dishes is added several drops of the alkaline Beam reagent, and to the
other, several drops of the acid Beam reagent. In a large number of tests, no samples which were negative to this modification were found to be positive by the original Beam test, or any of
a number of other modifications. It seems, therefore, that this is the most satisfactory modification of the Beam test.
Dr. Bouquet, in Tunis, has developed two amyl alcohol tests, one of which uses charcoal, and the other does not. His test consists of grinding a sample of Marihuana with potassium Hydroxide, and then adding a portion of alcohol, mixing thoroughly, and filtering, whereupon a purple color appears in the filtrate.
Then, to a portion of this filtrate he adds about a ten-fold volume of water, and extracts with one cc. of amyl alcohol. The purple color is extracted into the amyl alcohol.
In his test involving the use of charcoal, he adds a small amount of animal charcoal to the mixture of the alcohol, KOH, and Marihuana, presumably to remove chlorophyll, and then lets it stand for two hours before filtering.

The resultant filtrate is free of chlorophyll, and the tests obtained are better. We have found that his test, involving the use of charcoal, works better with activated charcoal, such as norit or Darco than with the animal charcoal prescribed.
Also, we find that we get results by filtering immediately after the addition of charcoal, instead of letting the mixture stand for two hours. We have not found any case of failure to respond upon immediate filtration, which would respond after standing.
Another modification which we have developed in our laboratory, and which may be considered a modification of the Bouquet test, is probably most convenient of the modifications to run, because it involves the least manipulation. It consists merely of shaking for a few seconds the sample of Marihuana with a two percent alcoholic solution of potassium hydroxide.
Add to this is an amount of activated charcoal equal in weight to the Marihuana. The mixture is shaken for a few seconds, and filtered immediately. The filtrate is purple and on dilution with water, the purple colored substance may be extracted by amyl alcohol.
Other modifications of the technique have been proposed by Dr. Myttanaere, Viehoever, Placencias and others.

As I said before, a large number of plants fail to respond to any of these modifications of the Beam tests. We have followed the appearance of the Beam test in plants with regard to age, parts of plant, and variety. In regard to age, some plants three inches tall have been found to
respond to the Beam test. Some plants, at all states of growth, up to their decadence, respond.
Many male plants, which are all withered, and consist of nothing but a skeleton with a few dried flowers sticking to the top still respond to the alkaline Beam test.
As far as an individual plant goes, the order of response is best in the top, both with the male and female plants, followed by the upper leaves, lower leaves, upper twigs, upper stalk, lower twigs, and lower stalk. In other words, as you go up towards the top of the plant you get the best response.
Where the top responds very strongly, you generally get a weak response in the skin of the lower stalk. If the tops respond very weakly, the upper leaves will probably be negative, or just give a trace of response. We have never been able to get any response at all from the pith or roots of the plant.

This last summer we tested six varieties of hemp, as Dr. Robinson indicated this morning. Three of them were Roumanian varieties; one of them was Italian, one, Manchurian; and one of Chinese origin. Of the three Roumanian varieties, about a percent could be positively identified by the alkaline Beam test, as Dr. Natchett pointed out this morning. The other three gave a trace of response to the test. The Italian variety was very close to this, having about 98 percent
attainable by the Beam test. Of the Manchurian and Chinese, only about 20 to 50 percent could be positively identified, and of these, practically none of the tests were as strong as the tests obtained from the Roumanian and Italian varieties. The largest number of the plants were absolutely negative, or showed merely faint trances of response, which we would not consider to be suitable for identification if the plants were unknown.
We studied the effect of heating the plants towards response to the alkaline Beam test. We found that heating parts of the plant at 100 degrees with air blowing over them for up to five hours did not have any harmful effects on the material response for the alkaline Beam test.
In fact, some of the plants showed better response after heating than before. Although some workers in Europe say in no case should the extract be heated. over 50 degrees Centigrade.
However, heating the plant at 150 degrees under the same conditions did prove to have a deleterious effect. Another treatment we tried was permitting the plant to mold. We subjected the tops of some of the plants, both negative and positive, to molding, in a very moist atmosphere, for a period of about five weeks. After this period, the whole plant was covered with slimy mold. Response to the alkaline Beam test was as good as it was originally.
Some negative plants which were molded remained negative after the molding period.
Oxidization is an essential part of the Beam test. Beam, in his original article, pointed out that if the tests be applied in the absence of air, a dirty brown color will result instead of the purple color. We have taken up some of the resin in alcohol, and added an equal volume of 2 per
cent alcoholic KOH solution. The resultant solution was colorless, but on passing oxygen through it, the characteristic purple color developed.
Shaking the solution with charcoal had the same effect. Presumably this is because oxidization is effected by the oxygen adsorbed on the charcoal. Oxidation must be done in alkaline solution. Charcoal plays a dual role in the test. In the case of the ethyl acetate test the effect is merely to
remove extraneous matter, since no oxidation is effected in this solution.
In certain solvents, such as petroleum ether, the activated charcoal will entirely remove from solution the material responsible for the Beam test, while animal charcoal does this to a much smaller extent. In fact, animal charcoal may be used in petroleum ether, although its effect as a
cleaning agent is not very large.
The second role of charcoal is that of an oxidizing agent. The oxygen adsorbed on activated charcoal is effective in producing the necessary oxidation of the material responsible for the alkaline Beam test. Animal charcoal is effective to a very much smaller extent, and is
therefore unsatisfactory for use in this process.

MR. WOLLNER: I think we will have a recess now for about five minutes before we resume.

(A short recess was taken, after which the proceedings were resumed as follows:)

MR. WOLLNER: Now, after that rather exhaustive treatise on the Beam test, I would like to have Mr. Benjamin tell us something about this so-called Duquenois test.
The reason time is being given on these tests is this: Every so often a new test is obtained, and this is the experience in every phase of chemical activity, and until several months or years have been put in on it, everyone gets highly enthusiastic about it.
Efforts are made to introduce it as witness material in court, and the first thing you know you run into a situation where you are yourself out on a peninsula and you can not possibly get back.
We have asked the Treasury Department not to employ any tests unless they are absolutely tests on a theory of so-called triangulation, so that when our men go to court to testify on a seizure that their evidence is sound.

Mr. Benjamin, will you tell us briefly about the Duquenois test and what our experience has been with that.

MR. BENJAMIN: I think this was proposed in the early part of 1938. Reagent number 1 is an alcoholic solution with vanillin and acetaldehyde. The second reagent is concentrated hydrochloric acid; The technique consists of extracting hemp with petroleum ether and
driving off the solvent by heat.
One then takes one cc. of the alcoholic reagent, adds it to the residue, and into the solution thus prepared, puts two c.c.Õs of concentrated hydrochloric acid.
There is a change in color from green to slate blue to violet blue. Now, since we have had this, we have tested approximately 165 substances, including some of the alkaloids, the essential oils, C. D. alcohols, everything I could get my hands on that might show up with this reagent.
So far, not one sample of hemp has failed to respond to this test. When I say "hemp" I am referring to solid extracts, fluid extracts, and ether available preparations. They all show positive.
Certain essential oils when treated by the test procedure ran through the same changes as the hemp, namely, oil of bay, geranyl acetate, rhodinyl acetate, and one or two others.
There was also a compound known as Denol, that gave practically the same reaction as hemp. That is used in C. D. alcohols. I do not know exactly what it is composed of, but I think a mixture of higher alcohols and ketones.

MR. SCHICKTANZ: Yes, higher alcohols.

MR. BENJAMIN: I must emphasize the fact that not one sample of Cannabis or fluid extract of Cannabis or solid extract has failed so far.

MR. WOLLNER: Would you recommend the test as an eliminative test for hemp; that is to say, hemp would have to give a positive test of the Duquenois agent in order to be considered hemp, and then proceed?

MR. BENJAMIN: Yes.

MR. WOLLNER: To apply another test to see whether it is another substance?

MR. BENJAMIN: I think the fact that ia the Duquenois test for Cannabis fails to respond, then one should hesitate to call the sample Cannabis.

MR. WOLLNER: That is predicated---

MR. BENJAMIN: (Interposing) On the 165.

MR. WOLLNER: Dr. Munch, you have had some experience with some other tests, which we do not think very much of.

DR. MUNCH: That is fine.

MR. WOLLNER: What is your experience with that test?

DR. MUNCH: I started about three or four years ago with the method developed by an official in the British pharmacopoeia for ergot, using paradimethyl amino benzaldehyde. The results I obtained I then dropped, because I got busy on something else, but I have had occasion this last spring to review and finish up that work, which was reported at Madison and the American Pharmaceutical Association. The manuscript is in the hands of the editor of the American
Pharmaceutical Association, and ought to be out next month.
In it I tried various modifications and found the 2-1/2 per cent solution of paradimethyl amino benzaldehyde in 65 per cent sulphuric acid, or in 65 per cent phosphoric acid, which is my principal reagent. The material to be tested is shaken with 10 parts, or approximately 10 parts, of low boiling petroleum ether, (below 40 degrees Centigrade) which has been redistilled and a half part of MerckÕs activated charcoal or norit, or any other U. S. P., activated carbon for five or ten minutes, then filtered. The filtrate is evaporated, and the reagent applied to the residue. The direct application of the reagent causes charring, or does not cause anything, according to how much material has been removed. But on the addition of a drop of water there is an immediate development of a blue color, shifting toward the violet end of the spectrum,
and disappearing within two seconds or two hours, depending on how much material is present.
Ergot gives a blue color similar to that of Cannabis, but the color shift is toward the red. The red is much slower than with that of Cannabis, and it persists for several days.
I went to two neighborhood drug stores, and got ten or eleven materials out of their prescription department, and sent them through the same tests. Many of them gave no color. Others gave colors of some sort. But, to make a long story short, none of them gave the same type of
color as the blue shift that I obtained with Cannabis. I have tried about 50 samples of Cannabis, so far, and every one of those gave the same reaction.
While my eye is not too good, still there is a symmetric trend between the potency on dogs and the degree of color developed here. By degree I mean intensity and duration, considering them together. But a product stronger on the dog has given me a stronger color.

MR. WOLLNER: Dr. Matchett, what is your experience with that Ghamrawy test?

DR. MATCHETT: We found too many other substances, which we regard as giving colors too similar to that given to Cannabis, to have any value in the hands [of] anyone not very definitely expert in the use of it. Even in such hands it is our judgment that colors given tobacco and certain other substances were still too near the color given by Cannabis for use in the case of extracts. Of course, where we have a plant to look at, we realize a different situation exists.

DR. MUNCH: Even with tobacco?

DR. MATCHETT: Tobacco was very close.

DR. MUNCH: Is that right?

DR. MATCHETT: This was not used with charcoal.

DR. MUNCH: If I do not use charcoal, I get inconclusive results along that line.

DR. MATCHETT: I am coming 'to that. It was our experience that the U.S.P. activated charcoal would remove either the hemp or tobacco test substance from petroleum ether. The result being that the test in hemp was about the same as it was in tobacco. I will ask Mr. Benjamin if that is a correct statement.

MR. BENJAMIN: That is correct.

MR. WOLLNER: Dr. Lancaster, have you had any experience with these tests in Canada?

MR. LANCASTER: Yes, Dr. [sic] Wollner. Some years ago our interest was aroused as to whether or not Canadian Cannabis would be at all active. As some of you know, the plant
thrives in all climates. We have it in all altitudes. There are no cultivated plants. But some years ago we wanted to satisfy ourselves about the activity of the Canadian produced Cannabis. Some was grown in western Canada for windbreak purposes, that is the only economic function it had with us. After applying the alkaline Beam test, as put out from Geneva we obtained positive reactions, and the pharmacologists tried it on dogs and we found it responded, so we concluded that some of the Canadian Cannabis was active.
From the standpoint of the administration of the Narcotic Act, our interest is entirely limited to the illicit traffic, and it is confined to the reefer or cigarette, where we have the advantage of a physical. diagnosis, rather than chemical.
The chemical work on this we intend to follow up as of extreme importance, because it is difficult to predict to what extent the extracts may come into use. There is a possibility of developing a non-reactive type of plant, but it is not of an immediate concern, although there again conditions of world trade and combined complications might become such that Canada might be asked to take up that problem again. Who knows?
So we can not afford to lose interest in these chemical phases of the testing.

Our experience with the Beam test has been that it is somewhat erratic, and does not always give the equivalent results in the hands of different operators, for some reason. Of course, there again, we have run some of our tests on plants where we know we have had them in storage for some years. It is rather puzzling there is no reaction there.

MR. WOLLNER: No reaction?

MR. LANCASTER: On prolonged storage.

MR. WOLLNER: Under what conditions was that hemp stored?

MR. LANCASTER: In a large glass stopped bottle. However, we have to check that again, because of the results of this vegetation which remained an open field, which is another puzzle.

MR. WOLLNER: That is where we all find ourselves at the moment. What I would like to hear from the group is something in the way of suggestions as to how this problem can be most competently attacked. What is to be done? The problem is not a simple one, although there is no indication as to its complexity. I say it is not a simple problem, because if material has not been advanced after several thousand years of experience there must be some barrier there.
Is there any technique which should be considered which has not been considered?
Dr. Hibben, have you any suggestions along this line?

DR. HIBBEN: That is rather a difficult question. I think we are proceeding correctly.
I think the first thing that has to be done is developing an adequate method for determining the content of the active principle, and until such a method is developed, there is not going to be very much room for improvement.

MR. WOLLNER: The only difficulty has been that this problem has been very much the problem of peeling an onion--the more you peel, apparently the more peels you can take off, until you peel away the onion and then there is nothing left.

DR. HIBBEN: That is quite true. But I do not think there has been any comprehensive, systematic work done on the problem, by an adequate chemical staff, under adequate direction.

MR. WOLLNER: The first thing seems to be to find the active principle. That is a different proposition.

DR. MUNCH: Has any work been done on the chlorophyl of the leaf?

DR. MATCHETT: All the work we know of is what you have done.

DR. MUNCH: The only thought I have is, if the chlorophyll of Marihuana happens to be different from all other chlorophylls in the universe, it can be identified microscopically.

MR. WOLLNER: Dr. Hibben, you have run some microscopic tests on Cannabis direct for the chlorophyll.

DR. HIBBEN: I did not look for chlorophyll. I say that is very desirable, but I say they would be very doubtful on the chlorophyll alone.

DR. MATCHETT: Of course, there are some points about that.

MR. VALAER: The chemistry of opium was very uncertain for a long, long time. We had a crude mass to work with, and gradually they pulled out one hundred pieces or more. I believe, after all, we have not been interested in this more than about two years. I believe the chemical crude resin Marihuana will work out in the same way. We have two stills upstairs. We have a good many people working on it in extracts and various ways, even now in this brief time.

MR. WOLLNER: Dr. Couch, do you have anything to offer?

DR. COUCH: I would like to say that this problem is in no more desperate condition than a great many other problems in which a solution has not been reached. They all present this picture before the real work is done in solving it which makes it all seem extremely baffling.
It is very curious that by plugging along and keeping infernally at it, that one of these days the problem is solved almost before it is realized, and it seems to me that the lines that have been projected here and the lines that have been followed are exactly those that should be followed, and will, in the course of time, lead to the solution of the problem; I mean the information that you desire to have. There is one thing that occurs to me that has not been mentioned, and
that is if any work has been done upon the smoke from Marihuana, as the smoke is physiologically active.
It may be different from the resin taken by mouth or injected into the veins. That is another matter. But it seems to me that there is something there that might be developed as a test for identity.

MR. SMITH: I think Dr. Hershfield did something on that chlorophyll work which he did two years ago.

DR. COUCH: Of course, along that line is also the possibility of reaction from the protein. It simply rests on the possibility and probability that that is, of course, the leafy tissue, and the extract probably would not serve.
The leafy portion would contain some protein. That with a water extract or salt extract of the plant itself, in a very short time would get positive results, one away or the other. The difficulty there is there may be present some protein that is also present in another plant. That difficulty arises, but the precipitation reactions are amazingly specific and amazingly direct. When one goes from one animal tissue to another he has to wash his hands extremely carefully as he changes over from one to another, so as not to spoil the test in the next tissue. There is that sort of delicacy. I simply offer that as a test. I presume there has already been a lot of thought discounted on that subject.

MR. WOLLNER: I do not know of that test. Dr. Loewe?

DR. LOEWE: If I may bring up the encroachment which is in doubt, it has been known thousands and thousands of years that sex cells contain an active principle. However, it has taken up to this century to get hold of those active principles, and the reason was not the difficulty with respect to chemistry, but the difficulty was that there was no test for the active principle only as an active principle, and no chemical tests were given.
This is the same situation in Cannabis with one exception, and we know the physiological test for the active principle is given. It is much more easily accessible. This biological test is much more easily accessible, so there is a test, and the thing which has not yet been done, at least not
yet systematically enough, is to dovetail the identification of the active principle by its active nature by a biological test. I think this specific picture of the dovetail work gives immediately
the solution, unless somebody is inclined to drop the whole thing. But I again remember that same situation in the female hormone and the male hormone, which involves just this one property to give a beautiful color reaction. However, it did not take more than five years after finding the right test and using the right test in the right way from the first isolation of the principle. I think this is the way prescribed.

MR. WOLLNER: Do you know of any experiments, Dr. Loewe, that have been performed on the smoke itself?

DR. LOEWE: No.

DR. BLATT: I know of where they took the Marihuana smoke and passed it through solvents.

DR. LOEWE: Through chloroform?

DR. BLATT: No, through water.

DR. LOEWE Water, rather than chloroform?

DR. BLATT: Yes.

COMMISSIONER ANSLINGER: There is a great deal of work being done at the present time with respect to the use of opium smoke. A paper has just been prepared by someone in a laboratory in London, which has just been issued. Are you familiar with Dr. Nicholls?

MR. LANCASTER: Yes, Dr. Nicholls was mentioned in connection with the research today.

COMMISSIONER ANSLINGER: Is that the same Nicholls who is on the Opium Assay Committee?

DR. BLATT: I was going to say very much the same thing as Dr. Loewe said; that is, that I can not see any reason for being discouraged as far as chemistry is concerned. Now, you have got a perfectly good point of attack. The amazing thing about this whole problem is that nothing has been done with the exception of this one matter in 1932, when it was established that crude Cannabinol behaves like a pure substance, and the customary high vacuum distillation technique fails to separate it into its constituents. The work has been apparently dropped from that time on. The surprising thing is that someone has not jumped into that.

MR. WOLLNER: Of course, there has been no commercial demand in that respect, .and that is one of the reasons for that.

DR. BLATT: It is difficult to get hold of the material.

DR. HIBBEN: If you want to get something on spectroscopic methods, when these chemists leave the point where there are spectroscopic methods for determination and for determining the structure, they would aid greatly in facilitating this problem.

MR. WOLLNER: It would aid greatly.

DR. MATCHETT: We would like to ask Dr. Hibben if there are not some such methods which may be correlated to the bioassay? The essential or volatile oils come out of this material very readily. It can be narrowed down to relatively few compounds. If we knew, separately, the spectra of these various materials, would it not help to follow the separations at that stage of operations?

DR. HIBBEN: Yes, I think it would. You can start in by that procedure on hormones.

DR. MATCHETT: And I believe also carotenoids.

DR. HIBBEN: Yes.

DR. MATCHETT: As the fractionation goes further and further, the number of bioassays increases almost without limit, and that is one reason we were particularly interested in it. Also, the quantitative phase has to be considered.

DR. LOEWE: But, as an economic matter, and the rational method is to proceed in an economical way, which can be done by carefully choosing fractions to test.

DR. MATCHETT: I do not believe we could quarrel about that.

DR. MUNCH: Doctor, there is the other thought, and that is that we have not been picking on any of the prisoners lately.

COMMISSIONER ANSLINGER: Doctor, we are not dealing with the same problem as opium, where we can take the addict to a hospital at Lexington and go through all of the experiments. There is a little danger that this drug might affect a man permanently. He might do something which we may be sorry for later. I think that must be given serious thought.

DR. WRIGHT: Dr. Wollner, I will not be very long now, but I just want to clarify a point, and it would seem from reports and other information that the tests are rather indicative of hemp rather than of the active principle.

I am saying that for this reason: We would like to be in a position to approach the development of strains that were free from the active principle, Now, until we have a test it seems to be that we can not do anything.

MR. WOLLNER: Yes, and no.

DR. WRIGHT: I will say it is possible that one of these tests may be useful from a breeding standpoint, but it seems to me it is working entirely on a guess, It would seem to me that any approach would be resolving the strains in pure breeding alone. In other words, approach the inbreeding situation in a hybrid manner; that is the approach we want, but it would seem to me that all we could do at this state would be to develop those lines at random.

In other words, set up as many facilities as we could for pure breeding lines. Any individual plant that would be tested would be very indefinite as to what its progeny would be, and it would seem to me that that is more or less a blind approach; shall I say a lick in the dark; and we would have to develop as many as we could. We can do that, develop as many lines as possible, like in Prussic acid in Sudan grass.

My point is, and I am mentioning it to you chemists, that we can get nowhere without a test of consequence. We might be lucky. About ten thousand chances to one, we might be lucky until we have a test.

MR. WOLLNER: In the last analysis, you are unquestionably correct about that. Really, before significant progress can be made by the agricultural people, we will have to provide you with a formula. Before we adjourn, I would like to invite any of the visiting friends present to come upstairs for a few minutes and see our laboratory set-up for tackling this job, the molecular stills, and extraction equipment, and I am quite sure you will enjoy it.

And I want to really express my appreciation and thanks for your kind cooperation in helping to clarify these issues.

(Whereupon, at 5:10 o'clock p.m., the Conference was adjourned sine die.)