Analytical separations of mixtures of hallucinogenic drugs

Sections

EXPERIMENTAL
Gas chromatography procedure
FIGURE 1. Gas chromatogram showing the effective resolution of a mixture of five hallucinogenic drugs.
Thin-layer chromatographic identification and separation
TABLE 2
DISCUSSION

Details

Author: Melvin LERNER , Mary Diane KATSIAFICAS
Pages: 47 to 51
Creation Date: 1969/01/01

Analytical separations of mixtures of hallucinogenic drugs

Melvin LERNER
Mary Diane KATSIAFICAS
U. S. Customs Laboratory, Baltimore, Maryland

A study was made of the separation of mixtures of dimethyltryptamine, mescaline, psilocybin, ibogaine, and lysergic acid diethylamide, using both gas chromatography and thin-layer chromatography. Temperature-programmed gas chromatography of the trimethylsilyl derivatives gave excellent separations, as did thin-layer chromatography of the original compounds, using a dual-solvent method.

During a time when men seek meaningfulness within reality and awareness beyond normal consciousness through the use of one of many hallucinogenic drugs, there is increasing use of mixtures of these drugs, hopefully to intensify the resulting "euphoria", but most commonly producing more bizarre effects. Among the most common of the hallucinogenics in use are "DMT" (N,N-dimethyltryptamine), mescaline (3,4,5,-trimethoxyphenethylamine), psilocybin (3-2'-dimethylaminoethylindol-4-yl phosphate), ibogaine (7-ethyl-6,6a,7,8,9,10,12,13-octahydro-2-methoxy-6-9 methano-5H-pyrido (1',2':1,2) azepino (4,5-b) indole), and "LSD" (d-lysergic acid diethylamide). The separation of these five drugs was studied using both thin-layer chromatography and gas chromatography.

DMT, simplest in structure of the four indole compounds we worked with, is obtained from the pulverized seeds of Piptadenia peregrina and Piptadenia macrocarpa, leguminous shrubs which grow in the Caribbean Islands and the Orinoco area of South America [ 1] . Indian tribes of this area inhale the pulverized seeds as a snuff through a small tube. With a dose of 70 mg the onset of hallucinatory effects is within 2-5 minutes, and the resulting condition subsides within an hour [ 2] , thus explaining its nickname, "the businessman's trip". This fast but brief influence implies direct action on the central nervous system and simultaneously rapid metabolism.

Mescaline, probably the most widely known of the hallucinogenic compounds, excluding LSD, is about 1/5th as active as DMT and requires a higher oral dose (0.3-0.6 gm), but the duration of effects is 5-12 hours with some reported cases of reactions up to 11 days in length [ 3] . Mescaline is found in the dried heads or "buttons" of the Peyote cactus, Lophophora williamsii, which grows in the southwestern United States and northern Mexico. Some Indians of this area use peyote as a sacrament in their religious rites, and are allowed to do so under U.S. law. In such tribes, it is interesting to note the almost total absence of alcoholism.

One hundred times as effective as mescaline is psilocybin, a naturally occurring indole containing phosphorus. Obtained from the mushrooms Psilocybe mexicana and Stropharia cubensis, which grow in Mexico, Central America and Thailand, "teonanacatl", as it is called by the Indians has an effective oral dose of 4-8 mg.

Ibogaine is the only one of the four naturally occurring drugs under study whose home environ is not the New World. It comes from the shrub Tabernanthe iboga, found in Africa. In small doses African natives use it while stalking game to combat fatigue [ 5] ; remaining motionless for as long as 2 days while still retaining mental alertness. In larger doses, mental confusion is produced, accompanied by hallucinations, though, as with many of these new-to-civilization compounds, the behavioural effects of the pure substance on man have not been reported.

LSD, 100 times stronger than psilocybin and 10,000 times as strong as mescaline, was synthetized by Dr. Albert Hofmann in 1938: Not until 1943 did he, by accident, first experience its hallucinatory effects. Today the so-called "acid ", potent in such small doses as 100 μg, is used in increasing quantities to unlock those fetchingly exotic and dangerously psychotic experiences which have been described by its users.

EXPERIMENTAL

We used gas chromatography and thin-layer chromatography to separate and identify these drugs alone and in mixtures

Gas chromatography procedure

A mixture of chloroform and methanol (3:1) was used as a general solvent for the pure compounds. Ibogaine gave a good, sharp peak; one microgram could be easily detected. The other compounds gave either asymmetrical peaks or relatively poor sensitivity and recourse was had to derivatives to enhance sensitivity and stability. We had previous success with the trimethylsilyl derivative for the gas chromatographic analysis of LSD [ 6] and we found that all compounds under study formed trimethysilyl derivatives with varying ease. Ibogaine and mescaline required a catalyst, 4,5,6, trimethylchlorosilane [ 7] for efficient conversion, and this catalyst was used routinely for the mixed compound studies. The solvent-reaction mixture was dimethylformanide, bis (trimethylsilyl) acetamide and 4,5,6, trimethylchlorosilane in the proportions 1:4:1. The compounds were heated with this solvent-reaction mixture for one hour at 70 °C. Substantially complete conversion to the trimethylsilyl derivatives occurred for all the compounds except ibogaine, for which 80 % conversion was obtained. When ibogaine was present in a complex mixture, less than 50 % was usually converted to the trimethylsilyl derivative but the converted and unconverted ibogaine appeared as an easily recognizable double peak on the gas chromatogram.

Occasional difficulty was incurred in producing trimethylsilyl derivatives because of the instability of the bis (trimethylsilyl) acetamide in the presence of moisture. Bis (trimethylsilyl) acetamide which was purchased in glass vials and then transferred to bottles was found to deteriorate rapidly in reactivity and yield unsatisfactory results. Bis (trimethylsilyl) acetamide should be purchased in and dispensed from hypoder mic-type vials to eliminate moisture contamination and premature decomposition. The mixture of drugs was first dissolved in 0.1 ml dimethylformamide, 0.4 ml of bis (trimethylsilyl) acetamide was then added followed by 0.1 ml 4,5,6 trimethylchlorosilane. The solution was then heated for one hour at 70 °C.

An F & M model 400 gas chromatograph equipped with a flame ionization detector and a 2% SE 52 column was used. A Hamilton microliter syringe was used to inject the desired amount. Table 1 shows the isothermal column temperature, flash heater temperature, and detector temperature for the respective drugs. The flow rate of helium was in all cases 100 ml/min.

The differences in column temperatures needed to identify the individual drugs necessitated temperature-programming the column to achieve separation of the drugs within a mixture. The temperature range was 130°-250 °C, with a rise of 3 °C/min following a four-minute delay interval. The flash heater and detector temperatures were 260 °C.

In Table 1 are presented the retention times for the drugs under various conditions both alone and in programmed mixtures. No difficulty was encountered in identifying the individual substances in the program. Figure 1 is the chromatogram obtained from a mixture of the five compounds. Peak 1 is a reagent peak. Peak 2 is the trimethylsilyl derivative of dimethyltryptamine, peak 3 the trimethylsilyl derivative of mescaline, and peak 4 the trimethylsilyl derivative of psilocybin. Peak 5 is ibogaine; the secondary peak is the trimethylsilyl derivative of ibogaine. Peak 6 is the trimethylsilyl derivative of LSD.

Positive identifications can be made by recovering the injected compounds on potassium bromide crystals and obtaining infra-red curves [ 8] .

FIGURE 1. Gas chromatogram showing the effective resolution of a mixture of five hallucinogenic drugs.

Full size image: 114 kB

TABLE 1

Compound

Column

Temperature°C (Flash heater)

Detector

Retention time (Minutes)

Dimethyltryptamine
140 190 170 8.0
Dimethyltryptamine (as the trimethylsilyl derivative)
140 190 170 10.0
Dimethyltryptamine (as the trimethylsilyl derivative)
Programmed
260 260 12.0
Mescaline
140 190 170 5.4
Mescaline (as the trimethylsilyl derivative)
140 190 170 32.8
Mescaline (as the trimethylsilyl derivative)
Programmed
260 260 19.7
Psilocybine
180 235 210 4.6
Psilocybine (as the trimethylsilyl derivative)
180 235 210 21.8
Psilocybine (as the trimethylsilyl derivative)
Programmed
260 260 30.6
Ibogaine
220 280 240 11.7
Ibogaine (as the trimethylsilyl derivative)
220 280 240 12.7
Ibogaine
Programmed
260 260 39.3
Ibogaine (as the trimethylsilyl derivative)
Programmed
260 260 39.9
Lysergic acid diethylamide
250 280 280 14.6
Lysergic acid diethylamide (as the trimethyl- silyl derivative)
245 260 280 11.8
Lysergic acid diethylamide (as the trimethyl- silyl derivative)
Programmed
260 260 47.6

Thin-layer chromatographic identification and separation

Prepared Merck silica gel plates (250 μ thick) were used. They were activated by heating for 2 hours at 100 °C and then stored in a desiccator until required for use.

The solvent system to be used was prepared the day before. After mixing and shaking the appropriate solvents (total volume - approximately 300 ml) in a 500 ml separatory funnel, they were allowed to separate into layers and equilibrate overnight. The following day, the developing chambers (Desaga) were lined with filter paper. The aqueous layer (if present) in the separatory funnel was discarded; the mobile phase put in the tanks; which were then sealed using a starch-glycerine paste. If the mobile phase contained ammonium hydroxide, a 50 ml beaker was partially filled with ammonium hydroxide and placed in the chamber after the mobile phase had been added.

The drug sample was spotted 3 cm from the bottom of the plate and at least 2 cm from its edges. Individual samples were run at least 2 cm apart. The area of the spot, which was applied with a 10 μl micropipette, did not exceed 24 cm [ 2] . To aid in the drying of the spot and to control size, a steady flow of nitrogen through a Teflon eye-dropper was applied. After allowing the chambers to equilibrate for 3 hours, the previously spotted plates were placed in the tanks for developing and removed when the solvent front reached the 15-cm mark. All separations were conducted in a constant temperature room. The plates were then allowed to air-dry, after which they were viewed under ultra-violet light and the results recorded. Following this observation, the plates were heated for 5 minutes at 110°C to remove any trace of alkaline vapours. They were then sprayed copiously with either a solution of p-dimethyl-aminobenzaldehyde (125 mg in 100 ml of 1:1 sulfuric acid and 2 drops of a 10% ferric chloride solution) for qualitative data or with potassium iodoplatinate for quantitative data, (3 ml of a 10% platinum chloride solution mixed with 97 ml water to which is then added 100 ml of a 6% solution of potassium iodide). With the p-dimethylaminobenzal- dehyde spray, following an additional 20 minutes of heating at 100 °C, characteristic colours of the individual drugs appear. The advantage of the iodoplatinate spray is that, though the resulting colours are only varia-tions of brown, a spot may be scraped from the plate and reduced with sodium sulfite [ 9] after which the drug may be appropriately redissolved. When sufficient material is available, simultaneous chromatograms can be run; the separated compounds localized on one chromatogram with a colour spray and the geometrically equivalent spots scraped from the unsprayed plates for subsequent solution. The solutions thus obtained may be used directly for gas chromatography or filtered and concentrated on a porous triangle of potassium bromide [ 10] for infra-red identification. Evaporation takes place preferentially at the apex of the porous triangle, depositing the sample at the tip which is then removed and pressed directly into a transparent disc suitable for infra-red spectrophotometry.

All of the drugs under consideration in this report except ibogaine have been previously investigated using thin-layer chromatography. The U.S. Food and Drug Administration [ 11] used an alcohol: ammonia (25 %)

(1:3) solvent system for dimethyltryptamine, giving an R f value of about 0.60, and an acetone: chloroform (4:1) system [ 12] which have an R f value for dimethyltryptamine of 0.11.

Cochin and Daly [ 13] reported R f values for mescaline using four different systems on silica gel plates:

  1. Ethyl alcohol: pyridine: dioxane: water (50:20:25:5)

R f = 0.18

  1. Ethyl alcohol: acetic acid: water (60:30:10)

R f = 0.81

  1. Ethyl alcohol: dioxane: benzene: ammonia (25%)(5:40:50:5)

R f = 0.30

  1. Methyl alcohol: n-butanol: benzene: water (60:15:10:15)

R f = 0.30

Leung et al. [ 14] have found n-propanol; ammonia (5%)(5:2) and n-butanol: acetic acid: water (2:1:1) to serve as effective mobile phases in identifying psilocybin (R f = 0.15 for both systems).

Many thin-layer systems have been reported for the identification of LSD. Sandoz Pharmaceuticals [ 15] used dichloromethane: methanol (93:7) for LSD-base (R f =0.6) while the U.S. Food and Drug Administration [ 16] prefers acetone: chloroform (4:1) as a developing system, which gives an R f value for LSD of about 0.4. Dal Cortevo et al. [ 17] obtained good results with 1,1,1-trichloroethane: methanol (90:10); their R f value for LSD-base was 0.55. E.G.C. Clarke [ 18] used a methanol-ammonium hydroxide system (100:1.5) and reported R f values of 0.04 for psilocybin, 0.23 for mescaline, 0.34 for dimethyltryptamine and 0.60 for LSD.

We found that most effective separations could be obtained if two systems were used. A benzene: ethyl acetate: diethylamine [ 19] (7:2:1) solvent system proved useful in separating dimethyltryptamine, mescaline, ibogaine, and LSD; while an n-propanol-ammonia (5%) [ 14] (5:2) system successfully separated psilocybin from the remaining four drugs. R f values for these systems are listed in Table 2. The R f values stated may only be considered approximate since many factors are involved in the affecting conditions. Therefore, for accurate identification and separation, standard samples must be simultaneously run along with the substance/mixture in question.

TABLE 2

System: benzene: ethyl acetate: diethylamine (7:2:1)

Compound

R f value

Colour with p-dimethylamino- benzaldehyde

Psilocybine
0.00
Navy blue
Mescaline
0.16
Light brown
LSD
0.29
Clear blue
Dimethyltryptamine
0.42
Deep blue-green
Ibogaine
0.55
Dirty yellow-green

System: n-propanol - ammonia (5%) (5:2)

Compound

R f value

Psilocybine
0.18
Mescaline
0.55
LSD
0.71
Dimethyltryptamine
0.70
Ibogaine
0.72

DISCUSSION

We believe that running a gas-chromatograph of the material recovered from a thin-layer chromatograph is a good method of identification for court purposes; especially if the thin-layer chromatograph can also be characterized by distinctive colour formation. When adequate amounts of material are available, infra-red characterization of the compounds recovered from the gas chromatograph and/or the thin-layer chromatogram is always advantageous.

References

001

M. S. Fish, N. M. Johnson and E. C. Horning, J. Am. Chem. Soc. 77, 5892 (1955).

002

S. Szara, Federation Proc. 20, 885 (1961).

003

I. Stevenson and T. W. Richards, Psychopharmacologia 1, 241 (1960).

004

A. Hofmann, R. Heim, A. Brack, H. Kobel, A. J. Frey, H. Ott and F. Troxler, Helv. Chim. Acta 42, 1557 (1959).

005

J. A. Schneider and E. B. Sigg, Ann. N.Y. Acad. Sci. 66, 765 (1957).

006

M. Lerner, U.N. Bulletin on Narcotics, XIX, 3 (1967).

007

E. C. Horning, M. G. Horning, N. Ikekawa, E. M. Chambaz, P. I. Jaakonmeki and C. J. W. Brooks, J. Gas Chromatog. 5, 283 (1967).

008

M. Lerner, A. L. Mills, S. F. Mount, J. of. Forensic Science 8, 126 (1963)

009

P. Schweda, personal communication, 1967.

010

H. Garner, Pittsburgh Conference on Analytical Chemistry, March, 1967; the porous triangles of potassium bromide are marketed under the trade-name "Wick-Stick" by the Harshaw Chemical Co.

011

J. C. Brucciani, private communication, Food and Drug Administration, U.S. Department of Health, Education and Welfare, 1965.

012

E. Beyer and D. D. Dechert, private communication, Food and Drug Administration, U.S. Department of Health, Education and Welfare, 1967.

013

J. Cochen and J. W. Daly, Experientia, 18, 294 (1962).

014

A. Y. Leung, A. H. Smith and A. G. Paul, J. Pharm. Sci. 54, 1576 (1965).

015

Sandoz Pharmaceuticals, private communication, 1965.

016

T. G. Alexander, private communication, Food and Drug Administration, U.S. Department of Health, Education, and Welfare, 1966.

017

L. A. Dal Cortivo, J. R. Broich, A. Dihrberg and B. Newman, Anal. Chem. 38, 1959 (1966).

018

E. G. C. Clarke, J. of Forensic Science Society 7, 46 (1967).

019

D. Waldi, K. Schnackerz and F. Munter, J. Chromatog. 6, 61 (1961).