LSD analysis in seizures

Abstract

In the United States, d-Lysergic acid diethylamide tartrate (LSD) has become an important offshoot of the narcotics traffic. "Acid" as it is commonly called is the most powerful hallucinatory drug known; as little as 20 micrograms can be distinguished by most people from a placebo and 100 micrograms is considered an average dose.

Details

Author: Melvin LERNER
Pages: 39 to 45
Creation Date: 1967/01/01

LSD analysis in seizures

Melvin LERNER
U.S. Customs Laboratory, Baltimore (Maryland)

In the United States, d-Lysergic acid diethylamide tartrate (LSD) has become an important offshoot of the narcotics traffic. "Acid" as it is commonly called is the most powerful hallucinatory drug known; as little as 20 micrograms can be distinguished by most people from a placebo and 100 micrograms is considered an average dose.

LSD was synthesized by A. Stoll and A. Hofmann of the Sandoz Laboratories in 1938 but it was not until April 1943 that Dr. Hofmann, in what is now a classic case of accidental discovery, found that microgram amounts of d-lysergic acid diethylamide tartrate produced " a not unpleasant delirium which was marked by an extreme degree of fantasy. In a sort of trance with closed eyes (I found the daylight unpleasantly glaring), fantastic visions of extraordinary vividness accompanied by a kaleidoscopic play of intense coloration continuously swirled around me. After two hours this condition subsided." [1] Later Dr. Hofmann took 250 micrograms of LSD and observed "visual distortions, the faces of those present appeared like grotesque colored masks confusion alternating with a clear appreciation of the situation; at times standing outside myself as a neutral observer and hearing myself muttering jargon or screaming half-madly." [1]

1. Gas chromatogram of LSD

Full size image: 81 kB, 1. Gas chromatogram of LSD

2. Gas chromatogram of trimethylsilyl derivative of LSD

Full size image: 67 kB, 2. Gas chromatogram of trimethylsilyl derivative of LSD

LSD, at present, seems to be obtainable in the same illicit market as marihuana and is being used by an increasing proportion of marihuana users. A sugar cube can be bought impregnated with 100-200 micrograms of LSD which will assure the user an eight-hour trip into an hallucinatory world. Tolerance to LSD is rapidly acquired and three or four days abstinence must elapse before tolerance for the drug is lost. This phenomenon has led to the practice of taking LSD only on week-ends while smoking marihuana the rest of the week.

The identification and quantification of LSD in seizures is complicated by the fact that LSD is not an extremely stable compound; both moisture and ultraviolet light produce breakdown products which respond to some colorimetric tests in a manner similar to LSD itself; in addition, illegal methods of manufacture often produce a product only part of which is LSD; the remainder a hodge-podge of compounds containing the lysergic acid moiety.

Thin-layer chromatography is extremely useful both for identification and for separation. The method of Sandoz Pharmaceuticals [2] involves extraction of the LSD base with chloroform from an aqueous solution made alkaline with sodium bicarbonate. Ten to twenty micrograms of LSD base are spotted on a silica gel G plate (0.25 mm thick) and placed in a chromatography tank with dichloromethane-methanol (93: 7).

LSD base has an R f value of about 0.6 and a blue fluorescence under long-wave ultraviolet light. The U.S. Food and Drug Administration uses a similar method [3] but prefers chloroform-acetone (1:4) as a developing solvent; LSD base has an R f value of about 0.4-0.5 in this system. Indicative results with thin-layer chromatography may be obtained with as little as 1 microgram of LSD. The separated spot, containing LSD, may be sprayed with a color-producing reagent such as p-dimethyaminobenzaldehyde (0.5 g) in hydrochloric acid (37%) (5 ml ) and ethanol (95 ml) [4] . A blue-violet spot is produced with LSD and other ergottype alkaloids.

3. Gas chromatogram of trimethylsilyl derivative of LSD after direct extraction with chloroform-methanol (9:1)

Full size image: 66 kB, 3. Gas chromatogram of trimethylsilyl derivative of LSD after direct extraction with chloroform-methanol (9:1)

The blue p-dimethylaminobenzaldehyde color can itself be used for LSD quantitation [5] . The blue color from a hundred micrograms of LSD dissolved in a freshly prepared solution of p-dimethylamonibenzaldehyde (1.2 mg) in sulfuric acid (6-ml) and water (4-ml) may be read in a suitable colorimeter or spectrophotometer at 550 mµ against a reference standard prepared in the same manner; the color should be read after standing 20 minutes in subdued light.

In pure form, LSD can be determined from its ultraviolet absorption maximum, in mildly acidic aqueous solution, at 312 mµ but many substances interfere and the method is not very sensitive. The most sensitive analytical method for LSD is the spectrophoto-fluoro- metric method [6] . The LSD is extracted from saltsaturated alkaline solutions into heptane containing 2 per cent isoamyl alcohol. The LSD is then returned to dilute hydrochloric acid solution, activated at 325 mµ and its fluorescence measured at 445 mµ; as little as 0.001 micrograms of LSD can be determined. This method is not specific for LSD and determines most ergot-type alkaloids and LSD breakdown products along with the LSD.

The rigid requirements of court testimony, as applied to LSD, have been competently evaluated by Genest & Farmilo [7] . They amplified the value of thin-layer chromatography by using it both before and after intensive ultraviolet (3660 A) irradiation of the LSD basespot; after irradiation an additional spot, which reacted to the p-dimethylaminobenzaldehyde spray, was produced having an R f value less than that of the unchanged LSD; this helps distinguish LSD from the more important ergot alkaloids. Genest & Farmilo also enhanced the specificity of the spectrophotofluorometric method by using thin-layer chromatography for the separation, methanol elution of the spot, and subsequent spectrofluorometric analysis of the diluted methanol solution; a range of 0.01-0.4 microgram LSD/ml was preferred.

4. Gas chromatogram of trimethylsilyl derivative of LSD after direct extraction with chloroform-methanol (9:1), sugar bands in early portion of chromatogram

Full size image: 70 kB, 4. Gas chromatogram of trimethylsilyl derivative of LSD after direct extraction with chloroform-methanol (9:1), sugar bands in early portion of chromatogram

Our laboratory has supplemented the above procedures with the use of gas chromatography and infra-red. Radecka and Negam [8] were unable to directly apply gas chromatography to LSD and used the more stable hydrogenated LSD, their method of gas chromatography after hydrogenation was found by them to be inapplicable to a sugar cube impregnated with 50 micrograms of LSD (thin-layer was found useful for this amount). Contrary to the experience of Radecka and Negam, we have been able to subject LSD directly to gas chromatography. The difference between our results and those of Radecka & Negam may stem from our use of an all-glass gas chromatographic system which minimizes decomposition [9] . Figure 1 is a gas chromatogram we obtained from 30 micrograms of LSD. A 2% SE 52 column was used at 250 °C with an inlet temperature of 280°C; some decomposition products are evident in the gas chromatogram. For greater sensitivity and stability in gas chromatography, we now prefer to use the trimethylsilyl derivative of LSD. A good reagent for the silylation is bis (trimethylsilyl) acetamide [10] . The reaction takes place readily in dimethylformamide solution (dimethylformamide is an excellent solvent for LSD). No separation of the reaction products is required for gas chromatography as both mono-and disilyl-substituted acetamide give sharp peaks with low retention times. Figure 2 shows a gas chromatogram obtained from 0.2 microgram of the trimethylsylil derivative of LSD. A 2% SE 52 column was used with a column temperature of 245 °C and an inlet temperature of 280°C. For small amounts of LSD we find a direct solution in chloroform: methanol (9:1) gives somewhat better recoveries than the usual partition method. Our procedure for solids, such as a sugar cube impregnated with LSD, a tablet, or the contents of a capsule, is to pour 5-ml portions of chloroform-methanol (9:1) through the crushed solids contained in a filter paper. The filtration and all subsequent operations are conducted in subdued light. The solution is evaporated under partial vacuum (with a slow inlet flow of nitrogen). When the solution has evaporated to about 1.0 ml, it is transferred to a 1 ml beaker and evaporated to dryness.

5. Infra-red curve of the trimethylsilyl derivative of LSD (recovered from the gas chromatograph)

Full size image: 44 kB, 5. Infra-red curve of the trimethylsilyl derivative of LSD (recovered from the gas chromatograph)

6. The top of figure 6 is the infra-red curve of LSD base and the bottom of figure 6 is the infra-red curve of LSD

Full size image: 82 kB, 6. The top of figure 6 is the infra-red curve of LSD base and the bottom of figure 6 is the infra-red curve of LSD

7. Gas chromatogram of trimethylsilyl derivative of LSD after aqueous extraction with chloroform from sodium bicarbonate solution

Full size image: 116 kB, 7. Gas chromatogram of trimethylsilyl derivative of LSD after aqueous extraction with chloroform from sodium bicarbonate solution

Two successive portions, 100 µl each, of chloroformmethanol (9:1) are then used to dissolve the LSD in the 1-ml beaker and transfer it to a small centrifuge tube; this operation minimizes the sugar content of the final solution and, at the same time, permits the use of the convenient micro-volume of the centrifuge tube tip. The transfer is conveniently performed with a hypodermic syringe; a small plug of cotton in the Luer tip serves as an intake filter (the liquid hold-up is approximately 6 µl). The 200 µl of solution in the centrifuge tube is then evaporated to dryness under partial vacuum (with a slow inlet flow of nitrogen through a capillary tube).

The residue is dissolved in I0 microliters of dimethylformamide, 30 microliters of bis (trimethylsilyl) acetamide is added, the centrifuge tube is stoppered and placed in a 55° C water bath for 30 minutes. One microliter is then withdrawn for gas chromatography. Figure 3 shows the gas chromatogram obtained from a tablet containing 50 micrograms of LSD; this represents onefortieth of the total amount recovered. Figure 4 shows a similar gas chromatogram demonstrating the presence of sugar as trimethylsilyl derivatives.

When an infra-red curve is required, one or more 10 µl portions can be injected into the gas chromatograph and recovered on potassium bromide crystals [11] . Figure 5 shows the infra-red curve of 50 micrograms of the trimethylsilyl derivative of LSD as obtained fromthe gas chromatograph. For reference purposes Figure 6 gives, on top, the infra-red curve of LSD base and, on the bottom, the infra-red curve of LSD.

When the LSD is in water solution, or, if it is desired to completely separate the LSD from water-soluble materials such as sugar, we use a standard chloroform extraction of the LSD from sodium bicarbonate solution. Extreme care must be taken that the chloroform is dry before subsequent evaporation; anhydrous sodium sulfate held in the outlet of the separating funnel by a small pledget of cotton will insure dryness. The dry chloroform extract is made acid with a methanol solution of tartaric acid and the chloroform evaporated under partial vacuum (with a slow inlet flow of nitrogen). When the solution has evaporated to about 0.5 ml, it is transferred to a small centrifuge tube and evaporated to dryness. The residue is dissolved in 10 microliters of dimethylformamide, 30 microliters of bis (trimethylsilyl) acetamide is added, the centrifuge tube is stoppered and placed in a 55°C water bath for 30 minutes. One microliter is then withdrawn for gas chromatography. Figure 7 shows a gas chromatogram obtained from one-fortieth of the total extract from an aqueous solution containing 70 micrograms of LSD.

We have also made an LSD derivative suitable for gas chromatography by the use of trifluoroacetic anhydride but this derivative is not stable and is inferior for analytical use to the trimethylsilyl derivative.

Our laboratory routinely uses thin-layer chromatography (before and after u.v. irradiation), p-dimethylaminobenzaldehyde color formation and gas chromatography of the trimethysilyl derivative for the identification and quantitation of LSD. When sufficient quantities are available, infra-red curves of the trimethylsilyl derivative of LSD (recovered from the gas chromatograph) are also obtained.

References

001

Stoll, W. A., Schweiz. Arch. Neurol. Psychiat., 60, 279 (1947).

002

Sandoz Pharmaceuticals, private communication, 1965.

003

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

004

Hellberg, H., Acta. Chem. Scand 11, 219 (1957).

005

Vining, L.C. and Taber, W.A., Can. J. Microbiol., 5.441 (1959).

006

Axelrod, J., Brady, R. O., Witkop, B. and Evarts, E.V., Nature, Lond., 178, 143 (1956).

007

Genest, K. and Farmilo, C. G., J. Pharm. Pharmacol., 16, 250 (1964).

008

Radecka, C. and Negam, I. C., J. Pharm. Sci., 55, 1861 (1966).

009

F. & M. Model 400 Bio-Medical Gas Chromatograph, flame ionization detector.

010

Klebe, J. F., Finkbeiner, H. and White, D. M., J. Am. Chem. Soc., 88, 3390, 1966.

011

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