Thin-layer and gas chromatographic analysis of hashish samples containing opium

Sections

Introduction

Experimental
Results and discussion
Acknowledgements

Details

Author: D. ESKES,* , A. M. A. VERWEY**, A. H. WITTE**
Pages: 41 to 47
Creation Date: 1973/01/01

Thin-layer and gas chromatographic analysis of hashish samples containing opium

D. ESKES,*
A. M. A. VERWEY**
A. H. WITTE**

Introduction

In the Netherlands, analyses for forensic purposes are performed both at the Laboratory of the Municipal Police of Amsterdam and at the Forensic Science Laboratory of the Ministry of Justice at the Hague.

At present, the number of cases related to infringements of the Dutch law on narcotic drugs is about two hundred a month, giving rise to the investigation of several hundreds of samples. In 75 per cent of these cases, the samples proved to be either marihuana or hashish.

The forensic examination of marihuana is based on the microscopic identification of the specific botanical elements of Cannabis sativa L., while the presence of cannabis resin is shown by the Duquenois reaction and by thin-layer chromatography. In this way, some information may also be obtained about the amounts of THC and THC acid in the hashish and marihuana samples. In certain cases, the content of some cannabinoids is determined by gas chromatography.

Though we are often requested to answer the question whether certain strong and strange effects experienced by hashish consumers, may have been caused by mixing the hashish with more dangerous drugs, such as S.T.P., mescaline or opium, we were never able to prove the presence of the above mentioned drugs in the specific cases. It was invariably found that these hashish samples did contain a high amount of the psychotropically active tetrahydrocannabinol and the effects could therefore be ascribed to an overdose of tetrahydrocannabinol.

However, in recent years there has been much talk in this country about hashish samples containing opium (1, 1 a), but little information was available in literature (2, 2 a). It was not until March 1971, after searching for one year, that we received, both in Amsterdam and The Hague, our first samples of hashish containing opium. In most of these samples, tea leaves could be detected and caffeine was found. Since that time we have received regularly small numbers of such samples, but much greater numbers were received in the months of August and September 1971.

In the figure below the numbers of these hashish samples received by us per month is plotted for that year. When we received these samples, the question was raised whether they came from the same source or whether they had been prepared at different places, either abroad or in this country.

In order to answer this question, the content of cannabinol, cannabidiol, tetrahydrocannabinol, morphine and caffeine in these samples was determined. The analysis of the first four substances was done by gas chromatography, while the caffeine content was determined by a combined thin-layer/spectrophotometric method of analysis.

* Laboratory of the Municipal Police of Amsterdam, Elandsgracht 117, Amsterdam.

** Forensic Science Laboratory of the Ministry of Justice, Volmerlaan 17, Rijswick.

Number of hashish samples received by us monthly in 1971

Full size image: 4 kB, Number of hashish samples received by us monthly in 1971

Experimental

A. PRELIMINARY INVESTIGATIONS

All the samples of hashish which we received from police seizures (about 250 monthly) were checked for opium alkaloids by means of thin-layer chromatography. To this end, about 300 mg of powdered air dried hashish was shaken with one ml of ethanol 70 per cent. Of this solution, about 50μl were spotted on a Merck Silica gel G plate. The plate was developed (3) in a saturated chamber with a mixture of toluene-acetone-ethanol-ammonia 25% (45 + 45 + 7 + 3) and subsequently sprayed with an iodo platinate solution. At least four of the most important opium alkaloids (including morphine) could be detected in this way. In the quantitative investigations, only morphine was determined. About 40 of the 65 samples found positive for opium were analysed during these investigations, using the criterion that only samples weighing more than 1,000 mg were analysed. Moreover, on microscopical examination of these hashish samples it appeared that most of them had a brittle layered structure in which tea leaves could be found. Some other samples were of a hard and firm constituency and tea leaves were not found in them. For this reason, all the hashish samples were examined qualitatively for the presence of caffeine. This analysis was done in the same way as for the detection of morphine. The plate was sprayed with Dragendorff reagent.

B. DETERMINATION OF THE CONTENT OF SOME CANNABINOIDS IN HASHISH

Gas chromatography is the method of choice for the quantitative determination of hashish constituents (4, 5, 6, 7). In this study, a Perkin-Elmer F 11 gas chromatograph, connected to a Perkin-Elmer D 26 integrator, was used in the experiments. The chromatographic conditions were: glass column 2 metres in length, filled with 2 % OV-17 on Chromosorb W-AW-DMCS; nitrogen as carrier gas at a flow rate of 40 ml/min; oven temperature 220 [ 0] C; injection port temperature 230 [ 0] C. Under these conditions, a good separation of cannabinol (CBN), from cannabidiol (CBD) and from tetrahydrocannabinol (THC) was obtained. Relative response factors for CBD, CBN and THC were estimated using squalane as the internal standard and weighed amounts of pure cannabinoids; these factors being known, the amounts of CBD, THC and CBN can easily be estimated.

Sample preparation: Prior to gas chromatography the samples (air dried) were pulverized in a mortar and 100 mg were weighed into an erlenmeyer flask, and shaken for 15 min with 10 ml of n-hexane. The solution was filtered through glass wool and the n-hexane evaporated in a drying box at 40 [ 0] C. The extract was weighed, then dissolved in n-hexane containing a weighed amount of squalane. This solution was again filtered through filter paper into a 10 ml measuring flask. Three μl of this solution were injected into the gas chromatograph at least twice. The results are given in table I. For full details of this analysis, we refer to (8).

C. DETERMINATION OF THE AMOUNT OF MORPHINE IN HASHISH

In the literature, the use of gas chromatography for the quantitative determination of morphine is well known (9, 10, 11, 12). In this work the same gas chromatographic method was used as in B. Chromatographic conditions were as follows: glass column two metres in length filled with 3 % SE-30 on Chromosorb W-AW-DMCS; nitrogen as carrier gas at a flow rate of 30 ml/min; oven temperature 200 [ 0] C; injection port temperature 210 [ 0] C.

Sample preparation: The samples were pulverized in a mortar, 100 mg were weighed into a small beaker to which 2 ml of glacial acetic acid were added. The acetic acid was partly evaporated on a water bath, while stirring the slurry. Next, the slurry was taken up in 10 ml of water, about 50 mg of ammonium nitrate were added and the pH of the solution was adjusted to 9.0 with ammonia 10 %, using a pH meter. The aqueous solution was then shaken in an extraction funnel with 10 ml of ether, in order to remove various extraneous substances. The layers were separated and the ether layer was checked gas chromatographically for morphine. No morphine could be detected. The aqueous layer, after filtration through filter paper, was shaken for 5 min with 10 ml of a mixture of dichloromethane-isopropanol (9 + 1). After separation of the layers, the organic layer was filtered through filter paper and the solvent, to which a known amount of the internal standard quinine had been added, was evaporated under a stream of nitrogen at room temperature. The residue, which had to be kept free from moisture, was taken up in 100 μl of N.0-bis-(trimethylsilyl)-acetamide (BSA). This solution was then allowed to stand for half an hour, after which 3 μl of the thoroughly homogenized solution were injected into the gas chromatograph at least twice.

Relative response factors for morphine, in a concentration range to be expected in the experiments, were estimated using morphine hydrochloride solutions in water and the extraction procedure described above. After adding a known amount of quinine to the organic solvent and evaporating under a stream of nitrogen, the residue was taken up in 100 μl BSA. Response factors were estimated by injecting 3 μl into the gas chromatograph several times. The reproducibility of the morphine determination was found to be better than 5 per cent. The results are to be found in table I.

TABLE I

Results of the analysis of some cannabinoids in hashish samples containing opium

Number

Date

% CBD

% THC

% CBN

Ratio of % CBD % THC % CBN resp. to total of percentages of CBD, THC and CBN

1.
23/4
1.54 4.68 0.36
21:73:7
2.
4/5
1.42 4.39 0.38
23:71:6
3.
11/5
1.31 4.54 0.30
21:73:6
4.
11/8
1.41 4.80 0.44
21:72:7
5.
14/8
1.67 4.99 0.54
23:69:8
6.
17/8
1.81 6.15 0.43
21:73:6
7.
27/8
1.54 4.58 0.35
23:71:6
8.
27/8
1.45 4.51 0.35
23:71:6
9.
27/8
1.55 5.13 0.43
22:72:6
10.
31/8
1.31 4.15 0.41
23:70:7
11.
31/8
1.11 3.35 0.32
23:70:7
12.
6/9
1.37 4.24 0.36
23:71:6
13.
13/9
1.60 5.10 0.47
22:71:7
14.
13/9
1.71 5.44 0.5l
22:71:7
15.
16/9
1.47 6.27 0.51
18:76:6
16.
21/9
2.63 7.96 0.71
23:70:7
17.
11/3
1.17 3.06 0.56
24:64:12
18.
1/6
1.01 2.48 0.47
26:63:11
19.
16/7
1.14 3.04 0.51
24:65:11
20.
16/7
1.43 3.99 0.59
24:66:10
21.
17/8
1.12 3.03 0.51
24:65:11
22.
17/8
1.01 3.02 0.35
23:69:8
23.
18/8
1.39 3.96 0.49
24:68:8
24.
23/8
1.17 3.38 0.35
24:69:7
25.
23/8
1.29 3.33 0.49
25:65:10
26.
23/8
1.32 3.32 0.47
26:65:9
27.
30/8
1.13 3.30 0.39
23:68:9
28.
22/9
1.51 3.43 0.40
28:64:8
29.
6/10
1.47 3.24 0.38
29:64:7
30.
25/10
1.35 3.09 0.37
28:64:8
31.
9/3
1.49 2.77 0.60
31:57:12
32.
22/3
1.12 2.16 0.59
29:56:15
33.
29/4
1.31 2.33 0.69
30:54:16
34.
24/9
1.66 2.77 0.56
33:56:11
35.
20/9
1.86 3.74 0.56
30:61:9
36.
28/4
1.11 10.06 0.97
9:83:8
37.
17/5
0.33 3.21 0.12
9:88:3
38.
24/9
0.70 4.44 0.23
13:83:4
39.
23/11
2.86 10.30 0.45
21:76:3
40.
23/11
1.46 6.18 0.18
19:79:2

D. DETERMINATION OF THE AMOUNT OF CAFFEINE IN THE SAMPLES

The content of caffeine was determined in the following way. Four hundred mg of finely powdered hashish were weighed into a weighing bottle and one ml of ethanol 70 per cent was added. The stoppered bottles were placed in a thermostat bath at 70 °C for one hour. After this, the bottles were set aside for several hours, with occasional shakings. Six μl of this solution were spotted on a Merck F254 plate and the plate was developed with toluene-acetone-ethanol-ammonia 25 per cent (45 + 45 + 7 + 3) in a cylinder jar. After development, the plate was examined under U.V. light and the caffeine spot marked and scraped off. This silica gel spot was transferred to a centrifuge tube and washed with 3 ml of ethanol 70 per cent, the silica gel was separated by centrifuging and the solution transferred to a cell of 10 mm path length. The extinction was measured at 279 μm using a Zeiss Opton spectrophotometer. The amount of caffeine was determined with the aid of a calibration graph. Reproducibility was 5 per cent or better. The results are given in table II.

TABLE II

Results of the analysis of morphine and caffeine in hashish samples containing opium

Number

% Morphine

% Caffeine

Extract weight in mg

Number

% Morphine

% Caffeine

Extract weight in mg

1.
0.16 0.87 10.2
21.
0.24 0.84 10.6
2.
0.20
pos.*
10.4
22.
0.25 0.73 9.3
3.
0.25 0.90 11.7
23.
0.22 0.70 10.7
4.
0.16 0.83 10.1
24.
0.22 0.82 13.5
5.
0.21 0.84 13.5
25.
0.27 0.83 11.6
6.
0.21
pos.
9.6
26.
0.22 0.90 10.2
7.
0.15 0.58 13.3
27.
0.15
0.90(0.94)* * *
10.7
8.
0.12 0.63 12.7
28.
0.18 0.67 11.6
9.
0.22
pos.
12.1
29.
0.11 0.83 11.3
10.
0.18 0.68 13.0
30.
0.11 0.82 10.9
11.
0.14 0.58 11.1
31.
0.23 0.84 21.0
12.
0.15 0.58 11.1
32.
0.17 0.84 8.4
13.
0.16
pos.
12.6
33.
0.09 0.65 8.9
14.
0.16
pos.
12.6
34.
0.08 0.82 11.4
15.
0.05
pos.
14.9
35.
0.13 0.82 17.3
16.
0.05
neg.* *
10.3
36.
0.01
neg.
19.3
17.
0.17 0.80 11.2
37.
0.01
neg.
6.6
18.
0.10 0.84 8.9
38.
0.05
neg.
10.3
19.
0.20 0.73 8.8
39.
0.05
neg.
19.5
20.
0.23 0.82 10.0
40.
0.03
neg.
18.4

* The amount of caffeine was not determined quantitatively, the qualitative reaction on caffeine was found positive.

** The amount of caffeine was not determined, as the qualitative reaction on caffeine was found negative.

*** In parentheses are the results of the gravimetric analysis of caffeine.

In order to check the accuracy of the method described above and to obtain some material for IR identification, the amount of caffeine in one sample was determined gravimetrically (13). For this purpose 10 gm of hashish (finely powdered) were taken up in 12 ml of ammonia 10 per cent. This solution was vigorously shaken in a separation funnel with 100 ml of chloroform. After separation of the layers, the ammoniacal aqueous layer was again extracted twice with chloroform. Then the chloroform layers were collected and the chloroform evaporated on a water bath. The residue was dissolved with mild heating in 20 ml ethanol 30 per cent and the solution obtained was diluted with 40 ml of water. This solution was allowed to stand for 24 hours. Afterwards, it was filtered through water moistened filter paper and the solvent was evaporated from the filtrate. An impure non-crystalline substance was obtained and it was purified in the following way (14). A hundred ml of boiling water was added to the residue and after cooling, 30 ml of a 1 per cent potassium permanganate solution were added. After one hour, a 3 per cent hydrogen peroxide solution in 1 per cent acetic acid was added dropwise until the intense permanganate colour disappeared. The remaining solution was heated on a water bath for half an hour and filtered hot through filter paper. Subsequently, the solvent was evaporated on a water bath. The residue obtained was taken up in 25 ml of chloroform, filtered through filter paper and the chloroform evaporated on a water bath. The pale yellow residue was dried to constant weight in a drying box at 105 °C. The purity of caffeine was checked by thin-layer chromatography and IR. A good agreement was found between the gravimetric and the thin-layer spectrophotometric methods of analysis (see table II).

Results and discussion

In tables I and II, the results of the various analyses are given. Table I also gives the dates when we received the samples and the weights of the samples after extraction with n-hexane. Besides this, the respective ratios of the percentages of CBD, THC and CBN to the total percentages of CBD, THC and CBN are given. Thus a series of numbers is obtained for a hashish sample which is independent of adulterating substances in the hashish (8). This series of figures can also be used for classification purposes. In table I the samples are classified on the basis of their content of THC and the above mentioned ratios. It appeared that four groups could be distinguished according to this criterion, namely: the samples 1-16, 17-30, 31-35 and 36-40. In the last group no caffeine was found, while the variation in both the caffeine and the morphine content is almost equal in the first three groups. However, it must be noted that as regards the THC content and the ratios of CBD to THC and to CBN there is a gradual change from this first group to the second group.

It is quite interesting that only four groups were found. A much greater variation in the ratios and the THC content was to be expected, as these hashish samples were probably prepared in this country (8) and it is assumed that no great amount of hashish was available for the preparation of this type of hashish.

In general, the THC content of these hashish samples is quite usual and samples with about the same THC content were found to form about fifty per cent of 200 random hashish samples (8).

The morphine content in the samples is found to be low, ranging from 0.01 per cent to 0.27 per cent. Considering this type of hashish was to be smoked in a dose of about 200 mg hashish per cigarette, not more than 0.6 mg morphine would then be available in each. This dose is considerably lower than the dose usually taken when smoking opium. Besides this, the chance of pyrolysing morphine in a cigarette will be greater than when opium is smoked in the usual way because of the higher temperature in the cigarette. In our opinion, the dose of morphine is too low to cause morphine addiction in the normal consumption of this kind of hashish.

It is, however, an open question whether or not a euphoric effect, due to morphine, is to be expected when smoking this kind of hashish. As far as we know, nothing is known about a possible interaction of the physiological effects caused by THC and morphine in man.

Nevertheless, the presence of tea leaves and the rather high content of caffeine in almost all the samples investigated (table II), as well as the easy way in which a suspension in water could be made with this kind of hashish, all point to the fact that the hashish was intended to be consumed as a drink. (As tea normally contains about 2 per cent of caffeine, the hashish must have been mixed with equal quantities of powdered tea leaves in the preparation process.) The presence of the small quantities of opium in these hashish samples may be explained by assuming that, during the preparation of this type of hashish, the same apparatus was used as in the preparation of smoking opium introducing, by this mode of operation, the small amounts of morphine as a mere impurity.

Acknowledgements

The authors wish to express their gratitude for the careful help of Miss Meggie M. Brunsmann, Miss Atie de Bruijne, Miss Georgine J. A. Bulterman, Mrs. Marja W. Smulders and Miss Rita M.Th. Twente in performing the experiments.

References

001

M. Schouten, "Marihuana en Hasjiesj ", A. W. Bruna, Utrecht Antwerpen, 1969, pp. 44.

002

H. C. Honnecker, Deut. Med. Wochenschrift, 95, pp. 2129-2131 (1970).

003

E. Stahl, "Chromatographische und Microscopische Analyse von Drogen ", G. Fischer Verlag, Stuttgart, 1970, pp. 52-56.

004

. Betts and P. J. Holloway, J. Pharm. Pharmcol., 19, Suppl. 97S-102S (1967).

005

M. Lerner and J. T. Zeffert, Bulletin on Narcotics, XX, 2, pp. 53-54.

006

K. D. Parker, J. A. Wright, A. F. Halpern and C. H. Hine, Bulletin on Narcotics, XX, 4, pp. 9-14.

007

P. Lerner, Bulletin on Narcotics, XXI, 3, pp. 39-42.

008

A. M. A. Verwey and A. H. Witte, Pharm. Weekblad, 107, pp. 153-164 (1972).

009

E. Brochmann-Hanssen and A. Baerheim Svendsen, J. Pharm. Sci. 52, pp. 1134-1136 (1963).

010

N. Ikekawa, K. Takayama, E. Hosoya and T. Oka, Anal. Biochem. 28, pp. 156-163 (1969).

011

A. Robinson and F. M. Williams, J. For. Sci. 16, pp. 135-139 (1971).

012

E. P. J. van der Slooten, H. J. van der Helm and P. J. Geerlings, J. Chromatogr, 60, pp. 131- 133 (1971).

013

Ned. Pharmacopee, 6 ed. Staatsdrukkerij en Uitgeversbedrijf, 's-Gravenhage; 1958, pp. 225.

000

Warenwet Koffie en Theebesluit, 14 ed. Tjeenk Willink, Zwolle, 1959, pp. 90.