ABSTRACT
Introduction
Discussion
Experimental
Results and discussion
Conclusions
Author: P. B. BAKER, T. A. GOUGH, B. J. TAYLOR
Pages: 31 to 40
Creation Date: 1980/01/01
Samples taken from seizures of illicitly imported cannabis and cannabis resin of known geographical origin have been examined by thin-layer chromatography. The chromatographic characteristics were considered in conjunction with the gross physical appearance of the materials and it was found possible to discriminate between samples of different origin. Thus by carrying out thin-layer chromatography and a visual inspection on a sample of unknown provenance, an opinion as to its geographical origin can be offered. Extensive use is made of a reference collection of samples.
Although Cannabis 1 is grown in many parts of the world, there is only a relatively small number of countries from which supplies of the illicit products (cannabis, cannabis resin and liquid cannabis) reach the United Kingdom. A means of identifying the geographical origin of samples of any of these materials would be extremely useful in the ensuing criminal investigations and in aiding international control [ 1] [ 2] [ 3] [ 4] .
1 The term Cannabis in this paper refers to Cannabis sativa L.; cannabis to marijuana; and cannabis resin to hashish.
It is well known that there are wide variations in the relative amounts of cannabinoids in Cannabis and that furthermore some plants contain additional cannabinoids [ 5] . Many factors have been considered in attempts to explain these variations. These include the genetic characteristics of the seedstock, the environment in which the plant is grown, the maturity, sex and part of the harvested plant and the time which has elapsed between harvesting and chemical analysis, as well as the conditions of storage of the plant. The mode of preparation will additionally affect the cannabinoid content, particularly in the case of liquid cannabis.
Several groups of workers are of the opinion that the major factor governing the amounts of cannabinoids in a plant is genetic [ 1] [ 6] [ 7] [ 8] [ 9] [ 10] . Bearing this in mind and assuming that seeds are freely transferred from one country to another, Ohlsson [ 9] concludes that there is little valid basis for attempts to correlate the cannabinoid content with country of origin. In addition, the majority of seizures of illicit cannabis received in the Laboratory of the Government Chemist contain fertile seeds, thus indicating little need for inter-country transfer of seeds. The effect of the environment upon the cannabinoid content of the products from Cannabis is widely considered to be small [ 11] [ 12] , but although the ratios between the cannabinoids may remain unchanged, the absolute quantities may vary [ 13] .
There is no conclusive evidence to show that changes in environment do not affect the cannabinoid content of successive generations of seeds of common stock. However, there are other authors who consider that the environment plays a major part in the determination of the cannabinoid content of Cannabis products [ 14] [ 15] [ 16] .
Until recently the male Cannabis plant was thought to be less pharmacologically active than the female. The main active constituent is Δ-9-tetrahydrocannabinol (THC), but this compound and several other cannabinoids are found in similar amounts in both sexes of plants grown under the same conditions [ 9] [ 12] [ 17] . In contrast, the cannabinoid ratios vary within a plant at different stages of its growth [ 11] [ 18] [ 19] [ 20] . Neither of these latter two factors is of great concern in examining cannabis imported into this country since the vast. majority of such seizures are of fully mature female plant material bearing fertile seeds.
On storage of samples of Cannabis products, the THC content gradually decreases as a result of oxidation to cannabinol (CBN) [ 21] . Similarly, the 3-propyl homologue, tetrahydrocannabivarin (THV), gives rise to cannabivarin (CBV) [ 22] . The two products CBN and CBV are absent in fresh cannabis or cannabis resin [ 9] and are not therefore relevant to the geographical origin of the sample. Although the presence of CBV may indicate that the sample originally contained THV, it cannot be regarded as conclusive as there may be other precursors. The formation of CBN from THC is not quantitative [ 23] [ 24] , neither is it useful for determining origin since all Cannabis products will originally have contained THC.
Cannabis has been broadly classified into fibre-type or drug-type [ 7] [ 25] [ 26] . The former is characterized by a high cannabidiol (CBD) to THC ratio (usually > 5) and the latter by a much lower ratio (usually < 0.2). Small and co-workers have introduced further classification, one type with a CBD:THC ratio close to unity [ 27] and a second in which cannabigerol methyl ether (CBGM) is present [ 13] , following discovery of this compound by Yamauchi and others [ 28] . Although Fairbairn has suggested that the causes of the differences between fibre and drug plants may be geographic [ 29] , the existence of varieties intermediate between these extremes makes this simple classification of little value in the determination of the origin of a given sample. Marshman and others found considerable variation in the quality of Jamaican cannabis [ 19] . Further, some plants are found to contain no detectable amounts of CBD [ 30] [ 31] [ 32] ]. The cannabivarins have been found in samples from different parts of the world and although Turner reports different amounts for different geographical origins, he does not believe these compounds alone to be reliable indications of origin [ 33] . A summary of published data on the presence or absence of CBD and THV [table 1, references [ 34] [ 35] [ 36] [ 37] [ 38] confirms that these compounds alone are not sufficiently diagnostic to establish origin. However, de Faubert Maunder, as a result of thin-layer chromatographic (TLC) studies, is of the opinion that the presence or absence of CBD and its ratio to THC is a useful criterion for indicating the country of origin provided that the gross appearance of the sample is taken into account [ 30] .
Table 1
|
Observation |
Product a |
Country or region of origin |
Reference |
|---|---|---|---|
|
Absence of CBD
|
C
|
Brazil
|
[10, 31, 35] |
|
C
|
Costa Rica
|
[34] | |
|
C
|
Cyprus
|
[35] | |
|
C
|
India
|
[10]
|
|
|
C
|
Mexico
|
[34] | |
|
C
|
Nigeria
|
[35] | |
|
C
|
South Africa
|
[32] | |
|
C
|
Southern Africa
|
[30] | |
|
Presence of THV
|
CR
|
India
|
[36] |
|
CR
|
Nepal
|
[22, 36] | |
|
CR
|
Pakistan
|
[36] | |
|
C
|
Congo
|
[38] | |
|
C
|
India
|
[37] | |
|
Presence of CBD
|
C
|
Mostly countries producing CR
|
[4] |
|
CR
|
b
|
[4] |
aC= cannabis; CR = cannabis resin.
bCBD was present in all samples of CR, whatever the country of origin.
Jenkins and Patterson [ 3] and Davis and others [ 39] , using gas-liquid chromatography (GLC), measured the amounts of THC, CBD and CBN in samples of cannabis and cannabis resin of various origins. The former authors use a 3 co-ordinate graphical plot of the proportion of each cannabinoid present in the total cannabinoid content, the latter a graph of CBD vs THC + CBN. The loss of information due to the on-column decarboxylation of the cannabinoid acids is a disadvantage and neither of these studies takes into account the occurrence and variations of the cannabivarins. The picture is further confused by the many studies, including some of those discussed above, using plant material which, although derived from seed of known geographical origin, has been grown in a second entirely different environment.
Components of Cannabis other than the cannabinoids might be useful indicators of origin. Hood and Barry studied the headspace volatiles of cannabis and cannabis resin of different origins by GLC but found no correlation between volatile constituents and geographical origin [ 40] . This study used cultivated cannabis and reservations on the use of this material have already been noted by de Faubert Maunder [ 4] . Stromberg [ 41] [ 42] [ 43] and de Zeeuw and others [ 44] consider that the minor non-cannabinoid components of cannabis products could be an important factor in determining geographical origin of cannabis and cannabis resin, but neither has studied large numbers of samples of known origin.
In view of all these considerations it is not surprising that the relationship between cannabinoid content and geographical origin has remained unclear. From the point of view of such a study the Laboratory of the Government Chemist is in the fortunate position of having available an extensive and continually expanding collection of Cannabis products. These are all derived from seizures of illicit imports, whose country of origin is known in most cases. This has provided the opportunity to study a large number of samples and this report is an updated and extended review based on these findings [ 4] [ 45] .
Each sample was analysed as received and TLC was carried out within one month of importation. The most probable country of origin was assigned by taking into account information from the carrier, from Officers of Her Majesty's Customs and Excise and from the route of importation. In addition, some samples were received wrapped in paper or plastic bearing indications of their country of origin and others had identifying marks indicating their purported country of origin. Some of the latter were known to be spurious, particularly on cannabis resin and presumably were designed either to confuse law enforcement agencies or to indicate a higher quality product to the buyer.
TLC was the method of choice for this study as it provides a means of rapidly and visually analysing a number of samples simultaneously. Unlike the comparison of two samples in order to assess whether they are the same (or different), when quantitative analysis is essential, assessment of origin is qualitative, since the sample under study and the reference samples are, in fact, different. There is thus little point in using more powerful techniques such as high-performance liquid chromatography as the quantitative information provided is likely to confuse the picture. The TLC system was designed to give information on the cannabivarins in addition to the more commonly studied cannabinoids. The principal cannabinoids observed using this system are given in table 2. Sample preparation and detection were as previously described [ 47] [ 48] . The solvent system used was a mixture of chloroform (ethanol-free) and l,l-dichloroethane (15:10) with simple ascending TLC on 10 x 20 cm silica gel pre-coated plates with a layer thickness of 0.25 mm (E. Merck, Darmstadt, G.F.R., Art. No. 5729) [ 46] . Each extract was analysed within one hour of preparation. Twenty different samples of herbal cannabis and cannabis resin from several different countries were subjected to hot-solvent extraction to simulate the illicit preparation of liquid cannabis. The resulting chromatograms were compared with those of the starting material.
|
Cannabidiol |
R f |
Colour (after detection) |
|---|---|---|
|
Cannabidiol (CBD)
|
0.63 |
Yellow
|
|
Tetrahydrocannabinol (THC)
|
0.60 |
Red
|
|
Cannabinol (CBN)
|
0.55 |
Purple
|
|
Tetrahydrocannabivarin (THV)
|
0.50 |
Red
|
|
Cannabivarin (CBV)
|
0.45 |
Purple
|
|
Cannabigerol (CBG)
|
0.41 |
Orange
|
|
Cannabichromene (CBCh)
|
0.38 |
Purple
|
|
Cannabinoid acids
|
0.00-0.25
|
Red-orange
|
Source: [46].
The visual features of cannabis and cannabis resin from the countries supplying the major proportion of these materials to the United Kingdom are listed in table 3. Although a preliminary opinion as to the geographical origin of a Sample can often be given simply from the physical appearance of a single isolated sample, this may be considerably reinforced by visual comparison with reference samples of established origin. Similarly, comparison of the TLC patterns (described in table 4) with the TLC characteristics of material of known origin is valuable supporting evidence.
Visual characteristics are based on more than 1,600 samples received in the period 1974-1979. TLC characteristics are based on 600 samples in the period 1978-1979. The survey by de Faubert Maunder [ 4] is mainly based on samples received prior to mid-1972 and, although it covers a wider range of countries than this report, it lacks the detail available from the improved TLC system. Despite the limited number of countries now covered, these supply over 95 per cent of the Cannabis products intercepted by Officers of HM Customs and Excise over the period of this study. Although some countries show similar appearances and TLC patterns consistent with those found in the previous survey, there is increasing evidence that more than one type of a given product can originate from a single country, the types being both visually and chemically distinguishable. This is particularly true of cannabis from southern Africa, where one variety contains significantly higher amounts of THV than the other (with little indication of an intermediate variety). Samples from Angola, Swaziland and Zimbabwe, although infrequently encountered, show a tendency to higher amounts of THV (one Zimbabwean sample had more THV than THC). It may be that THV is less stable than THC and that some part of the preparation procedure of the low-THV variety selectively decomposes THV. This seems unlikely, as elevated values of cannabivarin (CBV) are not found in this variety (oxidation to CBV being the most likely reaction). It is possible that the high THV is the result of early harvesting, although this too is unlikely in view of its remarkable consistency. The difference could also be the result of a distinct chemical race which gives rise to high concentrations of the cannabivarins.
|
Country |
Number of samples examined a |
Features |
|---|---|---|
|
A. Cannabis
|
||
|
Ghana
|
120 |
Brown; often compressed into blocks or slabs; usually leafy with few identifiable flowering or fruiting tops
|
|
India
|
20 |
Brown with clumped resinous tops - typical
Cannabis indica; dark green-brown and chopped or very immature green female plants (three varieties)
|
|
Jamaica
|
350 |
Brown and very coarse with a high percentage of stalk and seed; some recent samples as compressed blocks
|
|
Kenya
|
100 |
Green or brown material
|
|
Morocco
|
25 |
Light green to yellow green and very finely chopped; no recognizable seed or stalk
|
|
Nigeria
|
190 |
Brown with small, characteristically twisted stems; often as small pieces rather than whole (fruiting) tops: some recent samples as compressed blocks
|
|
South Africa
|
60 |
Green; often in small rolls in brown paper (this variety is usually immature); brown mature (fruiting) variety also occurs
|
|
Thailand
|
110 |
Green or brown sticks of several seedless tops tied around bamboo [45] with a number of sticks compressed into a slab; some recent seizures have been loose herb and/or of lower quality [49]
|
|
Zambia
|
40 |
Green or brown material; the compressed "corn-cob" shape wrapped in coarse vegetable fibre is characteristic
|
|
B. Cannabis resin
|
||
|
India
|
120 |
Dark-brown, as sticks (often in bundles), balls and irregular shapes and (rarely) as pale-brown "twists"; often mouldy; sometimes encountered as wafers with a glossy surface
|
|
Lebanon
|
100 |
Red-brown and powdery, a darker resin than Moroccan; as blocks (often 500 g) in white cotton bags with many different, often crudely made, ink stamps; resin bears imprint of cloth when unwrapped
|
|
Morocco
|
95 |
Yellow-brown and powdery in thin, rectangular slabs, wrapped in clear plastic; markings are rare, but include "12 Madeleines" and "6 Madeleines" and coin imprints; biscuity smell when fresh
|
|
Nepal
|
50 |
Dark-brown to black in colour; occasionally as large "Temple Balls" but more often as rough lumps (resembling dry soil) or thin, roughly prepared slabs in plastic; often mouldy with almost a perfumed smell and crumbles readily
|
|
Pakistan
|
250 |
Dense, dark-brown rectangular or square slabs with a greener interior; pliable with a pleasant smell when fresh, becoming hard, brittle and odourless with age; embossed marks on the resin in various languages or symbols [50]
|
|
Turkey
|
10 |
Usually as greenish-brown powder or (rarely) as small thin wafers of brittle material wrapped in thin plastic
|
aOriginal seizures of Cannabis varied from 100 g to 1,000 kg.
|
Country |
Number of samples examined |
TLC fetures a |
|---|---|---|
|
A. Cannabis
|
||
|
Ghana
|
50 |
Very little chemical difference; all lack CBD and have low
|
|
Jamaica
|
150 |
THV:THC ratios. Ghana often shows both CBG and CBCh > THV,
|
|
Nigeria
|
100 |
while Jamaica and Nigeria usually show THV > both CBG and CBCh
|
|
India
|
10 |
"Clumped" variety contains CBD and THC:THV 1. Green-brown variety resembles Ghana, Jamaica, Nigeria group (no CBD). Immature variety contains CBD. THC:THV = 1, but is very weak in cannabinoid content
|
|
Kenya
|
25 |
Both show two varieties, neither having CBD. One has THC:THV = I
|
|
Zambia
|
10 |
with low CBG and CBCh. The other variety resembles the Ghana, Jamaica, Nigeria group
|
|
Morocco
|
10 |
TLC pattern identical to Moroccan cannabis resin
|
|
South Africa
|
20 |
No CBD. Greenish varieties sometimes show THC:THV = 1. More usually, the TLC is similar to the Ghana, Jamaica, Nigeria group. A few samples have an unidentified orange spot with Rf similar to THV
|
|
Thailand
|
25 |
Normally only THC and THC acid. No CBD and negligible THV
|
|
B. Cannabis resin
|
||
|
India
|
35 |
Very variable. CBD:THC may vary from unity to very small CBG and CBCh THV = THC
|
|
Lebanon
|
25 |
CBD > THC THV is very low Acids may be high and grossly overload the TLC plate, running as a yellow streak
|
|
Morocco
|
15 |
CBD:THC 1:2 THV is low CBG and CBCh > THV A yellow spot R
f 0.9 is often noted before spraying The amount of cannabinoid acids is variable, appearing as a yellow streak after spraying
|
|
Nepal
|
20 |
CBD may be very low and almost absent THC > THV CBG and CBCh
|
|
Pakistan
|
100 |
A similar pattern to Morocco sample, but the cannabinoid acids are less marked CBG and CBCh > THV (fresh samples are very rare)
|
|
Turkey
|
5 |
CBD:THC 1:4 CBG and CBCh > THV Cannabinoid acids may be very high and seriously distort the TLC pattern, yellow streaking running to R
f 0.5 or more
|
aKey to abbreviations is in table 2.
The many different products from India show wide variations and may reflect either the different environmental conditions in a very large country or the existence of several chemical races of plants. There is evidence from this Laboratory's reference collection that cannabis from Bangladesh and southern India does not contain CBD and considerable evidence that most cannabis resin from India comes from the north and north-west of the country.
The characteristic appearance of "Thai Sticks" is of particular importance as this is a recent feature of cannabis from South-East Asia. When first encountered these were of consistently high quality, but this has fallen of late. Although there have been many significant qualitative changes in some cannabis products, it is of equal forensic importance that many retain the same physical characteristics over many years.
The TLC pattern of liquid cannabis was found to be closely related to that of the parent herbal material or resin. The heating process, as used in the clandestine preparation, decarboxylates the cannabinoid acids, but the basic TLC pattern remains the same, provided no further process has been performed on the sample. Liquid cannabis not containing CBD must have been prepared from herbal material, as all resin contains at least some CBD (table 4). The converse is, however, not true (table 4).
On the basis of physical appearance and thin-layer chromatographic characteristics it is possible to assign the most probable country of origin to the majority of Cannabis products illicitly entering the United Kingdom. TLC examination is a valuable indicator of the country of origin where there are no characteristic physical features, for instance, in the analysis of small amounts of material or in the case of liquid cannabis.
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