ANALYTICAL CHEMISTRY AT FORENSIC INSTITUTES 80 CHIMIA 2002, 56, No. 3

Chimia 56 (2002) 80–83 © Schweizerische Chemische Gesellschaft ISSN 0009–4293

Analysis of Cannabinoids in Hemp Plants

Christian Giroud*

Abstract: In 2001, guidelines concerning cannabis were issued by the Group of Forensic Chemistry of the Swiss Society of Forensic Medicine. These guidelines deal with the sampling of Cannabis plants and their processing before analysis. Some recommendations are also suggested for the determination of ∆9-tetrahydrocannabinol (∆9-THC). In this paper, the biosynthetic pathway of ∆9-THC is presented and the putative ecological function of the cannabinoids discussed. The taxonomy and botany of the hemp plant are also considered. The influence of age, sex, and plant part on the accumulation of ∆9-THC is discussed. The need for criteria aimed at the distinction between fiber hemp and cannabis belonging to the drug type is especially emphasized. The current Swiss situation and legislation are briefly presented. Furthermore, the sampling of cannabis material, its processing and analysis are described, alternative strategies for the characterization of hemp exhibits are also presented. Keywords: Cannabinoids · Cannabis · Forensics · Hemp analysis · Profiling of cannabis products · THC

Introduction of Cannabis sativa L. usually contain which is thought to be responsible of the cannabinoids in the form of their carbox- psychoactive effects must be determined Cannabis is the most commonly used il- ylic acid derivatives (Scheme) [3]. These alone. licit drug in Switzerland. Thorough clini- tetrahydrocannabinolic acids, named cal studies have demonstrated that among ∆9-THC acid A and ∆9-THC acid B, read- more than 70 different cannabinoids, ily decarboxylate into neutral ∆9-THC Biosynthesis and Function of ∆9-tetrahydrocannabinol (∆9-THC) is the when heated and consequently cannot be Cannabinoids main active compound responsible for examined directly by gas chromatogra- the psychological and physiological ef- phy. In summer, the temperature of the The biosynthetic pathway of ∆9-THC fects of cannabis in man [1]. Only ∆8-tet- plant parts is very likely high enough (fo- has been delineated recently: condensa- rahydrocannabinol, which is present in liar temperatures of 50Ð55 °C are quite tion of geranyl pyrophosphate with olive- very low concentrations in the plant, but common) to allow a partial decarboxyla- tol carboxylic acid by a hemp transferase can be obtained quite easily by chemical tion of some acid cannabinoids. Howev- yields cannabigerolic acid [5] which in synthesis or formed as an artifact from er, most of the neutral cannabinoids are turn is a substrate for ∆9-THC acid A syn- ∆9-THC [2], has shown similar psycho- probably formed slowly during the dry- thase [6]. In contrast to what was previ- active properties. Cannabinol, which is ing and the collection of the resinous ously postulated, cannabidiol is not a pre- essentially a chemical degradation prod- fraction of the plant. Likewise, cannabis cursor of ∆9-THC. Cannabidiol is very uct of ∆9-THC, has also shown some smoking results in the almost complete likely also formed as its acid derivative, activity; its concentration generally in- conversion of the acids into their decar- i.e. cannabidiolic acid [7]. Although non- creases as samples age. Fresh specimens boxylated active counterpart, i.e. ∆9-THC. psychoactive, this compound has been Foodstuffs or beverages containing can- shown to possess anticonvulsant and nabis or cannabis extracts cooked or anxiolytic properties. As far as we heated before administration (e.g. space know, the regulation of ∆9-THC acid A cake, hemp Swiss fondue, hemp milk de- synthesis and its interdependence with coction) contain only trace amounts of other pathways, e.g. the terpenoid path- the acids. The potency of a preparation way, are not yet elucidated and require is therefore better described by its total further studies. ∆9-THC concentration, usually as the Several hypotheses have been put for- % total ∆9-THC (i.e. neutral ∆9-THC + its ward for the biological function of can- acid counterparts) per dry weight of ma- nabinoids; most of them deal with an ad- terial. The taxonomy of cannabis also re- aptation to environmental stress. A rela- *Correspondence: Dr. C. Giroud lies on the relative proportions of canna- tion was suggested with drought and heat Laboratoire de toxicologie analytique Institut Universitaire de Médecine Légale binoids concentrations presented as the tolerance. Moreover, cannabinoids could Rue du Bugnon 21 sum of neutral compound and the corre- provide protection against UV radiation, CH-1005 Lausanne sponding carboxylic acids [4]. In the case pathogens (antibiotic effect) and preda- Tel.: +41 21 314 70 86 Fax: + 41 21 314 70 90 of oral administration of unheated plant tors, e.g. aphids which might become en- E-Mail: [email protected] material, the neutral fraction of ∆9-THC snared in the resinous cannabinoids. ANALYTICAL CHEMISTRY AT FORENSIC INSTITUTES 81 CHIMIA 2002, 56, No. 3

Scheme. Biosynthesis of ∆9-tetrahydrocannabi- nolic acid from triketoacid. The enzyme names are printed in italics.

[4]. Variation in the genus Cannabis is due in large part to selection by man. Two phenomena make easier the hybridi- zation and intermixing of hemp plants: the escape of cultivated plants to the wild and their wind-pollination. The cannabi- noid content is used to characterize the phenotypes or chemotypes belonging ei- ther to the ‘drug’ type or to the ‘fiber’ hemp type.

Variation in ∆9-THC Content versus Age, Sex, and Plant Part

The ∆9-THC is concentrated into a resin which is secreted into trichomes found on the small leaves (bracts) and bracteoles (leaf-like structure which en- closes the ovary) of the flowering tops of the female plant [9]. The male plant pro- duces an equivalent amount of active constituent which is found throughout the plant. The amount of resin found in the pistillate flowering tops is influenced by the climatic conditions of the plant, warm climates stimulate the synthesis of cannabinoids while cold weather lead to The Hemp Plant is densely covered with secretory and lesser amounts. The female hemp flowers cystolith hairs. Short days initiate flower- remain fertile during a short time period. Cannabis sativa L. grows outdoors as ing and plants are photoperiodically Hemp is known to build large amounts an annual, dioecious herb, i.e. it compris- adapted to terminate growth in relation to of new flowers as long as it does not es both staminate and pistillate plants. the length of their local growing season. become pollinated. Prevention of polli- Some varieties, however are monoe- There is no general agreement on the in- nation stimulates the formation of new cious, with both female and male flowers frageneric taxonomic treatment of canna- flowers and increases the yield of canna- on the same plant. The plant is widely bis. Together with hops (Humulus lupu- binoids. This strategy is known by grow- distributed throughout temperate and lus L.), the hemp plant belongs to the ers of drug cultivars as the ‘sinsemilla’ tropical zones. The hemp plant is charac- Cannabaceae family which is member of technique. terized by palmate leaves with a toothed the order of the Rosales. The leaf mor- GC-FID analysis of the main parts of margin, usually composed of five to nine phology was used to discriminate C. sati- the hemp plant showed that the parts de- narrow lanceolate leaflets. The male va L., C. indica Lam and C. ruderalis crease in ∆9-THC content in the follow- flowers are gathered in panicles while fe- Janischevsky [8]. Since there appear to ing order: bracts, flowers, leaves, smaller male flowers form compact cymes. Fe- be no barriers to successful hybridization stems, roots, and seed. All plant parts male flowers have five perianth segments within the genus, it seems obvious to contain cannabinoids [10][11]. Young and are surrounded by large, persistent consider hemp as monotypic with one floral leaves, those within the first nodes bracts. The abaxial surface of these bracts polymorphic species: Cannabis sativa L of the shoot apex, were found to contain ANALYTICAL CHEMISTRY AT FORENSIC INSTITUTES 82 CHIMIA 2002, 56, No. 3 higher concentrations of cannabinoids of ∆9-THC to cannabidiol higher than 1. mentioned levels can be used to deter- than older vegetative leaves [11]. Pollen According to [14], hemp could be classi- mine whether a product must be consid- grains and seeds were shown to contain fied as given in the Table. ered as a narcotic or not [20]. low levels of cannabinoids. When exam- Taking into account the possible Hemp has become a dual-use crop, ined by scanning electron microscopy, degradation of ∆9-THC into cannabinol, i.e. it may be both taken as an illicit nar- pollen grain samples were found to hemp has been classified into two major cotic drug (e.g. marijuana) and used le- contain epidermal glandular trichomes phenotypes according to the value of the gally in agriculture, for instance, for the (heads) intermixed with the pollen cannabinoid ratio: ([∆9-THC] + [CBN])/ production of fibers, foodstuffs, cosmet- grains. The existence of gland heads, [CBD]. If the ratio exceeded 1, hemp ics. Some materials can be used either as known to contain cannabinoids, suggests plants were classified as ‘drug pheno- a drug or as a legal article, typically, that the low level of cannabinoids repre- type’, otherwise as belonging to the ‘fiber hemp coins might be gathered by collec- sent a contaminant derived from glands phenotype’ [15]. tors fond of numismatics or used as a rec- rather than from pollen grains [11]. The reational or narcotic drug [21]. presence of most of the cannabinoids in seeds could be also explained by external The Current Swiss Situation: contamination. Indeed, the location of Legislation Cannabis Products ∆9-THC within the achenes was found to be mainly on the outside surface of the The cultivation of hemp is prohibited For drug purposes, either the resin seed coat, possibly the result of physical in many countries, except for ∆9-THC- (hashish) is used or the flowering tops interaction with the plant bracts during poor cultivars. In Switzerland, the culti- of the female plant (marijuana). The per- processing [12]. Since manicured sam- vation of hemp is not prohibited provided centage of resin in hashish determines its ples most likely contain different ratio of it is not done for the purpose of produc- color, consistency and ∆9-THC content. plant organs and tissues, cannabinoids ing narcotic drugs [16][17]. Anybody Hashish oil is an extraction residue ob- values therefore represent a mean esti- growing hemp with a ∆9-THC content tained after treatment of dried hemp tips mate of the total cannabinoid profile higher than 0.3% is however liable to in- with an organic solvent (e.g. 2-propanol). within the flowering portion of the plant. vestigation and prosecution. Since the Hashish can be obtained from pollen, Until recently it was widely thought that decision of the Swiss Federal Supreme also called in Holland pollem or polm. the intoxicant properties of the resin Court of 29 August 1991, Cannabis, even One should not mistake ‘pollen’ for of Cannabis were extremely subject to in large quantities ‘cannot endanger the the male reproductive parts of the hemp environmental modification. It now ap- health of many individuals’. Subsidies plant. ‘Pollen’ is extracted and sieved pears, however, that the intoxicant char- are provided for the cultivation of mechanically with a pollinator from the acteristics of the resin are reasonably ∆9-THC-poor strains of Cannabis by the blooming female plant after harvesting conservative [4]. Moreover, no signifi- Federal Office of Agriculture to develop and contains therefore no more than trac- cant fluctuation in the relative amounts new sources of renewable materials. A es of pollen! of the main cannabinoids ∆9-THC, can- list of saleable varieties of hemp seeds nabidiol and cannabichromene could be was drawn up in 1998 [18]. The list observed in the various types of Canna- which encompasses about a dozen va- Sampling of Cannabis Material bis sativa analyzed so far [13]. rieties, e.g. Fedora 19, Félina 34, Kom- polti, Uniko-B is periodically brought According to recent guidelines issued up-to-date. All these strains are charac- by the group of Forensic Chemistry of the Criteria Aimed at the Distinction terized by a ∆9-THC level lower than Swiss Society of Forensic Medicine, Between Fiber Hemp and Cannabis 0.3%. Regulations concerning the mar- Cannabis sampling must performed as Belonging to Drug Type keting of foods and beverages containing follows: hemp products were issued by the Swiss Studies and analysis of cannabis plants Federal Office of Public Health in 1998 a. Fields have indicated that cannabis strains or [19]. On the whole, the limit was estab- Thirty flowering tops from 30 differ- cultivars can be classified into two main lished between 0.00002 and 0.005% ent female plants are taken along the chemical phenotypes according to their ∆9-THC. For instance, alcoholic beverag- diagonal of the field. No sampling must cannabinoid content. Fiber hemp strains es containing more than 0.2 mg/kg no be performed on the sides of the planta- or varieties generally contain more can- longer constitute foodstuffs within the tion. The best time to collect the speci- nabidiol than ∆9-THC while resinous meaning of the Swiss Food Act. A recent mens is just before harvesting, in the cannabis plants belonging to the ‘drug settlement issued by the Swiss Federal afternoon of a dry day. The cannabinoid type’ are characterized by a weight ratio Supreme Court established that the afore- content of a mixed sample of 30 plants is considered to be a reliable approximation of the average content of the field. When possible, it is recommended to let the farmer harvest the crop and to take dried Table. Classification of hemp chemotypes into fiber and resinous Cannabis according to [14] samples from it for analysis. Formerly, the European Community procedure to 9 Phenotype ∆9-THC [%] CBD [%] ∆9-THC/CBD determine total ∆ -THC content in fiber hemp cultivars dictated a sample size of Fiber hemp < 0.5 > 0.5 < 1 500 individual plants to be analyzed as a Resinous Cannabis > 0.5 < 0.5 > 1 mixed sample [22]. The cutoff value was 0.3% total ∆9-THC in the dry matter. In ANALYTICAL CHEMISTRY AT FORENSIC INSTITUTES 83 CHIMIA 2002, 56, No. 3

2000, new guidelines were issued by the Retsch Grindomix) and sieved to yield a [6] F. Taura, S. Morimoto, Y. Shoyama, R. European Union. The size of the sample homogenous and fine powder. A small Mechoulam, J. Am. Chem. Soc. 1995, 117, was decreased from 500 plants to 50 and aliquot of the powder is extracted with an 9766. the value of the cutoff lowered to 0.2% organic solvent (e.g. petrol ether, metha- [7] R. Mechoulam, S. Ben-Shabat, Nat. Prod. [23]. Investigations carried out by [24] nol/dichloromethane 9:1 (v/v)) with or Rep. 1999, 16, 131. indicated that a mixed sample of 20 without sonication overnight or during a [8] L.C. Anderson, Harvard University Bo- tanical Museum leaflets 1980, 28, 61. plants could be considered to be a reliable few minutes only. According to [29], [9] E.S. Kim, P.G. Mahlberg, American J. approximation of the mean content of a some hashish and hash oil samples have Botany 1997, 84, 336. small plot of plants. exhibited the unusual property of being [10] P.S. Fetterman, E.S. Keith, C.W. Waller, insoluble in organic solvents, but soluble O. Guerrero, N.J. Doorenbos, M.W. b. Raw Material in water. In order to dissolve them, they Quimby, J. Pharmac. Sciences 1971, 60, Marijuana samples are directly blend- must be prepared by partitioning small 1246. ed with a mixer and the homogenous aliquots between chloroform and water. [11] J.K. Hemphill, J.C. Turner, P.G. Mahl- berg, J. Nat. Prod. 1980, 43, 112. powder analyzed as described below. All analyses must be performed in tripli- [12] S.A. Ross, Z. Mehmedic, T.P. Murphy, Two samples of at least 1 g each are taken cate. Squalane, 4-androstene-3,17-dione, M.A. ElSohly, J. Anal. Toxicol. 2000, 24, per sheet of hashish with a metal punch. tetracosane can be used as an internal 715. Sampling near the sides must be avoided. standard for cannabinoids determina- [13] I . Barni-Comparini, S. Ferri, F. Centini, The samples are then pulverized with a tions. ∆9-THC or cannabinol, which is For. Sci. Int. 1984, 24, 37. mixer. more stable, can be used as standards to [14] G. Fournier, M.R. Paris, Plant Med. Phy- set up calibration curves (Recommenda- tother. 1979, 13, 116. [15] P.S. Fetterman, E.S. Keith, C.W. Waller, c. Foodstuffs and Beverages tions for the analysis of hemp, Group of O. Guerrero, N.J. Doorenbos, M.W. These samples can be prepared as de- Forensic Chemistry, Swiss Society of Fo- Quimby, J. Pharmaceut. Sciences 1971, scribed in ref. [27]. rensic Medicine, 2001). 60, 1246. [16] C. Giroud, L. Rivier, TIAFT Bulletin Analysis of Cannabinoids in Hemp 1996, 26, 30. Plants and Derivatives Other Strategies Used for the [17] C. Giroud, A. Broillet, M. Augsburger, W. Capillary gas-chromatography with Classification of Hemp Plants Bernhard, L. Rivier, P. Mangin, Praxis 1999, 88, 113. flame ionization detection (GC-FID) and [18] Ordinance SR 916.151.6 of the Swiss Fed- mass spectrometry (GC-MS) and high- Many attempts have been made to eral Office of Agriculture issued Decem- performance liquid chromatography with characterize hemp cultivars of different ber 7, 1998. UV (HPLC-UV) or diode array detection origin. One strategy was to use electro- [19] Ordinance SR 817.021.23 of the Swiss (HPLC-DAD) are generally used to es- phoretic profiles of seed proteins [30]. Federal Office of Public Health on For- tablish the chemical profile of Cannabis Various DNA ‘profiling’ techniques eign substances and ingredients in food samples and to carry out cannabinoid have been shown to assess the genetic re- products on January 30, 1998. determinations. Without derivatization, latedness of species, varieties, cultivars [20] Swiss Federal Supreme Court of Appeal, judgment of the March 13, 2000. GC methods yield the so-called ‘total- and even individuals [31][32]. For in- [21] C. Giroud, A. Broillet, M. Augsburger, W. ∆9 -THC’ concentration that corresponds stance, RAPD (Random Amplified Poly- Bernhard, L. Rivier, P. Mangin, Toxico- to the total ∆9-THC (i.e. neutral ∆9-THC morphic DNA using the Polymerase rama 1998, 11, 51. + its acid counterparts). Formation of Chain Reaction) was used to obtain in- [22] G. Fournier, O. Beherec, S. Bertucelli, J.- the silylated or alkylboronate derivatives formative and reproducible fragments, P. Mathieu, Annales de Toxicologie Ana- stabilize the acids and allow the separate that showed clear differences between lytique 2001, 13, 275. determinations of ∆9-THC and of its acid samples from different sources [32] [23] Règlement (CE) n°2860/2000 de la Com- counterparts (∆9-THC-A and ÐB) [25][26]. mission du 27 décembre 2000. Journal Officiel des Communautés européennes For food control purposes, HPLC is gen- Received: January 29, 2002 28/12/2000. erally preferred over GC techniques. [24] E.P.M. de Meijer, H.J. van der Kamp, A solvent-programmed reversed-phase F.A. van Eeuwijk, Euphytica 1992, 62, HPLC method with UV and fluorescence 187. detection for the determination of [25] S. Billets, F. El-Feraly, P.S. Fetterman, ∆9-THC-A and ∆9-THC in foods such as C.E. Turner, Org. Mass Spectrom. 1976, edible oil, herb-teas, herbal hemp or 11, 741. hempseed has been presented recently [26] D.J. Harvey. Biomed, Mass Spectrom. 1977, 4, 88. [27]. The method was adapted from [28]. [27] O. Zoller, P. Rhyn, B. Zimmerli, J. Chro- One main drawback of these methods [1] R. Mechoulam, A. Shani, H. Edery, Y. matogr. A 2000, 872, 101. is that no certified reference standard of Grunfeld, Science 1970, 169, 611. [28] T. Lehmann, R. Brenneisen, J. Liquid 9 ∆ -THC-A is commercially available, [2] S. Agurell, S. Levander, M. 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