Analysis of Cannabinoids in Hemp Plants

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 50–55 °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-tetrahydrocannabinolic 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 hybridization and intermixing of hemp plants: the escape of cultivated plants to the wild and their wind-pollination. The cannabinoid content is used to characterize the phenotypes or chemotypes belonging either 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.

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