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Variations of ∆9-THC Content in Single Plants of Hemp Varieties.
Klemens Mechtler, Josef Bailer and Karl de Hueber
Federal of ce and research centre of agriculture, A-1226 Vienna, Spargelfeldstraße 191, Austria. www.b .at; kmechtler@b .at; jbailer@b .at
Within a given plant population, the concentration of any constituent is expected to vary within a certain band- width. To test the distribution of ∆9-Tetrahydrocannabinol (THC) in hemp populations, a number of single plants were taken from populations of 5 well-known hemp accessions (Fasamo, Beniko, Bialobrzeskie, Félina 34, Kompolti) and a Hungarian provenance. The quantitative analysis of single plants delivered a set of 30 to 61 THC and Cannabidiol (CBD) values for each of the six hemp accessions under consideration. The distribu- tion of THC within a number of hemp plants often shows no Gaussian distribution, the different varieties have quite characteristic distributions of THC. Most single plant values are close together, the variation, however, differing from variety to variety. In addition, single plants are found with THC-values far outside this band- width.
Keywords: Hemp; Cannabis; ∆9-Tetrahydrocannabinol (THC); Cannabidiol (CBD) 1. Introduction
The presence of psychoactive components in the hemp plant has no adverse affects on bre quality, but gives rise to some practical problems, mainly concerning the public acceptance. A stimulant level low enough to make the cultivars unsuitable for drug use has been a breeding goal for years, and therefore, modern hemp varieties are not only superior in vegetative growth characteristics and resistance to pests and diseases, but also their psychoactive potency is well below acceptable limits.
The psychoactive potency of hemp cultivars is expressed in the ∆9-THC content of a sample prepared by col- lecting the upper part of hemp plants, drying and removing stems and seeds, and grinding. The THC-content of this preparation is limited to 0.2% by EU regulations (VO (EG) Nr. 1420/98) and settled by law also in many countries outside the European Union. Within a given plant population, the concentration of any constituent is expected to vary within a certain bandwidth. A sample representative for the whole population is obtained if the number of plants taken is big enough to assure the average concentration of the constituent in the sample is the same as in the whole population. The minimum number of plants to be sampled is easily calculated from data on the variability of the constituent, presuming the variation follows a normal, Gaussian, distribu- tion. Based on literature (de Meijer 1992), a sample size of 20 plants was expected to be suf cient for reliable analyses. When these results were questioned, we decided to repeat the experiment with more varieties and more single plants per variety.
2. Material and Methods
2.1. Plant material:
The test was performed with 5 well-known hemp accessions and one variety with THC content above the tolerated level. The accessions used vary with respect to their vegetative growth characteristics, and, despite the fact that most modern accessions used are monoecious, there is also a dioecious variety under test.
1. 2. 3. 4. 5. 6.
Fasamo (Germany), monoecious, very early.
Beniko (Poland), monoecious, early.
Bialobrzeskie (Poland), monoecious, early to medium. Félina 34 (France), mainly monoecious, medium. Kompolti (Hungary), dioecious, late.
grown in Fuchsenbigl (dry growth area), Kompolti and Bialobrzeskie in Grabenegg (more humid growth area). Both trial sites are located in Lower Austria.
When the plants reached the end of owering, a number of approx. 30 to 60 plants were sampled. Each plant was cut to the upper third and air-dried at ambient temperature to a residual humidity of less than 12% before it was brought to the laboratory. With the aid of test sieves (mesh 1.4mm), stems and seeds were separated from the dried plant material manually, the resulting material (“laboratory sample”) being weighed and grinded in a mortar.
2.2. THC determination:
The chemical analysis followed the former Austrian national method for THC screening (Flachsbeihilfeverordnung, BGBl. II Nr. 156/1999). 100mg laboratory sample with 4-Androsten-3,17-dion as internal standard was extracted 3 times with 5ml hexane with an ultrasonic disintegrator for 10 min (Branson Soni er, the sample dipped into a beaker with cold tap water for cooling), the solvent phases combined in a volumetric ask and brought to 25ml with hexane.
GC instrument settings: HP 5890 II+, Column HP-5MS, 30m x 0.25mm, lm thickness 0.25μm, mobile phase helium 1 ml/min, oven program gradient from 200° to 280°C. Direct injection of the diluted extract, injection volume 2 μl, auto injector HP 6890, injection block temperature 280°C, split 1:50. A quadruple mass spectrometer (HP 5989 B) was used for detection, EI-mode, transfer capillary 260°C, ion source 250°C, quadrupole 120°C, SIM-mode using the fragments 314 and 299 to quantify THC, 314 and 231 for Cannabidiol (CBD) and 286 and 124 for the internal standard.
were grown in 1999 on test elds of the research centre in two different regions in Austria. Fasamo, Beniko, Félina and the Hungarian provenance were
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