CBD-Rich Cannabis – the Legal Marijuana
Cannabinoid Concentrations in Confiscated Cannabis Samples
- Fig. 1: Colorimetric on-site test (vial 1: cannabis sample with solution 1; vial 2: cannabis sample with solutions 1+2; vial 3: solution 1 as reference). Blue coloration indicates THC-rich cannabis; pink coloration indicates CBD-rich cannabis.
- Fig. 2: HPLC-UV chromatogram of a CBD-rich cannabis sample. Detection at 210 nm (AU = absorption units). The compounds elute in the following order: CBD-acid (tR: 5.72 min), CBD (tR: 6.84 min), CBN (not detected, expected tR: 9.02 min), THC (tR: 10.80 min), THC-acid A (tR: 12.53 min).
- Fig. 3: Distribution of confiscated cannabis samples according to their total THC and CBD concentrations (logarithmic axis).
The availability and the consumption of cannabis products rich in cannabidiol (CBD), legally accessible in Switzerland if the total Δ9-tetrahydrocannabinol (THC) content is lower than 1%, is a widely debated issue in the media and poses a challenge for the judicial authorities. In this short review, we address analytical issues related to the distinction of THC- and CBD-rich cannabis, establish classification thresholds for the different chemotypes and evaluate a newly introduced on-site test kit as rapid alternative to the laborious liquid or gas chromatography (LC or GC)-based techniques.
Cannabis, one of the oldest plants cultivated as a source of fiber, food, and oil, for ritual use or as an intoxicant drug, has regained a lot of interest and popularity in the general public as well as in the research community . This, however, is not because of its principal psychoactive compound Δ9-tetrahydrocannabinol (THC), which is responsible for the so-called “high” including a number of adverse effects (anxiety, cognitive deficits, paranoia, chronic psychosis, and dependence), but which also shows a variety of therapeutic attributes including anti-inflammatory, antiemetic, appetite stimulant, analgesic, and antispasmodic effects [2-4]. In recent years, the focus of research and legislation has shifted to other cannabinoids, the most predominant being the non-euphoriant compound cannabidiol (CBD), which is thought to alleviate the adverse effects of THC by modulating its psychoactivity. More important, however, are CBD’s auspicious therapeutic activities per se, such as anti-inflammatory, anticonvulsive, anxiolytic, analgesic, neuroprotective, anticancer, and antioxidant effects [5,6].
Classification of Cannabis and Legal Situation
Cannabis can be classified according to its THC/CBD ratio, resulting in three different chemotypes: THC-rich, intermediate, and CBD-rich. Genetic factors (strain type) essentially determine the ratio of the different cannabinoids, but environmental and harvesting conditions (e.g., growing and storage conditions, state of maturity at harvest) also have an influence .
Until recent years, there was no demand among recreational cannabis users to breed plants with high CBD concentrations.
On the contrary, seeking the ultimate “high”, they selectively bred cannabis plants with the highest THC and lowest CBD levels. This changed, however, with the growing interest in the therapeutic effects of CBD and cannabis breeders started to cultivate CBD-rich strains with a total CBD concentration of up to 25% and less than 1% total THC (typically 0.3–0.7%) . The total THC and CBD contents (in % of dry weight) are determined by the sum of the free cannabinoid and its pharmacologically inactive carboxylic acid precursor (THC-acid A and CBD-acid, respectively), corrected for molecular weight of the carboxylic acid groups.
The European Union stipulates that cannabis plants, which are legally used for the cultivation of fibers and seeds, must be proved to have a total THC content of less than 0.2% and must not lead to psychoactive effects. In Switzerland, the legislation is somewhat laxer, as only plant material with a total THC concentration of 1% or higher is controlled by the narcotic legislation.
For more than a year now, CBD-rich/THC-poor cannabis products have been on the Swiss legal market as tobacco substitutes and the popularity of such products, for both therapeutic and recreational purposes, is still increasing. Only five companies were registered at the beginning of 2017, whereas now there are already more than 500 (personal communication by Swiss Federal Customs Administrations). As CBD-rich/THC-poor cannabis cannot be differentiated olfactorily and/or morphologically from THC-rich/CBD-poor cannabis, the availability and the consumption of these products have posed a challenge for public health and especially to the judicial authorities (police and forensic experts/toxicologists).
Colorimetric On-site Test Kit for Differentiation of CBD- versus THC-cannabis
Originally, the distinction between CBD- and THC-cannabis resulted in high financial and administrative costs, as it was only possible by laborious analytical methods (mostly GC- or LC-based techniques). Since last year, a liquid colorimetric test can be used by police forces to directly conduct an analysis and take a decision on-site within a very short time and thus greatly reduce these costs .
To execute this test, a small sample of dried or fresh cannabis plant material is transferred into a glass vial and 1–2 mL of solution 1 (0.2 mg/mL 4-aminophenol in ethanol/isopropanol [95:5; v/v], acidified with 0.5% hydrochloric acid [HCl 2 N]) and four drops of solution 2 (30 mg/mL sodium hydroxide in ethanol/water [70:30; v/v]) are added. After shaking and leaving to stand for 2 min to allow reaction, the color of the solution is visually evaluated: THC-rich cannabis is indicated by a blue coloration, CBD-rich cannabis by a pink coloration. The typical difference of the solution’s coloration between THC-rich and CBD-rich cannabis can be seen in figure 1.
HPLC-analysis of Cannabis Products for Determination of THC, THC-acid A, CBD, CBD-acid and Cannabinol (CBN)
The HPLC-DAD method used to analyze the cannabinoid content of cannabis products has been validated and is used in the daily routine of the accredited laboratory. It has already been published, but has recently been updated by adding CBD-acid to the calibrators and the control samples, in the same concentrations as THC-acid A [10,11].
For sample preparation, approximately 500 mg of a dried and ground cannabis sample is weighed into a test tube and 10 mL of methanol/n-hexane (9:1; v/v) is added. The samples are briefly vortexed and then placed in an ultrasonic bath for 20 min for extraction. Subsequently, the samples are vortexed again and left to stand for about 5 min until the undissolved substances have settled. The clear supernatant is diluted 1:20 with methanol/n-hexane (9:1; v/v) in a disposable test tube. An aliquot thereof is filled into an HPLC vial and is ready for injection.
The samples are analyzed on an HPLC Alliance 2695 system with photo-diode-array-detector 2996 (Waters), using the following equipment for chromatographic separation: column holder: manu-CART “4” (Merck 1.50078); pre column: LiChrospher 60, RP-select B, 5 µm (Merck 50963); column: LiChroCart 125-4, LiChrospher 60, RP-select B, 5 µm (Merck 50829). Separation is achieved using an isocratic mode with 36% A (TEAP buffer 25 mmol/L in Milli-Q-water) and 64% B (acetonitrile) and a flow rate of 1.0 mL/min. Calibrators, quality controls and samples are injected with a volume of 10 µL each and detection is at a wavelength of 210 nm (fig. 2).
Evaluation of Confiscated Cannabis Samples
Approximately 800 confiscated cannabis samples have been analyzed for cannabinoids since January 2017. For the evaluation of the THC and CBD contents, only samples containing dried or fresh cannabis flowers, which have been analyzed with the new method already including CBD-acid, were considered (total N = 531).
The examination of this data on a scatterplot shows three quite characteristic groups (fig. 3). These could be classified further according to their ratio of total THC/total CBD: samples with a ratio ≥ 3 were defined as THC-rich/CBD-poor cannabis (n = 294, 55%); samples with a ratio ≤ 0.33 as CBD-rich/THC-poor cannabis (n = 205, 39%). This observation of a THC-rich and a CBD-rich chemotype agrees with Potter , as also an intermediate type with a total THC/total CBD ratio between 3 and 0.33 has been detected (n = 32, 6%).
The colorimetric on-site test has been successfully applied to distinguish between the two major chemotypes “THC-rich” and “CBD-rich”. If the THC concentrations are significantly higher than the CBD concentrations or vice versa, the test results are indisputable and reliable. Problems occurred, however, when applying this test to samples with similar THC and CBD concentrations. There was a higher possibility for false negative results (26%), indicating a pink coloration after 2 min, whereas the correct result (blue coloration) became visible after two more minutes.
Even if the on-site test indicates a pink coloration and thus in favor of legal CBD-rich cannabis, the sample should sometimes be tested in the laboratory. The analyses determined that 4% of the CBD-rich samples showed THC concentrations between 1 and 1.7%, rendering them illegal according to the Swiss narcotics regulations. However, it can be assumed that these samples would have passed the on-site test, because the CBD concentrations were at least 4.5 times higher than their corresponding THC concentrations.
Conclusion and Outlook
The introduction of a new colorimetric on-site test as alternative to the costly analytical techniques showed promising results for the differentiation of CBD- and THC-cannabis, but needs further validation. Moreover, it was possible to propose a classification of cannabis chemotypes based on the analysis of a large collective of confiscated cannabis samples: plant material with a ratio of total THC/total CBD ≥ 3 is considered THC-rich, whereas samples with a ratio ≤ 0.33 are graded as CBD-rich. For further information, including toxicological issues, please refer to our recent original article .
Tim J. Gelmi1 and Wolfgang Weinmann1
1Institute of Forensic Medicine, Department of Forensic Toxicology and Chemistry, University of Bern, Switzerland
Tim J. Gelmi
University of Bern
Institute of Forensic Medicine
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