Characteristics of cannabinoids composition of Cannabis plants grown in Northern Thailand and its forensic application

The Thai government has recognized the possibility for legitimate cultivation of hemp. Further study of certain cannabinoid characteristics is necessary in establishing criteria for regulation of cannabis cultivation in Thailand. For this purpose, factors affecting characteristics of cannabinoids composition of Thai-grown cannabis were investigated. Plants were cultivated from seeds derived from the previous studies under the same conditions. 372 cannabis samples from landraces, three different trial fields and seized marijuana were collected. 100g of each sample was dried, ground and quantitatively analyzed for THC, CBD and CBN contents by GC-FID. The results showed that cannabis grown during March-June which had longer vegetative stages and longer photoperiod exposure, had higher cannabinoids contents than those grown in August. The male plants grown in trial fields had the range of THC contents from 0.722% to 0.848% d.w. and average THC/CBD ratio of 1.9. Cannabis in landraces at traditional harvest time of 75 days had a range of THC contents from 0.874% to 1.480% d.w. and an average THC/CBD ratio of 2.6. The THC contents and THC/CBD ratios of cannabis in second generation crops grown in the same growing season were found to be lower than those grown in the first generation, unless fairly high temperatures and a lesser amount of rainfall were present. The average THC content in seized fresh marijuana was 2.068% d.w. while THC/CBD ratios were between 12.6 and 84.09, which is 10-45 times greater than those of similar studied cannabis samples from the previous study. However, most Thai cannabis in landraces and in trial fields giving a low log(10) value of THC/CBD ratio at below 1 may be classified as intermediate type, whereas seized marijuana giving a higher log(10) value at above 1 could be classified as drug type. Therefore, the expanded information provided by the current study will assist in the development of criteria for regulation of hemp cultivation in Thailand.     

First systematic evaluation of the potency of Cannabis sativa plants grown in Albania

Cannabis products (marijuana, hashish, cannabis oil) are the most frequently abused illegal substances worldwide. Delta-9-tetrahydrocannabinol (THC) is the main psychoactive component of Cannabis sativa plant, whereas cannabidiol (CBD) and cannabinol (CBN) are other major but no psychoactive constituents. Many studies have already been carried out on these compounds and chemical research was encouraged due to the legal implications concerning the misuse of marijuana. The aim of this study was to determine THC, CBD and CBN in a significant number of cannabis samples of Albanian origin, where cannabis is the most frequently used drug of abuse, in order to evaluate and classify them according to their cannabinoid composition. A GC-MS method was used, in order to assay cannabinoid content of hemp samples harvested at different maturation degree levels during the summer months and grown in different areas of Albania. This method can also be used for the determination of plant phenotype, the evaluation of psychoactive potency and the control of material quality. The highest cannabinoid concentrations were found in the flowers of cannabis. The THC concentrations in different locations of Albania ranged from 1.07 to 12.13%. The influence of environmental conditions on cannabinoid content is discussed. The cannabinoid content of cannabis plants were used for their profiling, and it was used for their classification, according to their geographical origin. The determined concentrations justify the fact that Albania is an area where cannabis is extensively cultivated for illegal purposes.     

Characterization of Algerian-Seized Hashish Over Eight Years (2011–2018). Part II: Chemical Categorization

In Algeria, large quantities of hashish are seized every year. This study aimed to investigate the total content of major cannabinoids in the illicit seized hashish in Algeria over an 8-year period (2011–2018) in order to establish the chemical profile of North African hashish. A total of 3265 hashish samples were analyzed using a validated high-performance liquid chromatography–diode array detection (HPLC-DAD) method, allowing the simultaneous quantification of both the acidic and the neutral forms of Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN). The results revealed a slight upward trend in the mean THC content, from 7.0% in 2011 to 9.4% in 2018, with an overall mean value of 8.4%. The overall means of CBD and CBN content were 3.5% and 0.8%, respectively. The number of high-potency hashish samples gradually increased to reach 6% in 2018. Two distinct hashish chemotypes were identified: the highly populated chemotype II, corresponding to the traditional medium-potency hashish ([THC + CBN]/CBD ~ 2.16), and chemotype I, containing hashish samples of relatively high THC levels and low levels of CBD (ratio ~ 4.90). Both chemotypes I and II were characterized in the ternary plot, and the proportions (THC:CBD:CBN) were about 85%:13%:2% and 60%:35%:5%, respectively.     

Weather-induced changes in cannabinoid content of hair

Authentic hair samples from Cannabis users and a drug free hair sample which was separately spiked with tetrahydrocannabinol (THC), cannabidiol (CBD) or cannabinol (CBN) were exposed outside as well as to natural sunlight at prevailing and elevated humidity in quartz glass tubes during 8 weeks. In addition, authentic and spiked hair samples were exposed to xenon arc radiation in a light exposure cabinet for 24 hours. Stability of THC, CBD and CBN in authentic samples differed from that of the spiked hair. The radiation experiment revealed that CBN could not be measured in hair which had been spiked with THC. Under all conditions chosen the concentrations of THC, CBD and CBN decreased. At high humidity the concentrations declined more rapidly. In both authentic and spiked samples THC was most unstable compared to CBD and CBN. Therefore, in hair analysis determination of CBD and CBN seems promising to detect Cannabis exposure even under unfavorable conditions.     

Potency of delta 9-THC and other cannabinoids in cannabis in England in 2005: implications for psychoactivity and pharmacology

Gas chromatography was used to study the cannabinoid content ("potency") of illicit cannabis seized by police in England in 2004/5. Of the four hundred and fifty two samples, indoor-grown unpollinated female cannabis ("sinsemilla") was the most frequent form, followed by resin (hashish) and imported outdoor-grown herbal cannabis (marijuana). The content of the psychoactive cannabinoid delta 9-tetrahydrocannabinol (THC) varied widely. The median THC content of herbal cannabis and resin was 2.1% and 3.5%, respectively. The median 13.9% THC content of sinsemilla was significantly higher than that recorded in the UK in 1996/8. In sinsemilla and imported herbal cannabis, the content of the antipsychotic cannabinoid cannabidiol (CBD) was extremely low. In resin, however, the average CBD content exceeded that of THC, and the relative proportions of the two cannabinoids varied widely between samples. The increases in average THC content and relative popularity of sinsemilla cannabis, combined with the absence of the anti-psychotic cannabinoid CBD, suggest that the current trends in cannabis use pose an increasing risk to those users susceptible to the harmful psychological effects associated with high doses of THC.     

Distribution of Chemical Phenotypes (Chemotypes) in European Agricultural Hemp (Cannabis sativa L.) Cultivars

In Europe, more than 50 approved cultivars of fiber hemp (Cannabis sativa L.) are in agricultural production. Their content of psychoactive tetrahydrocannabinol (THC) is legally restricted to <0.2% (%w/w in the dry, mature inflorescences). Cannabis strains with much higher THC contents are also grown, illegally or under license for drug production. Differentiation between these two groups relies on biochemical quantification of cannabinoid contents in mature floral material. For nonflowering material or tissue devoid of cannabinoids, the genetic prediction of the chemical phenotype (chemotype) provides a suitable method of distinction. Three discrete chemotypes, depending on the ratio of THC and the noneuphoric cannabidiol (CBD), can be distinguished: a “THC-predominant” type, a “CBD-predominant” type, and an intermediate chemotype. We present a systematic genetic prediction of chemotypes of 62 agricultural hemp cultivars grown in Europe. The survey reveals the presence of up to 35% B_T allele-carrying individuals (representing either a THC-predominant or an intermediate chemotype) in some cultivars—which is unexpected considering the legal THC limit of 0.2% THC. The fact that 100% of the seized drug-type seeds in this study revealed at least one B_T allele, reflects that plant breeding efforts have resulted in a fixation of the B_T allele in recreational Cannabis. To guarantee a sincere forensic application based on a genetic chemotype prediction, we recommend not to classify material of unknown origin if the samples size is below nine genetically independent individuals.     

Evolution of the content of THC and other major cannabinoids in drug-type cannabis cuttings and seedlings during growth of plants

In Europe, authorities frequently ask forensic laboratories to analyze seized cannabis plants to prove that cultivation was illegal (drug type and not fiber type). This is generally done with mature and flowering plants. However, authorities are often confronted with very young specimens. The aim of our study was to evaluate when the chemotype of cannabis plantlets can be surely determined through analysis of eight major cannabinoids content during growth. Drug-type seedlings and cuttings were cultivated, sampled each week, and analyzed by high-performance liquid chromatography with diode array detection. The chemotype of clones was recognizable at any developmental stage because of high total Δ(9)-tetrahydrocannabinol (THC) concentrations even at the start of the cultivation. Conversely, right after germination seedlings contained a low total THC content, but it increased quickly with plant age up, allowing chemotype determination after 3 weeks. In conclusion, it is not necessary to wait for plants' flowering to identify drug-type cannabis generally cultivated in Europe.     

Measurement of delta 9-tetrahydrocannabinol (THC) in whole blood samples from impaired motorists

The major psychoactive cannabinoid in marihuana, delta 9-tetrahydrocannabinol (THC) was measured in 1792 randomly selected blood specimens from erratic motorists arrested for impairment who submitted to blood alcohol sampling. Of these specimens, 14.4% were positive for THC (greater than or equal to 5.5 ng/mL). In those erratic driver specimens negative for alcohol THC positives rose to 23%. Drivers who used marihuana covered a broad age range. Aliquots of hemolyzed blood (10 microL) were analyzed by a sensitive radioimmunoassay (RIA) not requiring extraction. RIA accuracy and specificity were validated by gas liquid chromatography/mass spectroscopy (GLC/MS) split pair analysis (correlation coefficient = 0.93). This initial experience should facilitate and amplify a program designed to set forth the epidemiology of marihuana use in motorists and possible behavioral correlates.     

A preliminary investigation on the distribution of cannabinoids in man

An LC/MS/MS procedure to determine THC along with its major metabolites 11-OH-THC, THC-COOH and its glucuronide as well as the cannabinoids CBD and CBN was applied to 5 post mortem cases to study their distribution into some less commonly studied matrices. Analytes were determined in fluids and tissue homogenates following protein precipitation and liquid-liquid extraction. Gall bladder fluid exhibited maximum concentrations of all analytes except THC, which was detectable in high concentrations in muscle tissue along with CBD. THC was also present in lung specimens, whereas its concentration in liver samples was low or not detectable at all. Liver und kidney specimens contained appreciable amounts of THC-COOglu. Findings from bile support extensive enterohepatic recirculation of the glucuronide. Muscle tissue seems an interesting specimen to detect multiple cannabis use, and brain may serve as an alternative specimen for blood; nevertheless, the present findings should be substantiated by further investigations.     

THC content of cannabis and cannabis resin in samples seized by the Swedish police

The THC-content in 1100 samples of cannabis resin and 600 of marihuana has been determined. The content of THC varied much, in the resin groups mostly depending on the origin and age of the resin and in the marihuana groups depending on the origin and race of the seeds.     

A survey of the potency of Japanese illicit cannabis in fiscal year 2010

In recent years, increased 'cannabis potency', or Δ(9)-tetrahydrocannabinol (THC) content in cannabis products, has been reported in many countries. A survey of Japanese illicit cannabis was conducted from April 2010 to March 2011. In Japan, all cannabis evidence is transferred to the Minister of Health, Labour and Welfare after criminal trials. The evidence was observed at Narcotics Control Department offices in major 11 cities. The total number of cannabis samples observed was 9072, of which 6376 were marijuana. The marijuana seizures were further classified, and it was found that in terms of the number of samples, 65.2% of them were seedless buds, and by weight 73.0% of them were seedless buds. Seedless buds were supposed to be 'sinsemilla', a potent class of marijuana. THC, cannabinol (CBN) and cannabidiol (CBD) in marijuana seizures exceeding 1g were quantified. The number of samples analyzed was 1115. Many of them were shown to contain CBN, an oxidative product from THC. This was a sign of long-term storage of the cannabis and of the degradation of THC. Relatively fresh cannabis, defined by a CBN/THC ratio of less than or equal to 0.1, was chosen for analysis. Fresh seedless buds (335 samples) contained an average of 11.2% and a maximum of 22.6% THC. These values are comparable to those of 'high potency cannabis' as defined in previous studies. Thus, this study shows that highly potent cannabis products are distributed in Japan as in other countries.     

The effect of electrical lighting power and irradiance on indoor-grown cannabis potency and yield

The floral development and potencies [Δ(9) -tetrahydrocannabinol (THC) contents] of cannabis plants were compared when grown indoors under high-pressure sodium lamps consuming electrical power at three densities (270, 400, and 600 W/m(2)). After a 3-week vegetative phase, plants were grown for 8 weeks, with lamps maintaining an artificial day length of 12 h. Foliar and floral yields were measured. Gas chromatography was used to measure the content of the psychoactive cannabinoid THC. Mean yields per unit of electrical power in each lighting regime ranged from 0.9 to 1.6 g/W, the highest being achieved in the lowest irradiance regime. The individual potencies of the separated leaf and flower materials were not affected by increasing irradiance. However, there was a corresponding increase in the overall potency of the aerial plant tissue. This was because of the plants in brighter conditions producing a higher proportion of floral material.     

Validation of the 4‐aminophenol color test for the differentiation of marijuana‐type and hemp‐type cannabis

The analysis of cannabis plant material submitted to seized‐drug laboratories was significantly affected by the signing of the Agricultural Improvement Act of 2018, which defined hemp and removed it from the definition of marijuana in the Controlled Substances Act. As a result, field law enforcement personnel and forensic laboratories now are in need of implementing new protocols that can distinguish between marijuana‐type and hemp‐type cannabis. Colorimetric tests provide a cost‐effective and efficient manner to presumptively identify materials prior to submission to a laboratory for analysis. This work presents the validation of the 4‐aminophenol (4‐AP) color test and demonstrates its utility for discriminating between marijuana‐type and hemp‐type cannabis (i.e., typification). Validation studies included the testing of numerous cannabinoid reference materials, household herbs, previously characterized cannabis plant samples, and real‐case samples. The 4‐AP test reliably produces a pink result when the level of Δ⁹‐tetrahydrocannabinol (THC) is approximately three times lower than the level of cannabidiol (CBD). A blue result is generated when the level of THC is approximately three times higher than that of CBD. Inconclusive results are observed when the levels of THC and CBD are within a factor of three from each other, demonstrating the limitations of the test under those scenarios.     

A contribution to the improvement of accuracy in the quantitation of THC.

The accuracy of a quantitative analysis is highly dependent on the quality of the reference standard. Although reference standards are more and more supplied with a certificate, laboratories may feel the need for additional acceptance testing. In general, confirmation of the purity of many solid reference substances can be obtained by a number of simple tests. However, verification of the true content of reference solutions may be complicated. A number of problems with the THC quantitation caused our interest for a verification method for the THC reference solution. The quantitation of THC is performed by gas chromatography with flame ionisation detector. The effective carbon number concept was used to predict GC/FID response factors. Equations and data are presented to calculate theoretical response ratios of cannabinoids. The experimental data for CBD and CBN were in excellent agreement with the theoretical ones. The paper shows that the response factors of CBD and/or CBN can be used for the calculation of the THC content of either reference solutions or cannabis samples.     

Determination of cannabinoids in biological samples using a new solid phase micro-extraction membrane and liquid chromatography-mass spectrometry

A simple, rapid and relatively solventless method for extraction of objective compounds would be useful for forensic, judicial and clinical purposes. Solid phase micro-extraction membrane (SPMEM) is one such extraction technique that integrates sampling, extraction and concentration into a single step, and combines the advantages of both the solid phase micro-extraction (SPME) and membrane separation. In this study, a new kind of membrane was prepared using polyamide and Tenax compounds, and applied to solid phase micro-extraction. Characteristics of the membrane such as adsorption capacity were tested. Extraction conditions such as adsorption time, desorption solvents, desorption time and assisted desorption treatment methods were studied and optimized. Tetrahydrocannabinol (THC) and cannabidiol (CBD) in blood and brain of the injected male mice, and in spiked human urine were extracted using this solid phase micro-extraction membrane method. The extracted THC and CBD were further determined with LC-MS using APCI. Ions analyzed in single ion monitoring mode were 315 for THC and CBD, and 318 for the deuterated THC internal standard.     

Chemical identification and optimization of the 4-aminophenol colorimetric test for the differentiation between hemp-type and marijuana-type cannabis plant samples

The 4- Aminophenol (4-AP) colorimetric test is a fast, easy-to-use, and cost-effective presumptive assay of cannabis plant material producing different chromophores with THC-rich cannabis (blue color) and with CBD-rich cannabis (pink color). The main drawback of the 4-AP test is a brief observation window where the color rapidly changes to black, limiting the utility of the test. We now report for the first time, the identification of the product chromophores between 4-AP and CBD/THC as well as propose an explanation and a solution for the color degradation of the chromophores. The identification of the chromophores is provided by spectroscopic (UV–Vis), chromatography, and mass spectrometry (TLC and LC-QToF-MS). Oxidation of excess 4-AP (Reagent A) in the presence of NaOH (Reagent B) produces the black color observed for the previously reported 4-AP tests and reported in the literature. The adjustment of reactants concentrations and volumes of 4-AP:THC/CBD to a 1:1 ratio significantly reduces the black oxidation by-product and increases the observation window up to 2 h instead of the previously reported 5–10 min. For the first time, mass spectrometry and chromatography confirmed that the reaction of THC and CBD with 4-AP produced chromophores with m/z (M + H) = 420, consistent with proposed indophenol structures. The TLC method developed confirmed the separation between CBD and THC chromophores. The specificity of the test is also reported, showing false positive results for the presence of THC (blue color) for samples of thyme and oregano. LDA and SIMCA models showed that the optimized 4-AP procedure performs better than the previously reported 4-AP color test.     

Incidence and toxicological aspects of cannabis and ethanol detected in 1394 fatally injured drivers and pedestrians in Ontario (1982-1984)

A comprehensive epidemiological study of the involvement of cannabis and ethanol in motor vehicle fatalities in the Province of Ontario, Canada, is described. The study is based on toxicological analyses of blood and, when available, urine specimens. Ethanol was determined by headspace gas chromatography (GC). For cannabis, the methods employed were radioimmunoassays (RIAs) for screening and gas chromatography/mass spectrometry (GC/MS) for the determination of delta-9-tetrahydrocannabinol (THC) in blood. The study sample consisted of 1169 drivers and 225 pedestrians. THC was detected in the blood of 127 driver victims (10.9%) in concentrations ranging from 0.2 to 37 ng/mL, with a mean of 3.1 +/- 5.0 ng/mL. Ethanol was found in 667 driver victims (57.1%), in concentrations ranging from 9 to 441 mg/100 mL, with a mean of 165.8 +/- 79.5 mg/100 mL. For pedestrians, the incidence of THC and ethanol in the blood was 7.6 and 53.3%, respectively. The incidence of THC in the driver victims in this study constitutes an approximately threefold increase over the results of an Ontario study completed in 1979. At least a part of the increase may be attributed to interstudy differences in analytical methodology for cannabinoids.     

Dragon's Blood incense: misbranded as a drug of abuse?

An unknown red substance was being sold and used with other drugs of abuse in Virginia (often being used in conjunction with marihuana). The red substance was identified as Dragon's Blood incense from Daemonorops draco. In bioassays, Dragon's Blood incense exhibited a low, but measurable cytotoxicity in in vitro cell lines. Dragon's Blood incense or Volatilized Dragon's Blood had no adverse effect on mouse motor performance based on the inclined screen and rotorod tests. delta(9)-Tetrahydrocannibinol (THC) produced a dose-related decline in mouse performance on the rotorod test. The combination of Dragon's Blood incense or Volatilized Dragon's Blood with delta(9)-THC did not contribute further to the impairment of the mice on the rotorod. This data suggests that the abuse potential for Dragon's Blood incense alone or in combination with marihuana is minimal.     

Screening for cannabinoids in blood using EMIT: concentrations of delta-9-tetrahydrocannabinol in relation to EMIT results.

The EMIT d.a.u. cannabinoid assay of methanolic extracts of blood was found to be usable as a screening method in cases of suspected impairment by cannabis, when delta-9-tetrahydrocannabinol (THC) was analysed in the subsequent assay. A prerequisite is that the blood sample is taken some time after cannabis smoking. When a cut-off limit corresponding to 50 nM delta-9-tetrahydrocannabinol carboxylic acid (17 ng/ml) was used, 86% of the EMIT positive blood samples contained THC concentrations above the cut-off limit of 1 nM (0.3 ng/ml). A high EMIT result gave a high probability of finding a high THC concentration in the subsequent confirmation analysis.     

Deposition of cannabinoids in hair after long-term use of cannabis

Hair analysis has shown great potential in the detection and control of drug use. Whether an assay is of quantitative value roughly corresponding to the amount of drug consumed, is still a matter of debate. The present investigation was aimed at a possible relationship between the cannabinoid concentration in hair and the cumulative dose in regular users of cannabis. Hair samples from the vertex region of the scalp were obtained from 12 male regular users of cannabis, and 10 male subjects with no experience of cannabis served as controls. None of the subjects had his hair permed, bleached or colored. Cannabis users provided information on drug use such as the current cannabis dose per day, the cumulative cannabis dose of the last 3 months, as well as the frequency of cannabis use during the last year. The concentration of delta-9-tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD) in hair was determined using gas chromatography-mass spectrometry. Cannabinoids were present in any hair sample of cannabis users, but were not detectable in control specimens. An increase in the amount of cannabinoids in hair with increasing dose was evident. The concentration of major cannabinoids (sum of THC, CBD and CBN) was significantly correlated to either the reported cumulative cannabis dose during the last 3 months or to the cannabis use during the last 3 months estimated from the daily dose and the frequency per year (r=0.68 or 0.71, p=0.023 or 0.014). A significant relationship between THC and the amount of cannabis used could not be established. As a conclusion, the sum of major cannabinoids in hair of regular users may provide a better measure of drug use than THC.