Cannabis Sativa Samples Research Articles

An initial exploration of machine learning for establishing associations between genetic markers and THC levels in Cannabis sativa samples

Cannabis sativa, a globally commercialized plant used for medicinal, food, fiber production, and recreation, necessitates effective identification to distinguish legal and illegal varieties in forensic contexts. This research utilizes multivariate statistical models and Machine Learning approaches to establish correlations between specific genotypes and tetrahydrocannabinol (Δ9-THC) content (%) in C. sativa samples. 132 cannabis leaves samples were obtained from legal growers in Piedmont, Italy, and illegal drug seizures in Turin. Samples were genetically profiled using a 13-loci STR multiplex and their Δ9-THC content was detected through quantitative GC-MS analysis. This study aims to assess the use of supervised classification modelling on genetic data to distinguish cannabis samples into legal and illegal categories, revealing distinct clusters characterized by unique allele profiles and THC content. t-distributed Stochastic Neighbor Embedding (t-SNE), Random Forest (RF) and Partial Least Squares Regression (PLS-R) were executed for the machine learning modelling. All the tested models resulted effective discriminating between legal samples and illegal. Although further validation is necessary, this study presents a novel forensic investigative approach, potentially aiding law enforcement in significant marijuana seizures or tracking illicit drug trafficking routes.

Profiling of seized Cannabis sativa L. flowering tops by means of microwave-assisted hydro distillation and gas chromatography analyses

This research aimed to support police forces in their battle against illicit drug trafficking by means of a multi-technique approach, based on gas chromatography. In detail, this study was focused on the profiling of volatile substances in narcotic Cannabis sativa L. flowering tops. For this purpose, the Scientific Investigation Department, RIS Carabinieri of Messina, provided 25 seized samples of Cannabis sativa L. The content of Δ9-tetrahydrocannabinol (THC), useful to classify cannabis plant as hemp (≤ 0.2 %) or as marijuana (> 0.2 %), was investigated. Essential oils of illicit drug samples were extracted using a microwave-assisted hydro-distillation (MAHD) system; GC–MS and GC-FID analytical techniques were used for the characterization of the terpenes and terpenoids fingerprint. Furthermore, the enantiomeric and carbon isotopic ratios of selected chiral compounds were investigated using a heart-cutting multidimensional GC (MDGC) approach. The latter exploited a combination of an apolar column in the first dimension, and a chiral cyclodextrin-based column in the second one, prior to parallel isotope-ratio mass spectrometry (C-IRMS) and MS detection. Finally, all the data were gathered into a statistical model, to demonstrate the existence of useful parameters to be used for the classification of seized samples.

Assessing Intraspecific Genetic Variability in Cannabis sativa Populations of Punjab Pakistan Using RAPD and DNA Barcode Markers

Cannabis sativa possess substantial amount of omega-3 fatty acids and several medicinal benefits with potential secondary metabolites. This study employed rbcL, matK, and trnH-psbA barcode markers and RAPD marker to explore genetic diversity in C. sativa populations in Punjab, Pakistan. In population genetic study using DNA barcode markers, cladogram showed investigated samples were genetically similar; however, demonstrated significant genetic diversity with the available DNA sequences of this plant from other regions and countries. The nucleotide sequence variation (% divergence) indicated high intraspecific variation in plants of different geographical zones. However, the RAPD marker analysis displayed genetic diversity among collected Cannabis sativa samples. The principal component analysis reinforced these findings by providing a spatial representation of genetic distances among the samples that revealed a considerable genetic diversity among the collected material and significant intraspecific variation was observed, indicating diverse genetic profiles across geographical regions. This research highlights the potential of molecular markers in germplasm selection for breeding programs.

Is turbo-extraction an efficient method for obtaining cannabinoids?

Cannabis-based products are still not produced in many countries and the great need makes them imported, and often expensive. As an objective, a low-cost extraction method was proposed to facilitate access to the medicinal effects of cannabidiol. The samples of Cannabis sativa used were obtained from in vitro cultivation or from commercially purchased products. Two extraction techniques (ultrasonic extraction and turbo-extraction) and two solvent systems (methanol:chloroform and ethanol) were evaluated. The levels of cannabinoids, total phenolics and total flavonoids were evaluated. The best results were obtained with the turbo-extraction. Ethanol showed a higher yield of cannabinoids, in addition to being a solvent accepted in international conventions in reducing the use of toxic solvents to the environment. Turbo-extraction with ethanol proved to be effective, economical and in line with environmental concerns in the use of ecologically sustainable solvents. As future possibilities, it is proposed to use the information obtained in this study as part of the process of obtaining and subsequent analysis of Cannabis extracts for medicinal purposes.

Evaluation of genetic markers for the analysis of THC levels of Cannabis sativa samples using principal component analysis – A preliminary study

Cannabis sativa is a worldwide commercial plant used for medicinal purposes, food and fiber production, and also as a recreational drug. Therefore, the identification and differentiation between legal and illegal C. sativa is of great importance for forensic investigations. In this study, principal component analysis (PCA), an exploratory data analysis technique, was tested to correlate the specific genotype with the concentration of tetrahydrocannabinol (THC) in the samples. C. sativa samples were obtained from legal growers in Piedmont, Italy, and from illegal drug seizures in the Turin region. DNA was extracted, quantified, amplified with a 13-loci multiplex STR and finally analyzed with an automated sequencer. The results showed a trend in the analyzed samples as they differed by their THC content and allele profiles. PCA yielded two clusters of samples that differed by specific allele profiles and THC concentrations. Further validation studies are needed, but this study could provide a new approach to forensic investigation and be a valuable aid to law enforcement in significant marijuana seizures or in tracing illicit drug trafficking routes.

Kynurenine and kynurenic acid: Two human neuromodulators found in Cannabis sativa L.

L-Kynurenine (KYN) and kynurenic acid (KYNA) are products of the metabolism of L-tryptophan (TRP) in the central nervous system of animals, but they are not commonly found in plants. In particular, KYNA is known for its interesting pharmacological properties (anti-oxidative, anti-inflammatory, hypolipidemic, and neuroprotective), which suggest a potential functional food ingredient role. The three compounds were identified in samples of Cannabis sativa L. by means of high-performance liquid chromatography coupled to high-resolution mass spectrometry using an untargeted metabolomics approach. Their concentrations were evaluated using a targeted metabolomics method in three organs of the plant (roots, stem, and leaves) in soil at two different growth stages and in hydroponics conditions. The distribution of TRP, KYN and KYNA was found tendentially higher in leaves compared to stem and roots and changed over time. Moreover, the levels of KYNA found in this study are unprecedentedly high compared to those found so far in other plant species, suggesting that Cannabis sativa L. could be a promising alternative source of this metabolite.

A Ge.F.I. – ISFG European collaborative study on DNA identification of Cannabis sativa samples using a 13-locus multiplex STR method

Cannabis sativa is the most used controlled substance in Europe. With the advent of new and less restrictive European laws on cannabis sale for recreational use (including in Italy), an increase in indoor cannabis crops were observed. This increase was possible due to the availability of cannabis seeds through the internet market. Genetic identification of cannabis can link seizures and if in possession then might aid in an investigation. A 13-locus multiplex STR method was previously developed and validated by Houston et al. A collaborative exercise was organized by the Italian Forensic Geneticists – International Society of Forensic Genetics (Ge.F.I. – ISFG) Working Group with the aim to test the reproducibility, reliability and robustness of this multiplex cannabis STR kit. Twenty-one laboratories from three European countries participated in the collaborative exercise and were asked to perform STR typing of two cannabis samples. Cannabis DNA samples and the multiplex STR kit were provided by the University of Barcelona and Sam Houston State University. Different platforms for PCR amplification, capillary electrophoresis (CE) and genotyping software were selected at the discretion of the participating laboratories. Although the participating laboratories used different PCR equipment, CE platforms and genotyping software, concordant results were obtained from the majority of the samples. The overall genotyping success ratio was 96%. Only minor artifacts were observed. The mean peak height ratio was estimated to be 76.3% and 78.1% for sample 1 and sample 2, respectively. The lowest amount of −1 / + 1 stutter percentage produced, when the height of the parent allele was higher than 8000 RFU, resulted to be less than 10% of the parent allele height. Few common issues were observed such as a minor peak imbalance in some heterozygous loci, some artifact peaks and few instances of allelic drop-out. The results of this collaborative exercise demonstrated the robustness and applicability of the 13-locus system for cannabis DNA profiling for forensic purposes.

Macro and Trace Elements in Hemp (Cannabis sativa L.) Cultivated in Greece: Risk Assessment of Toxic Elements.

The accumulation of hazardous contaminants in Cannabis sativa L. raises warning signs regarding possible adverse effects on human health due to the consumption of herbal medicines and/or other herbal edible products made from cannabis. Thus, there is an urge to investigate the levels of hazardous contaminants, such as heavy metals, in cannabis plant. In the present study, 29 macro and trace elements, including both beneficial and toxic elements (heavy metals and metalloids), were investigated in 90 samples of Cannabis sativa L. collected from Greece. According to the results, the detected concentrations of macro elements in the leaves/flowers of cannabis ranged between 28 and 138,378 ppm, and of trace elements between 0.002 and 1352.904 ppm. Although the concentrations of elements varied among the samples, their accumulation pattern was found to be similar, with the contribution of toxic elements to the total concentration of trace elements being below 1%. The detected levels of the most toxic elements were below the prescribed limits established by the WHO, while the calculated THQ and CR values showed no risk (non-carcinogenic and carcinogenic) for the population exposed to the current cannabis samples. Positive correlation between the concentration of elements and cannabis geographical origin and variety was observed. Cannabis leaves/flowers were more contaminated with trace and macro elements than seeds.

APPLICATION OF MULTIVARIATE ANALYSIS ON DIGITAL IMAGES OF CANNABIS SATIVA L EXTRACTS

Cannabis sativa L is one of the most used drugs in the world. Information about the plant’s age and storage can help forensic scientists to identify and to track samples. The ratio between the cannabinoids tetrahydrocannabinol (THC) and cannabinol (CBN) has been related to the degradation of cannabis with time. Thus, this study aimed to test Multivariate Image Analysis (MIA) to evaluate cannabis extracts concerning its colors. Initially, 52 samples of Cannabis sativa L. extracts were analyzed by Gas Chromatography coupled to Flame Ionization Detector (GC/FID) to quantify THC and CBN. Afterwards, the extract samples were photographed and analyzed by two different multivariate analysis tools: ChemoStat®, a free chemometrics software, and PhotoMetrix PRO®, an app for mobile devices. Using exploratory analysis of principal component analysis (PCA) and hierarchical cluster analysis (HCA). It was observed that the more intense the color for an extract, the higher concentration of THC and CBN it has, while the lighter color extracts correspond to samples with no THC. The results suggest to propose a simple method for previous clustering of samples that may precede chromatographic analyzes, assist in chemical profile studies or simply aggregate samples of similar profiles for analyzed together.

Evaluation of two 13-loci STR multiplex system regarding identification and origin discrimination of Brazilian Cannabis sativa samples.

According to the Brazilian Federal Police (BFP), the Brazilian Cannabis sativa illicit market is mainly supplied by drugs originated from Paraguay and Northeastern Brazil (Marijuana Polygon region). These two known routes, the increasing indoor cultivations (supported by online market), and drugs from Uruguay are also in BFP's sight. Forensic tools to aid police intelligence were published in the past years. In genetics, microsatellites have gained attention due to their individualization capability. This study aims to evaluate the effectiveness and efficiency of two STR multiplex systems previously proposed in 94 Cannabis sativa samples seized in Brazil. Principal coordinate analyses (PCoA), forensic parameters, and genetic structure analysis were executed. Both panels were effective in individualizing and origin discriminating all samples, and the system proposed in 2015 demonstrated better results. For this marker set, the probability of identity for a random individual is approximately one in 65 billion; also, the PCoA shows a clear genetic distinction among samples according to its origin. Bayesian inference populational structure analysis indicated a significant genetic diversity among seizure groups, matching with its origin. Overall, the STR multiplex systems were able to achieve its purpose in individualizing and differentiating, according to geographic region, Brazilian Cannabis sp. samples.

Piezoelectric peptide-hpDNA based electronic nose for the detection of terpenes; Evaluation of the aroma profile in different Cannabis sativa L. (hemp) samples

A piezoelectric peptide-hpDNA based gas sensor array has been used for the detection of terpenes coming from Cannabis sativa samples. The array consisted in 11 sensors, 6 having pentapeptides and 5 having hairpin DNA as binding elements. The volatile composition of 28 Cannabis sativa samples, assessed by GCMS analysis, allowed their classification into 2 groups having as monoterpenes and sesquiterpenes in different amounts. The response of the gas sensor array to the same samples demonstrated that both type of sensors are sensitive to the terpenes and contribute to classification. A satisfactory classification (79 % of correctly identified samples) was found using a PLS-DA approach. Using the same dataset and a simple ANN approach the headspace analytical profile of the two different groups was predicted with an average prediction error ≤1 %.

13-loci STR multiplex system for Brazilian seized samples of marijuana: individualization and origin differentiation.

It is known that Cannabis in Brazil could either originate from Paraguay or be cultivated in Brazil. While consumer markets in the North and Northeast regions are maintained by national production, the rest of the country is supplied with Cannabis from Paraguay. However, the Brazilian Federal Police (BFP) has exponentially increased the seizure number of Cannabis seeds sent by mail. For this reason, the aim of the study was to assess the 13-loci short tandem repeat (STR) multiplex system proposed by Houston et al. (2015) to evaluate the power of such markers in individualization and origin differentiation of Cannabis sativa samples seized in Brazil by the BFP. To do so, 72 Cannabis samples seized in Brazil by BFP were analyzed. The principal coordinate analysis (PCoA) and probability identity (PI) analysis were computed. Additionally, the Cannabis samples' genotypes were subjected to comparison by Kruskal-Wallis H, followed by a multiple discriminant analysis (MDA). All samples analyzed revealed a distinct genetic profile. PCoA clearly discriminated the seizure sets based on their geographic origin. A combination of seven loci was enough to differentiate samples' genotypes, and the PI for a random sample is approximately one in 50 billion. The Cannabis samples were 100% correct as classified by Kruskal-Wallis H, followed by an MDA. The results of this study demonstrate that the 13-loci STR multiplex system successfully achieved the aim of sample individualization and origin differentiation and suggest that it could be a useful tool to help BFP intelligence in tracing back-trade routes.

Metals and organic compounds in the biosynthesis of cannabinoids: a chemometric approach to the analysis of Cannabis sativa samples

Illicit production and trade of Cannabis sativa affect many societies. This drug is the most popular and easy to produce. Important information for the authorities is the production locality and the indicators of a particular production. This work is an attempt to recognise correlations between the metal content in the different parts of C. sativa L., in soils where plants were cultivated and the cannabinoids content, as a potential indicator. The organic fraction of the leaves of Cannabis plants was investigated by GC-FID analysis. In addition, the determination of Cu, Fe, Cr, Mn, Zn, Ca and Mg was realised by spectroscopic techniques (FAAS and GFAAS). In this study, numerous correlations between metal content in plants and soil, already confirmed in previous publications, were analysed applying chemometric unsupervised methods, that is, principal component analysis, factor analysis and cluster analysis, in order to highlight their role in the biosynthesis of cannabinoids.

Resolution of co-eluting compounds of Cannabis Sativa in comprehensive two-dimensional gas chromatography/mass spectrometry detection with Multivariate Curve Resolution-Alternating Least Squares

Comprehensive Two Dimensional Gas Chromatography - Mass Spectrometry (GC × GC/qMS) analysis of Cannabis sativa extracts shows a high complexity due to the large variety of terpenes and cannabinoids and to the fact that the complete resolution of the peaks is not straightforwardly achieved. In order to support the resolution of the co-eluted peaks in the sesquiterpene and the cannabinoid chromatographic region the combination of Multivariate Curve Resolution and Alternating Least Squares algorithms was satisfactorily applied. As a result, four co-eluting areas were totally resolved in the sesquiterpene region and one in the cannabinoid region in different samples of Cannabis sativa. The comparison of the mass spectral profiles obtained for each resolved peak with theoretical mass spectra allowed the identification of some of the co-eluted peaks. Finally, the classification of the studied samples was achieved based on the relative concentrations of the resolved peaks.

Origin of samples of Cannabis sativa through insect fragments associated with compacted hemp drug in South America

Origin of samples of Cannabis sativa through insect fragments associated with compacted hemp drug in South America. Insects associated with a seizure of Cannabis sativa L. may indicate the origin of the illicit drug. Nevertheless, no work regarding this subject has been previously published for South America. In the present investigation, seven kilograms of vegetal material (C. sativa) were inspected for insect fragments. Three species were identified and used to test the origin of the seizure of cannabis plant material: Euschistus heros (Fabricius, 1794), Thyanta perditor (Fabricius, 1794) (Heteroptera, Pentatomidae), and Cephalotes pusillus (Klug, 1824) (Hymenoptera, Formicidae). These insect species restricted the geographic origin of the drug to the Neotropical region, and their distribution patterns showed an overlap of the State of Mato Grosso (Brazil), Argentina, and Paraguay. Based on this information, two of the three major C. sativa growing areas in South America were excluded: (1) the Colombian territory and (2) northeastern Brazil.

Field Testing of Collection Cards for Cannabis sativa Samples With a Single Hexanucleotide DNA Marker*,†

The validity and feasibility of using DNA collection cards in the field for preservation and analysis of Cannabis sativa genotypes were investigated using a highly specific hexanucleotide marker. Collection cards were submitted to the National Marijuana Initiative, which selectively trained and managed the collection of specific types of samples from a variety of participating agencies. Samples collected at seizure sites included fresh marijuana leaf samples, dried "dispensary" samples, U.S. border seizures, and hashish. Using a standardized PCR kit with custom-labeled oligonucleotide primers specific to marijuana, collection cards produced eight genotypes and 13 different alleles, extremely low baselines, and no cross-reactivity with control plant species. Results were produced from all sample types with the exception of hashish. Plant DNA collection cards represent an easily implementable method for the genetic identification and relatedness of C. sativa street and grow site-seized samples with applications for databasing and market disruption.

STUDY OF CANNABINOIDS CONTENT IN MARIHUANA SAMPLES (<I> Cannabis sativa </I> L.)CULTIVATED IN SEVERAL REGIONS OF COLOMBIA

The present study analyzes the cannabinoids content in samples of Cannabis sativa L. cultivated illicitlyin Colombia. The physicochemical conditions are optimized for the extraction and quantification ofCannabidiol (CBD), 9-Tetrahidrocannabinol (THC) and Cannabinol (CBN) starting from a vegetablesample using gas chromatography with flame ionization detector (GC-FID), validating the respectiveanalytic method. Samples coming from four diferent Colombian regions are analyzed (Llanos Orientales,LL; Cauca, CA; Sierra Nevada de Santa Marta, SN; Eje Cafetero, EC). The study let determination ofCBD, THC and CBN samples quantitatively. The average content of THC in the samples of the regionLL is of 15.74% ± 2.92, in the region CA is of 10.98% ± 6.70, in the region SN is of 2.81% ± 1.72 andin the region EC of 1.87 % ± 1.25. The higher content of THC in vegetable samples of Cannabis from the Llanos Orientales (LL) and Cauca (CA) could be indicative of the employment of improved varieties. This fact generates a great concern about the potential effects produced by the drug in consumers.

DNA Fingerprinting of Cannabis sativa Using Inter-Simple Sequence Repeat (ISSR) Amplification

Chemical analysis of cannabinoid, and Inter-Simple Sequence Repeat (ISSR) fingerprinting of DNA were used to identify different samples of Cannabis sativa L. for forensic purposes. Three samples were classified into two types, tetrahydrocannabinol (THC) and cannabidiol (CBD) chemo-types, by high performance liquid chromatography (HPLC). The two samples of the CBD type were not distinguished by their HPLC patterns. ISSR fingerprinting identified polymorphic DNA patterns between these samples. ISSR fingerprinting clearly differentiated between cannabis samples that could not be achieved by HPLC analysis.

Monoclonal antibody against tetrahydrocannabinolic acid distinguishes Cannabis sativa samples from different plant species

The cross-reaction of anti-Δ 1-THCA MAb against other cannabinoids was very wide. However, other naturally occurring and synthetic phenolics including opium alkaloids did not react to the MAb. Using this ELISA, this paper reports application of the competitive ELISA for detection of marijuana samples. The ELISA described here was very sensitive to the ether extracts of marijuana samples when compared to those of other plants. The assay provided a sensitive method useful for the judge of marijuana samples.

RAPD analysis distinguishes Cannabis sativa samples from different sources

Samples of the seeds and seedlings of Cannabis sativa, and its dried leaves and flowerheads (marijuana), could be reliably distinguished by RAPD-PCR (Random Amplified Polymorphic DNA using the Polymerase Chain Reaction). DNA was best extracted from fresh tissues using buffers and the detergent cetyltrimethylammonium bromide; poorly dried tissue or inviable seed yielded coloured samples of degraded DNA. DNA was isolated from 51 C. sativa and two Humulus lupulus (hops) samples. Of the C. sativa samples 43 were from Australia (ten from Canberra gardens, eight from a New South Wales crop and 25 from two Queensland crops) and eight were from Papua-New Guinea (P-NG). A total of 102 different bands were obtained using four 10-nucleotide primers with arbitrarily chosen sequences. Banding patterns were compared by calculating pairwise distances using various algorithms, and presented using the neighbour-joining tree and multidimensional scaling methods. These showed a clear difference between C. sativa and H. lupulus, and separated the samples of the latter into three distinct groups; one group comprised all the P-NG samples, another the Canberra samples, and the third, the three crop samples.