Validation and Quantitation of Fifteen Cannabinoids in Cannabis and Marketed Products Using High-Performance Liquid Chromatography-Ultraviolet/Photodiode Array Method.

Background: Cannabis sativa is a psychoactive plant indigenous to Central and South Asia, traditionally used both for recreational and religious purposes, in addition to folk medicine. Cannabis is a rich source of natural compounds, the most important of which are commonly known as cannabinoids that cause a variety of effects through interaction with the endocannabinoid system. Materials and Methods: In this study, a high-performance liquid chromatography-ultraviolet/photodiode array (PDA) method was developed and validated for the analysis of 15 cannabinoids in cannabis plant materials and cannabis-based marketed products. These cannabinoids are cannabidivarinic acid, cannabidivarin, cannabidiolic acid, cannabigerolic acid, cannabigerol, cannabidiol, delta-9-tetrahydrocannabivarin, delta-9-tetrahydrocannabivarinic acid, cannabinol, delta-9-tetrahyrocannabinol, delta-8-tetrahyrocannabinol, cannabicyclol, cannabichromene, delta-9-tetrahyrocannabinolic acid A, and cannabichromenic acid. The separation was carried out using a reversed-phase Luna® C18(2) column and a mobile phase consisting of 75% acetonitrile and 0.1% formic acid in water. A PDA detector was used, and data were extracted at λ=220 nm. Principal component analysis of cannabis four varieties was performed. Results: The method was linear over the calibration range of 5-75 µg/mL with R2>0.999 for all cannabinoids. This method was sensitive and gave good baseline separation of all examined cannabinoids with limits of detection ranging between 0.2 and 1.6 µg/mL and limits of quantification ranging between 0.6 and 4.8 µg/mL. The average recoveries for all cannabinoids were between 81% and 104%. The measured repeatability and intermediate precisions (% relative standard deviation) in all varieties ranged from 0.35% to 9.84% and 1.11% to 5.26%, respectively. Conclusions: The proposed method is sensitive, selective, reproducible, and accurate. It can be applied for the simultaneous determination of these cannabinoids in the C. sativa biomass and cannabis-derived marketed products.

Development and Validation of a GC-FID Method for the Quantitation of 20 Different Acidic and Neutral Cannabinoids.

For decades, Cannabis sativa had been illegal to sell or consume around the world, including in the United States. However, in light of the recent 2018 Farm Bill and the legalization of hemp across the US, various cannabis preparations have flooded the market, making it essential to be able to quantitate the levels of the different acidic and neutral cannabinoids in C. sativa and to have a complete cannabinoid profile of the different chemovars of the cannabis plant. A GC-FID method was developed and validated for the analysis of 20 acidic and neutral cannabinoids as trimethylsilyl (TMS) derivatives. The analyzed cannabinoids include cannabidivarinic acid (CBDVA), cannabidiolic acid (CBDA), cannabinolic acid (CBNA), cannabielsoic acid (CBEA), cannabicyclolic acid (CBLA), cannabichromenic acid (CBCA), trans-Δ9-tetrahydrocannabivarinic acid (Δ9-THCVA), trans-Δ9-tetrahydrocannabinolic acid A (Δ9-THCAA), cannabigerolic acid (CBGA), cannabidiol (CBD), cannabicyclol (CBL), cannabidivarin (CBDV), trans-Δ9-tetrahydrocannabivarin (THCV), cannabichromene (CBC), trans-Δ8-tetrahydrocannabinol (Δ8-THC), trans-Δ9-tetrahydrocannabinol (Δ9-THC), cannabigerol (CBG), cannabinol (CBN), cannabicitran (CBT), and cannabielsoin (CBE). The method limit of detection (LOD) was as low as 0.1 µg/mL, while the limit of quantitation ranged from 0.25 µg/mL to 0.5 µg/mL. The precision (%RSD) was < 10%, while trueness ranged from 90 - 107%. The developed method is simple, accurate, and sensitive for the quantitation of all 20 acidic and neutral cannabinoids. Finally, the proposed method was successfully applied to the quantitation of the cannabinoids in different cannabis chemovars grown at the University of Mississippi.

Microbial Biotransformation of Cannabidiol (CBD) from Cannabis sativa.

Microbial biotransformation of cannabidiol was assessed using 31 different microorganisms. Only Mucor ramannianus (ATCC 9628), Beauveria bassiana (ATCC 7195), and Absidia glauca (ATCC 22 752) were able to metabolize cannabidiol. M. ramannianus (ATCC 9628) yielded five metabolites, namely, 7,4″ß-dihydroxycannabidiol (1: ), 6ß,4″ß-dihydroxycannabidiol (2: ), 6ß,2″ß-dihydroxycannabidiol (3: ), 6ß,3″α-dihydroxycannabidiol (4: ), and 6ß,7,4″ß-trihydroxycannabidiol (5: ). B. bassiana (ATCC 7195) metabolized cannabidiol to afford six metabolites identified as 7,3″-dihydroxycannabidivarin (6: ), 7-hydroxycannabidivarin-3″-carboxylic acid (7: ), 3″-hydroxycannabidivarin (8: ), 4″ß-hydroxycannabidiol (9: ), and cannabidivarin-3″-carboxylic acid (10: ) along with compound 1: . Incubation of cannabidiol with A. glauca (ATCC 22 752) yielded three metabolites, 6α,3″-dihyroxycannabidivarin (11: ), 6ß,3″-dihyroxycannabidivarin (12: ), and compound 6: . All compounds were evaluated for their antimicrobial and antiprotozoal activity.

Cannabinoids, Phenolics, Terpenes and Alkaloids of Cannabis.

Cannabis sativa is one of the oldest medicinal plants in the world. It was introduced into western medicine during the early 19th century. It contains a complex mixture of secondary metabolites, including cannabinoids and non-cannabinoid-type constituents. More than 500 compounds have been reported from C. sativa, of which 125 cannabinoids have been isolated and/or identified as cannabinoids. Cannabinoids are C21 terpeno-phenolic compounds specific to Cannabis. The non-cannabinoid constituents include: non-cannabinoid phenols, flavonoids, terpenes, alkaloids and others. This review discusses the chemistry of the cannabinoids and major non-cannabinoid constituents (terpenes, non-cannabinoid phenolics, and alkaloids) with special emphasis on their chemical structures, methods of isolation, and identification.

Propagation of Cannabis for Clinical Research: An Approach Towards a Modern Herbal Medicinal Products Development.

Cannabis has been reported to contain over 560 different compounds, out of which 120 are cannabinoids. Among the cannabinoids, Δ9-tetrahydrocannabinol and cannabidiol are the two major compounds with very different pharmacological profile and a tremendous therapeutic potential. However, there are many challenges in bringing cannabis from grow-farms to pharmaceuticals. Among many, one important challenge is to maintain the supply chain of biomass, which is consistent in its cannabinoids profile. To maintain this process, male plants are removed from growing fields as they appear. Even with that practice, still there are fair chances of cross fertilization. Therefore, controlled indoor cultivation for screening, selection of high yielding female plants based on their cannabinoids profile, and their conservation and multiplication using vegetative propagation and/or micropropagation is a suitable path to ensure consistency in biomass material. In this chapter, the botany and propagation of elite cannabis varieties will be discussed.

Plant microbiome-dependent immune enhancing action of Echinacea purpurea is enhanced by soil organic matter content.

We previously demonstrated that extracts from Echinacea purpurea material varied substantially in their ability to activate macrophages in vitro and that this variation was due to differences in their content of bacterial components. The purpose of the current study was to identify soil conditions (organic matter, nitrogen, and moisture content) that alter the macrophage activation potential of E. purpurea and determine whether these changes in activity correspond to shifts in the plant-associated microbiome. Increased levels of soil organic matter significantly enhanced macrophage activation exhibited by the root extracts of E. purpurea (p < 0.0001). A change in soil organic matter content from 5.6% to 67.4% led to a 4.2-fold increase in the macrophage activation potential of extracts from E. purpurea. Bacterial communities also differed significantly between root materials cultivated in soils with different levels of organic matter (p < 0.001). These results indicate that the level of soil organic matter is an agricultural factor that can alter the bacterial microbiome, and thereby the activity, of E. purpurea roots. Since ingestion of bacterial preparation (e.g., probiotics) is reported to impact human health, it is likely that the medicinal value of Echinacea is influenced by cultivation conditions that alter its associated bacterial community.

Analysis of Terpenes in Cannabis sativa L. Using GC/MS: Method Development, Validation, and Application.

Terpenes are the major components of the essential oils present in various Cannabis sativa L. varieties. These compounds are responsible for the distinctive aromas and flavors. Besides the quantification of the cannabinoids, determination of the terpenes in C. sativa strains could be of importance for the plant selection process. At the University of Mississippi, a GC-MS method has been developed and validated for the quantification of terpenes in cannabis plant material, viz., α-pinene, ß-pinene, ß-myrcene, limonene, terpinolene, linalool, α-terpineol, ß-caryophyllene, α-humulene, and caryophyllene oxide. The method was optimized and fully validated according to AOAC (Association of Official Analytical Chemists) guidelines against reference standards of selected terpenes. Samples were prepared by extraction of the plant material with ethyl acetate containing n-tridecane solution (100 µg/mL) as the internal standard. The concentration-response relationship for all analyzed terpenes using the developed method was linear with r2 values > 0.99. The average recoveries for all terpenes in spiked indoor cultivated samples were between 95.0 - 105.7%, with the exception of terpinolene (67 - 70%). The measured repeatability and intermediate precisions (% relative standard deviation) in all varieties ranged from 0.32 to 8.47%. The limit of detection and limit of quantitation for all targeted terpenes were determined to be 0.25 and 0.75 µg/mL, respectively. The proposed method is highly selective, reliable, and accurate and has been applied to the simultaneous determination of these major terpenes in the C. sativa biomass produced by our facility at the University of Mississippi as well as in confiscated marijuana samples.

Cannabis and cannabinoid drug development: evaluating botanical versus single molecule approaches.

Accumulating evidence suggests that the endocannabinoid system is a promising target for the treatment of a variety of health conditions. Two paths of cannabinoid drug development have emerged. One approach is focused on developing medications that are directly derived from the cannabis plant. The other utilizes a single molecule approach whereby individual phytocannabinoids or novel cannabinoids with therapeutic potential are identified and synthesized for pharmaceutical development. This commentary discusses the unique challenges and merits of botanical vs single molecule cannabinoid drug development strategies, highlights how both can be impacted by legalization of cannabis via legislative processes, and also addresses regulatory and public health considerations that are important to consider as cannabinoid medicine advances as a discipline.

Detection and Quantification of Cannabinoids in Extracts of Cannabis sativa Roots Using LC-MS/MS.

A liquid chromatography-tandem mass spectrometry single-laboratory validation was performed for the detection and quantification of the 10 major cannabinoids of cannabis, namely, (-)-trans-Δ9-tetrahydrocannabinol, cannabidiol, cannabigerol, cannabichromene, tetrahydrocannabivarian, cannabinol, (-)-trans-Δ8-tetrahydrocannabinol, cannabidiolic acid, cannabigerolic acid, and Δ9-tetrahydrocannabinolic acid-A, in the root extract of Cannabis sativa. Acetonitrile : methanol (80 : 20, v/v) was used for extraction; d3-cannabidiol and d3- tetrahydrocannabinol were used as the internal standards. All 10 cannabinoids showed a good regression relationship with r2 > 0.99. The validated method is simple, sensitive, and reproducible and is therefore suitable for the detection and quantification of these cannabinoids in extracts of cannabis roots. To our knowledge, this is the first report for the quantification of cannabinoids in cannabis roots.

Determination of Acid and Neutral Cannabinoids in Extracts of Different Strains of Cannabis sativa Using GC-FID.

Cannabis (Cannabis sativa L.) is an annual herbaceous plant that belongs to the family Cannabaceae. Trans-Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) are the two major phytocannabinoids accounting for over 40% of the cannabis plant extracts, depending on the variety. At the University of Mississippi, different strains of C. sativa, with different concentration ratios of CBD and Δ9-THC, have been tissue cultured via micropropagation and cultivated. A GC-FID method has been developed and validated for the qualitative and quantitative analysis of acid and neutral cannabinoids in C. sativa extracts. The method involves trimethyl silyl derivatization of the extracts. These cannabinoids include tetrahydrocannabivarian, CBD, cannabichromene, trans-Δ8-tetrahydrocannabinol, Δ9-THC, cannabigerol, cannabinol, cannabidiolic acid, cannabigerolic acid, and Δ9-tetrahydrocannabinolic acid-A. The concentration-response relationship of the method indicated a linear relationship between the concentration and peak area ratio with R2 > 0.999 for all 10 cannabinoids. The precision and accuracy of the method were found to be ≤ 15% and ± 5%, respectively. The limit of detection range was 0.11 - 0.19 µg/mL, and the limit of quantitation was 0.34 - 0.56 µg/mL for all 10 cannabinoids. The developed method is simple, sensitive, reproducible, and suitable for the detection and quantitation of acidic and neutral cannabinoids in different extracts of cannabis varieties. The method was applied to the analysis of these cannabinoids in different parts of the micropropagated cannabis plants (buds, leaves, roots, and stems).

Cannabis cultivation: Methodological issues for obtaining medical-grade product.

As studies continue to reveal favorable findings for the use of cannabidiol in the management of childhood epilepsy syndromes and other disorders, best practices for the large-scale production of Cannabis are needed for timely product development and research purposes. The processes of two institutions with extensive experience in producing large-scale cannabidiol chemotype Cannabis crops-GW Pharmaceuticals and the University of Mississippi-are described, including breeding, indoor and outdoor growing, harvesting, and extraction methods. Such practices have yielded desirable outcomes in Cannabis breeding and production: GW Pharmaceuticals has a collection of chemotypes dominant in any one of eight cannabinoids, two of which-cannabidiol and cannabidivarin-are supporting epilepsy clinical trial research, whereas in addition to a germplasm bank of high-THC, high-CBD, and intermediate type cannabis varieties, the team at University of Mississippi has established an in vitro propagation protocol for cannabis with no detectable variations in morphologic, physiologic, biochemical, and genetic profiles as compared to the mother plants. Improvements in phytocannabinoid yields and growing efficiency are expected as research continues at these institutions. This article is part of a Special Issue entitled "Cannabinoids and Epilepsy".

In Vitro Propagation of Cannabis sativa L. and Evaluation of Regenerated Plants for Genetic Fidelity and Cannabinoids Content for Quality Assurance.

Cannabis sativa L. (Marijuana; Cannabaceae), one of the oldest medicinal plants in the world, has been used throughout history for fiber, food, as well as for its psychoactive properties. The dioecious and allogamous nature of C. sativa is the major constraint to maintain the consistency in chemical profile and overall efficacy if grown from seed. Therefore, the present optimized in vitro propagation protocol of the selected elite germplasm via direct organogenesis and quality assurance protocols using genetic and chemical profiling provide an ideal pathway for ensuring the efficacy of micropropagated Cannabis sativa germplasm. A high frequency shoot organogenesis of C. sativa was obtained from nodal segments in 0.5 µM thidiazuron medium and 95 % in vitro rhizogenesis is obtained on half-strength MS medium supplemented with 500 mg/L activated charcoal and 2.5 µM indole-3-butyric acid. Inter Simple Sequence Repeats (ISSR) and Gas Chromatography-Flame Ionization Detection (GC-FID) are successfully used to monitor the genetic stability in micropropagated plants up to 30 passages in culture and hardened in soil for 8 months.

Assessment of total phenolic and flavonoid content, antioxidant properties, and yield of aeroponically and conventionally grown leafy vegetables and fruit crops: a comparative study.

A comparison of the product yield, total phenolics, total flavonoids, and antioxidant properties was done in different leafy vegetables/herbs (basil, chard, parsley, and red kale) and fruit crops (bell pepper, cherry tomatoes, cucumber, and squash) grown in aeroponic growing systems (AG) and in the field (FG). An average increase of about 19%, 8%, 65%, 21%, 53%, 35%, 7%, and 50% in the yield was recorded for basil, chard, red kale, parsley, bell pepper, cherry tomatoes, cucumber, and squash, respectively, when grown in aeroponic systems, compared to that grown in the soil. Antioxidant properties of AG and FG crops were evaluated using 2,2-diphenyl-1-picrylhydrazyl (DDPH) and cellular antioxidant (CAA) assays. In general, the study shows that the plants grown in the aeroponic system had a higher yield and comparable phenolics, flavonoids, and antioxidant properties as compared to those grown in the soil.

Functional identification of valerena-1,10-diene synthase, a terpene synthase catalyzing a unique chemical cascade in the biosynthesis of biologically active sesquiterpenes in Valeriana officinalis.

Valerian is an herbal preparation from the roots of Valeriana officinalis used as an anxiolytic and sedative and in the treatment of insomnia. The biological activities of valerian are attributed to valerenic acid and its putative biosynthetic precursor valerenadiene, sesquiterpenes, found in V. officinalis roots. These sesquiterpenes retain an isobutenyl side chain whose origin has been long recognized as enigmatic because a chemical rationalization for their biosynthesis has not been obvious. Using recently developed metabolomic and transcriptomic resources, we identified seven V. officinalis terpene synthase genes (VoTPSs), two that were functionally characterized as monoterpene synthases and three that preferred farnesyl diphosphate, the substrate for sesquiterpene synthases. The reaction products for two of the sesquiterpene synthases exhibiting root-specific expression were characterized by a combination of GC-MS and NMR in comparison to the terpenes accumulating in planta. VoTPS7 encodes for a synthase that biosynthesizes predominately germacrene C, whereas VoTPS1 catalyzes the conversion of farnesyl diphosphate to valerena-1,10-diene. Using a yeast expression system, specific labeled [(13)C]acetate, and NMR, we investigated the catalytic mechanism for VoTPS1 and provide evidence for the involvement of a caryophyllenyl carbocation, a cyclobutyl intermediate, in the biosynthesis of valerena-1,10-diene. We suggest a similar mechanism for the biosynthesis of several other biologically related isobutenyl-containing sesquiterpenes.

Photosynthetic response of Cannabis sativa L., an important medicinal plant, to elevated levels of CO2.

The effect of elevated CO2 concentrations (545 and 700 µmol mol(-1)) on gas exchange and stomatal response of four high Δ(9)-THC yielding varieties of Cannabis sativa (HPM, K2, MX and W1) was studied to assess their response to the rising atmospheric CO2 concentration. In general, elevated CO2 concentration (700 µmol mol(-1)) significantly (p < 0.05) stimulated net photosynthesis (P N), water use efficiency (WUE) and internal CO2 concentration (C i), and suppressed transpiration (E) and stomatal conductance (g s) as compared to the ambient CO2 concentration (390 µmol mol(-1)) in all the varieties whereas, the effect of 545 µmol mol(-1) CO2 concentration was found insignificant (p < 0.05) on these parameters in most of the cases. No significant changes (p < 0.05) in the ratio of internal to the ambient CO2 concentration (C i/C a) was observed in these varieties under both the elevated CO2 concentrations (545 and 700 µmol mol(-1)). An average increase of about 48 %, 45 %, 44 % and 38 % in P N and, about 177 %, 157 %, 191 % and 182 % in WUE was observed due to elevated CO2 (700 µmol mol(-1)) as compared to ambient CO2 concentration in HPM, K2, MX and W1 varieties, respectively. The higher WUE under elevated CO2 conditions in Cannabis sativa, primarily because of decreased stomatal conductance and subsequently the transpiration rate, may enable this species to survive under expected harsh greenhouse effects including elevated CO2 concentration and drought conditions. The higher P N, WUE and nearly constant C i/C a ratio under elevated CO2 concentrations in this species reflect a close coordination between its stomatal and mesophyll functions.

Genetic identification of female Cannabis sativa plants at early developmental stage.

Sequence-characterized amplified region (SCAR) markers were used to identify female plants at an early developmental stage in four different varieties of Cannabis sativa. Using the cetyl trimethylammonium bromide (CTAB) method, DNA was isolated from two-week-old plants of three drug-type varieties (Terbag W1, Terbag K2, and Terbag MX) and one fiber-type variety (Terbag Fedora A7) of C. sativa grown under controlled environmental conditions through seeds. Attempts to use MADC2 (male-associated DNA from Cannabis sativa) primers as a marker to identify the sex of Cannabis sativa plants were successful. Amplification of genomic DNA using MADC2-F and MADC2-R primers produced two distinct fragments, one with a size of approximately 450 bp for female plants and one for male plants with a size of approximately 300 bp. After harvesting the tissues for DNA extraction, plants were subjected to a flowering photoperiod (i.e., 12-h light cycle), and the appearance of flowers was compared with the DNA analysis. The results of the molecular analysis were found to be concordant with the appearance of male or female flowers. The results of this study represent a quick and reliable technique for the identification of sex in Cannabis plants using SCAR markers at a very early developmental stage.

Potency trends of Δ9-THC and other cannabinoids in confiscated cannabis preparations from 1993 to 2008.

The University of Mississippi has a contract with the National Institute on Drug Abuse (NIDA) to carry out a variety of research activities dealing with cannabis, including the Potency Monitoring (PM) program, which provides analytical potency data on cannabis preparations confiscated in the United States. This report provides data on 46,211 samples seized and analyzed by gas chromatography-flame ionization detection (GC-FID) during 1993-2008. The data showed an upward trend in the mean Δ(9)-tetrahydrocannabinol (Δ(9)-THC) content of all confiscated cannabis preparations, which increased from 3.4% in 1993 to 8.8% in 2008. Hashish potencies did not increase consistently during this period; however, the mean yearly potency varied from 2.5-9.2% (1993-2003) to 12.0-29.3% (2004-2008). Hash oil potencies also varied considerably during this period (16.8 ± 16.3%). The increase in cannabis preparation potency is mainly due to the increase in the potency of nondomestic versus domestic samples.

High frequency plant regeneration from leaf derived callus of high Δ9-tetrahydrocannabinol yielding Cannabis sativa L.

An efficient in vitro propagation protocol for rapidly producing Cannabis sativa plantlets from young leaf tissue was developed. Using gas chromatography-flame ionization detection (GC-FID), high THC yielding elite female clone of a drug-type CANNABIS variety (MX) was screened and its vegetatively propagated clones were used for micropropagation. Calli were induced from leaf explant on Murashige and Skoog medium supplemented with different concentrations (0.5, 1.0, 1.5, and 2.0 µM) of indole- 3-acetic acid (IAA), indole- 3- butyric acid (IBA), naphthalene acetic acid (NAA), and 2,4-dichlorophenoxy-acetic acid (2,4-D) in combination with 1.0 µM of thidiazuron (TDZ) for the production of callus. The optimum callus growth and maintenance was in 0.5 µM NAA plus 1.0 µM TDZ. The two-month-old calli were subcultured to MS media containing different concentrations of cytokinins (BAP, KN, TDZ). The rate of shoot induction and proliferation was highest in 0.5 µM TDZ. Of the various auxins (IAA, IBA, and NAA) tested, regenerated shoots rooted best on half strength MS medium (1/2 - MS) supplemented with 2.5 µM IBA. The rooted plantlets were successfully established in soil and grown to maturity with no gross variations in morphology and cannabinoids content at a survival rate of 95 % in the indoor growroom.

Assessment of the genetic stability of micropropagated plants of Cannabis sativa by ISSR markers.

Inter-simple sequence repeat (ISSR) markers were used to evaluate the genetic stability of the micropropagated plants of Cannabis sativa over 30 passages in culture and hardening in soil for 8 months. A total of 15 ISSR primers resulted in 115 distinct and reproducible bands. All the ISSR profiles from micropropagated plants were monomorphic and comparable to mother plants, confirming the genetic stability among clones and mother plants. Chemical analysis of cannabinoids, using gas chromatography/flame ionization detection (GC/FID), was done to further confirm whether the qualitative and quantitative differences in the major secondary metabolites exist between the mother plant and micropropagated plants. Six major cannabinoids - Delta(9)-THC, THCV, CBD, CBC, CBG, and CBN - were identified and compared with the mother plant. Our results clearly showed a similar cannabinoid profile and insignificant differences in THC content between the two types of plants. These results suggest that the micropropagation protocol developed by us for rapid IN VITRO multiplication is appropriate and applicable for clonal mass propagation of C. SATIVA.

Assessment of cannabinoids content in micropropagated plants of Cannabis sativa and their comparison with conventionally propagated plants and mother plant during developmental stages of growth.

Gas chromatography-flame ionization detection (GC-FID) was used to assess the chemical profile and quantification of cannabinoids to identify the differences, if existing, in the chemical constituents of in vitro propagated plants (IVP), conventionally grown plants (VP) and indoor grown mother plants (MP-Indoor) of a high THC yielding variety of Cannabis sativa L. during different developmental stages of growth. In general, THC content in all groups increased with plant age up to a highest level during the budding stage where the THC content reached a plateau before the onset of senescence. The pattern of changes observed in the concentration of other cannabinoids content with plants age has followed a similar trend in all groups of plants. Qualitatively, cannabinoids profiles obtained using GC-FID, in MP-indoor, VP and IVP plants were found to be similar to each other and to that of the field grown mother plant (MP field) of C. sativa. Minor differences observed in cannabinoids concentration within and among the groups were not found to be statistically significant. Our results confirm the clonal fidelity of IVP plants of C. sativa and suggest that the biochemical mechanism used in this study to produce the micropropagated plants does not affect the metabolic content and can be used for the mass propagation of true to type plants of this species for commercial pharmaceutical use.