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

Abstract

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, β-pinene, β-myrcene, limonene, terpinolene, linalool, α-terpineol, β-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.

Absence of entourage: Terpenoids commonly found in Cannabis sativa do not modulate the functional activity of Δ9-THC at human CB1and CB2 receptors

“…Cannabis also contains a large number of monoterpene and sesquiterpene compounds (together called terpenoids), the most common of which include α-pinene, β-pinene, linalool, limonene and β-myrcene (monoterpenes) and β-caryophyllene and caryophyllene oxide (sesquiterpenes). 3 Terpenoids are volatile compounds that are synthesised alongside phytocannabinoids mainly in the trichomes of the cannabis plant, and provide cannabis with its distinctive aroma and flavour. 4 Terpenoids are often lost if the extraction process involves heating.…”

Terpenoids, Cannabimimetic Ligands, beyond the Cannabis Plant

“…Terpineol (2-(4-methylcyclohex-3-en-1-yl)propan-2-ol) is a volatile monoterpene alcohol present in the essential oil of Cannabis sativa [120], but also in several medicinal plants, such as Punica granatum L., Rosmarinus officinalis L., and Psidium guajava L. Until this moment, there is no evidence in the literature about the interaction of terpineol with CBR. Nonetheless, this compound shows different pharmacological properties that include antinociceptive [121], antifungal [122], anti-inflammatory [123], and antidiarrheal [124].…”

“…Alpha-pinene is considered a natural compound present not only in Cannabis sativa but also in essential oils of many aromatic plants, such as Lavender angustifolia, Rosmarinus officinalis, and coniferous trees [149]. Alpha-pinene is a bicyclo[3.1.1]hept-2-ene that contains a reactive 4-membered ring structure and exhibits antioxidant, antimicrobial, anti-tumor, hypnotic, and anxiolytic activities [83,120,[150][151][152]. There are different biological properties described to α-pinene, as well as essential oils containing this compound have been used to treat several diseases [153], although no affinity towards CBRs have been described [154].…”

A High-Throughput Method for the Comprehensive Analysis of Terpenes and Terpenoids in Medicinal Cannabis Biomass

“…As Leghissa and colleagues note [23], conclusively identifying terpenes in cannabis is challenging due to the large variety of possible candidates and a lack of commercial standards for a large number of them. With the increased interest in medicinal cannabis and the contribution of terpenes to the entourage effect, methods to quantify terpenes in medicinal cannabis biomass and products are becoming more available in the literature [24][25][26][27]. Often, these methods require large quantities of biomass (1-5 g), use large quantities of organic solvents (up to 100 mL/sample), and include separations with gradient runtimes exceeding 60 min.…”