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. 2022 May 5;27(9):2965.
doi: 10.3390/molecules27092965.

Direct Quantitation of Phytocannabinoids by One-Dimensional 1H qNMR and Two-Dimensional 1H-1H COSY qNMR in Complex Natural Mixtures

Evangelos Dadiotis  1 , Vangelis Mitsis  2 , Eleni Melliou  1 , Prokopios Magiatis  1
Affiliations

Direct Quantitation of Phytocannabinoids by One-Dimensional 1H qNMR and Two-Dimensional 1H-1H COSY qNMR in Complex Natural Mixtures

Evangelos Dadiotis et al. Molecules. .

Abstract

The widespread use of phytocannabinoids or cannabis extracts as ingredients in numerous types of products, in combination with the legal restrictions on THC content, has created a need for the development of new, rapid, and universal analytical methods for their quantitation that ideally could be applied without separation and standards. Based on previously described qNMR studies, we developed an expanded 1H qNMR method and a novel 2D-COSY qNMR method for the rapid quantitation of ten major phytocannabinoids in cannabis plant extracts and cannabis-based products. The 1H qNMR method was successfully developed for the quantitation of cannabidiol (CBD), cannabidiolic acid (CBDA), cannabinol (CBN), cannabichromene (CBC), cannabichromenic acid (CBCA), cannabigerol (CBG), cannabigerolic acid (CBGA), Δ9-tetrahydrocannabinol (Δ9-THC), Δ9-tetrahydrocannabinolic acid (Δ9-THCA), Δ8-tetrahydrocannabinol (Δ8-THC), cannabielsoin (CBE), and cannabidivarin (CBDV). Moreover, cannabidivarinic acid (CBDVA) and Δ9-tetrahydrocannabivarinic acid (Δ9-THCVA) can be distinguished from CBDA and Δ9-THCA respectively, while cannabigerovarin (CBGV) and Δ8-tetrahydrocannabivarin (Δ8-THCV) present the same 1H-spectra as CBG and Δ8-THC, respectively. The COSY qNMR method was applied for the quantitation of CBD, CBDA, CBN, CBG/CBGA, and THC/THCA. The two methods were applied for the analysis of hemp plants; cannabis extracts; edible cannabis medium-chain triglycerides (MCT); and hemp seed oils and cosmetic products with cannabinoids. The 1H-NMR method does not require the use of reference compounds, and it requires only a short time for analysis. However, complex extracts in 1H-NMR may have a lot of signals, and quantitation with this method is often hampered by peak overlap, with 2D NMR providing a solution to this obstacle. The most important advantage of the COSY NMR quantitation method was the determination of the legality of cannabis plants, extracts, and edible oils based on their THC/THCA content, particularly in the cases of some samples for which the determination of THC/THCA content by 1H qNMR was not feasible.

Keywords: COSY NMR; cannabichromene (CBC); cannabidiol (CBD); cannabielsoin (CBE); cannabigerol (CBG); cannabinoids; cannabinol (CBN); nuclear magnetic resonance spectroscopy (NMR); qNMR; tetrahydrocannabinol (THC).

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Conflict of interest statement

One of the authors (V.M.) is owner of the Ekati Alchemy Lab, SL. The other authors declare no conflict of interest.

Figures

Figure 1
Figure 1
1H-NMR spectra of pure cannabinoids CBD, CBDA, CBG, CBGA, CBC, CBCA, Δ9-THC, Δ9-THCA, CBN, Δ8-THC, and CBE dissolved in CDCl3. Selected signals of Table 1 are marked with asterisks.
Figure 2
Figure 2
1H-NMR spectra of pure cannabinoids CBD, CBDV, CBDA, CBDVA, Δ9-THCA, Δ9-THCVA CBG, CBGV, Δ8-THC, and Δ8-THVC dissolved in CDCl3. The selected signals of CBD and CBDV are marked with black dots, the selected signals of CBDA and CBDVA are marked with blue dots, and the selected signals of THCA and THCVA are marked with red dots. The encircled peaks highlight the similarities between CBG and CBGV and between Δ8-THC and Δ8-THCV.
Figure 3
Figure 3
Cannabidiol integration in COSY NMR for calibration curve construction: (a) The integration of tyrosol (IS) cross peak (7.11 ppm/6.80 ppm) was set as 100; (b) The integrations of CBD cross peak (4.68 ppm/1.65 ppm) in various increasing concentrations of 1 mg/0.75 mL, 2 mg/0.75 mL, and 5 mg/0.75 mL, with stable IS concentration (1 mg/0.75 mL) are 12.26, 25.66, and 58.22, respectively.
Figure 4
Figure 4
1H-NMR spectrum of hemp extract after distillation.
Figure 5
Figure 5
Δ9-THC quantitation with 1H-1H-COSY qNMR in plant material: (a) 1H-NMR spectrum of hemp plant; (b,c) detection of the presence of Δ9-THC in the material; and (d,e) Integration of the selected cross peak and quantitation of Δ9-THC & Δ9-THCA with 1H-1H COSY qNMR.
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