Development and Optimization of Supercritical Fluid Extraction Setup Leading to Quantification of 11 Cannabinoids Derived from Medicinal Cannabis

Abstract

In this study, the optimal setup of supercritical fluid extraction (SFE) was designed and developed, leading to the quantitation of 11 distinct cannabinoids (cannabidivann (CBDV), tetrahydrocannabivann (THCV), cannabidiol (CBD), cannabigerol (CBG) cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabinol (CBN), delta 9-tetrahydrocannabinol (Δ⁹-THC), delta 8-tetrahydrocannabinol (Δ⁸-THC), cannabichomere (CBC) and delta 9-tetrahydrocannabinol acid (THCA-A)) extracted from the flowers of medicinal cannabis (sp. Sativa). Supercritical carbon dioxide (scCO₂) extraction was performed at 37 °C, a pressure of 250 bar with the maximum theoretical density of CO₂ (893.7 kg/m³), which generated the highest yield of cannabinoids from the flower-derived extract. Additionally, a cold separator (separating chamber) was used and positioned immediately after the sample containing chamber to maximize the yield. It was also found that successive washing of the extract with fresh scCO₂ further increased yields. Ultra-high performance liquid chromatography coupled with DAD (uHPLC-DAD) was used to develop a method for the quantification of 11 cannabinoids. The C18 stationary phase was used in conjunction with a two solvent system gradient program resulting in the acquisition of the well-resolved chromatogram over a timespan of 32 min. The accuracy and precision of isolated cannabinoids across inter-and intra-day periods were within acceptable limits (<±15%). The assay was also fully validated and deemed sensitive from linearity, LOQ, and LOD perspective. The findings of this body of work are expected to facilitate improved conditions for the optimal extraction of select cannabinoids using scCO₂, which holds promise in the development of well-characterized medicinal cannabis formulations. As to our best knowledge, this is the first study to report the uHPLC quantification method for the analysis of 11 cannabinoids from scCO₂ extract in a single run with more than 1 min peak separation.

Keywords: SFE Helix unit; SFE Nottingham unit; cannabis flowers; neutral cannabinoids (sp. Sativa); supercritical carbon dioxide (scCO2); supercritical extraction.

Figure 1

The schematic representation of the cannabis sample preparation and analysis.

Figure 2

Schematic representation of the SFE Nottingham unit for the experimental setup A (A); Sample containing chamber was inverted and placed on stirrer for the depressurization (B).

Figure 3

Schematic representation of the Helix unit for setup E extraction.

Figure 4

Schematic representation of the Helix unit for setup B extraction.

Figure 5

Schematic representation of glass wool after experimental setup A (A) and SFE Nottingham unit extraction (B).

Figure 6

Schematic representation of bottom floor of the sample holding chamber in Helix unit. (A): bottom filter was blocked during depressurization of CO₂ from bottom outlet. (B): cannabis sample placed at the top of cone.

Figure 7

Throttling effect during the extract collection, (A): dry ice formation at sudden drop in pressure (250 bar to 0 bar), (B): Collection of extract at low pressure drop (50 bar to 0 bar).

Figure 8

HPLC profile of 11 cannabinoids using an analytical column, (A) ACE 5 C18-AR (250 mm × 4.6 mm ID, 5 μm) and (B) conventional c-18 column [20]. Elution order: 1-CBDV, 2-CBDA, 3-CBGA, 4-CBG, 5-CBD, 6-THCV, 7-CBN, 8-d9THC, 10-CBC, and 11-THCA. Reprinted with permission from Ref. [20]. Copyright 2021 Copyright Ciolino.

Figure 9

HPLC profile of 11 cannabinoids by using Luna C-18 analytical column [21]. Elution order: 1-I.S, 2-CBDA, 3-CBGA, 4-CBG, 5-CBD, 6-THCV, 7-CBN, 8-d9THC, 9-d8THC, 10-CBL, 11-CBC, and 12-THCA. Reprinted with permission from ref. [21]. Copyright 2021 Copyright Gul.

Figure 10

Peaks identification of eleven cannabinoids. (a) uHPLC profile of CBDV; (b) uHPLC profile of THCV; (c) uHPLC profile of CBD; (d) uHPLC profile of CBG; (e) uHPLC profile of CBDA; (f) uHPLC profile of CBGA; (g) uHPLC profile of CBN; (h) uHPLC profile of Δ⁹-THC; (i) uHPLC profile of Δ⁸-THC; (j) uHPLC profile of CBC; (k) uHPLC profile of THCA-A; (l) uHPLC profile of 11 cannabinoid mixture.

Figure 10

Peaks identification of eleven cannabinoids. (a) uHPLC profile of CBDV; (b) uHPLC profile of THCV; (c) uHPLC profile of CBD; (d) uHPLC profile of CBG; (e) uHPLC profile of CBDA; (f) uHPLC profile of CBGA; (g) uHPLC profile of CBN; (h) uHPLC profile of Δ⁹-THC; (i) uHPLC profile of Δ⁸-THC; (j) uHPLC profile of CBC; (k) uHPLC profile of THCA-A; (l) uHPLC profile of 11 cannabinoid mixture.

Figure 11

uHPLC profile of cannabis strains: (A) with dominant CBD + CBDA (around 90% w/w); and (B) with 55% w/w CBD+CBDA and 35% THC + THCA in the cannabinoid mixture.