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

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. <i>Sativa</i>). 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.