Comparing Extraction Methods in Sample Preparation for the Quantification of Cannabinoids in Industrial Hemp

Industrial hemp is legally defined in the United States by the Agriculture Improvement Act of 2018 (2018 Farm Bill) as Cannabis containing <0.3% total tetrahydrocannabinol (THC). The 2018 Farm Bill does not, however, specify standard methods for sample preparation or quantification of cannabinoids (including THC) in Cannabis. Extraction efficiency of phytochemicals is well-known to depend on the solvent and extraction method used. In this project, we evaluated the effect of sample preparation extraction methods on the quantitative analysis of five cannabinoids found in industrial hemp with regulatory or commercial significance: cannabidiol (CBD), cannabidiolic acid (CBDA), delta-9-tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THCA), and cannabinol (CBN). Extraction methods evaluated include: QuEChERS, diethyl ether, ethanol, and methanol. Extracts obtained via these methods were subject to quantitative cannabinoid analysis by UPLC/PDA. Standard curves for quantification of each cannabinoid were constructed using authentic standards for quantification. The concentrations of each cannabinoid in the plant material determined via each of the extraction methods were compared using one-way ANOVA followed by Tukey's HSD (significant difference defined as p <0.05). All extraction methods evaluated returned different concentrations of total THC in the plant material. The QuEChERS extraction resulted in the highest calculated concentrations of THC, THCA and CBDA, reporting three to four times greater than obtained via other extractions evaluated. Classification of the starting plant material as hemp or marijuana depended on the extraction method used. These findings clearly and quantitatively demonstrate the need for standardization of extraction methods for hemp analysis and regulation.