Extraction and determination of tetrahydrocannabinol in oil

Abstract

Tetrahydrocannabinol (THC) is responsible for the psychoactive effects of cannabis. As interest in medicinal cannabis increased, extraction and determination of THC gained importance. The aim of this study was to develop an effective, efficient, and reproducible procedure for the extraction and determination of THC from oil. Reversed-Phase High Performance Liquid Chromatography (HPLC) was performed using an Agilent 1260 Infinity system with an ACE 5 C18 column (250 x 4.6mm id) using OpenLab software. Chemicals and reagents used included LGC standard solution of THC 0.1mg/ml in methanol, Sigma-Aldrich internal standard of ibuprofen, Natures aid MCT oil and Primadonna Extra Virgin Olive oil. Honeywell HPLC grade acetonitrile and methanol were used. Methods of physical extraction were tested at different velocities and times using Vortex-Genie 2, Langford Sonomatic 1400 Ultrasonic Bath and Eppendorf Minispin centrifuge. Method development was conducted at the department of Pharmacy, University of Malta. A rapid HPLC method for analysis and determination of THC, CBD and CBN in methanol was first developed. HPLC parameters that repeatedly resulted in good results were mobile phase: phosphate buffer (pH2.5) and acetonitrile (80:20, v/v) at flow rate of 2ml/min and UV detector wavelength of 220nm. Analyses were conducted in triplets to ensure reproducibility and precision of results. Resolution of chromatograph, peak shape and retention time of THC were observed while area under the peak of THC was measured. Selected method for extraction of THC from oil involved: vortex mixing for 30 seconds, sonication for 15 minutes, followed by centrifugation for 15 minutes at 6000rpm. Two immiscible layers were yielded. The top layer, methanol and THC was extracted using a micropipette, refrigerated for 12 hours, and centrifuged for 15 minutes. Analyte was passed through a syringe filter prior to HPLC analysis. Method development was initially conducted using MCT oil as a carrier, this method was not found efficient or reproducible due to blockage and damage of the stationary phase and long elution time of MCT oil. Method development was hence continued using Extra Virgin Olive oil as a carrier. HPLC parameters that repeatedly resulted in good THC peak shape and resolution following extraction of THC from Extra Virgin Olive oil were mobile phase: phosphate buffer (pH2.5) and acetonitrile (70:30, v/v) at flow rate of 2ml/min, UV detector wavelength of 220nm and column temperature of 40°C.