Separating and Quantifying THC and THCA Using High Performance Liquid Chromatography

Abstract

A method was developed for a High Performance Liquid Chromatography (HPLC) instrument coupled with a UV-Vis detector to separate and quantify Δ9tetrahydrocannabinol (THC) and Δ9-tetrahydrocannabinolic acid (THCA). Initial conditions for method parameters were chosen from literature, and then an experimental approach was employed to optimize every parameter that had a significant impact on the method. The final method has a gradient mobile phase at flow rate 0.8 mL/min and an oven temperature of 40 °C with an Alltima HP C18 AQ column. Mobile phase A is 0.1% formic acid in water and mobile phase B is 0.1% formic acid in acetonitrile. Mobile phase B is pumped from 65% to 80% following a linear 8-min time gradient, followed by a linear 1-min step gradient back down to 65%, with total collection ending after 11 minutes. This method produces a peak for THC around 3.60 minutes and a peak for THCA around 4.67 minutes. Calibration curves were made from standards, where the Limit of Detection (LOD) and Limit of Quantification (LOQ) is calculated for THC as 30 ppm and 100 ppm, respectively, and calculated for THCA as 9.1 ppm and 30 ppm, respectively. A hemp oil sample was acquired from Amazon and has an undetectable amount of THC and a calculated THCA concentration of 47.2 ppm. The hemp oil sample was spiked with 10 µL of THC and 10 µL of THCA and has an undetectable amount of THC and a calculated THCA concentration of 56 ppm. Future work includes obtaining higher concentrations of THC and THCA to test using this method and identifying other peaks found in the hemp oil chromatograms.