Quantifying 16 Cannabinoids in Hemp Flower and Leaf using LC/MS/MS

A Fast, Sensitive and Comprehensive Assay to Quantify 16 Cannabinoids in Hemp Flower and Leaf using LC/MS/MS

Introduction

Marijuana is defined as any cannabis sativa plant that has greater than 0.3 percent tetrahydrocannabinol (THC). In comparison, hemp is a phenotype of the Cannabis sativa plant species that has 0.3 percent or less THC. In 2018, AOAC published a method to quantify 10 cannabinoids (CBDs) with a range of 0.500-10.0 μg/mL in finished goods. However, there are more than 100 CBDs isolated from cannabis in addition to these 10 CBDs in the AOAC method. In 2019, Dyad Labs validated an LC/MS/MS method for 16 CBDs in distillate, extract, and other finished products. In this paper, Dyad Labs has successfully developed and validated another fast, sensitive, and comprehensive LC/MS/MS assay to quantify 16 cannabinoids in the hemp leaf and flower samples.

Methodology

The hemp sample was homogenized into powder by the Resch Mixer Mill MM400. 0.500 gram of hemp powder was weighed and extracted with ethanol: water (90:10, v/v) in an amber container, followed by filtration with a 0.45 μm membrane filter. The internal standards, including cannabidiol-d3, cannabinol-d3, and delta9-THC-d3, were used in this method. Extracts were injected for analysis on Shimadzu Nexera UPLC system with an ARC-18 column at 35 °C.

Results and Methods

Stability

In the AOAC method, the standard solutions are expensive and have a stability of only 3 days. To better understand the stability of CBD, we comprehensively investigated the stability of all CBDs under various parameters, including light, heat, and pH. The study indicated that CBDs are sensitive to light, heat, and pH. Neutral condition with light protection and low temperature was adopted during sample preparation. Dyad Labs has successfully established stability up to 149 days.

Chromatographic Separation

Some CBDs are isomers and have the same MRM transitions, thus chromatographic separation is very critical in this method. We have investigated different columns, mobile phases, and buffers to achieve enough separation, a sharp and symmetric peak shape. A special C18 column provided sufficient separation.

Accuracy and Precision

The accuracy and precision were investigated by post-spiking all sixteen CBDs in hemp leaf and flower at the lower, medium, and high regions of the established calibration curve range (20.0-2000 mg/mL).

Conclusion

This method is a fast, sensitive, comprehensive, and accurate method and is the first validated method to simultaneously quantify 16 major CBDs in hemp flower and leaf samples using UPLC‐MS/MS.