Cannabinoid Analysis of Hemp: Developing an Efficient HPLC Method Workflow

Abstract

Several different HPLC method options are explored for the efficient and time effective analysis of cannabinoids in a sample of hemp flower. The differences between gradient and isocratic analyses are examined, and the two methods compared in determining the weight percentage of cannabinoids.

Introduction

Consumption of both marijuana and hemp products for medical use stems from the proposed benefits of cannabinoids and other natural compounds present in the plants. In the case of marijuana, the need for cannabinoid testing, often referred to as “potency testing”, is driven by the need to assure product quality and safety. Cannabinoid testing of hemp and hempderived products is also on the rise. Hemp, classified as a strain of Cannabis sativa with low THC content, was made legal on a federal level in the U.S. with the passing of the Agriculture Improvement Act of 2018, also known as the “2018 Farm Bill”.¹ This resulted in the establishment of the U.S. Domestic Hemp Production Program by the United States Department of Agriculture (USDA). Guidelines issued under this program as the “interim final rule” (IFR) designate that for a product to be classified as hemp, its THC content must be < 0.3%.² This has subsequently driven not only quality testing of hemp for cannabinoid content, but also the need for accurate THC measurement to ensure that the product meets legal requirements.

The two major cannabinoids of interest in both marijuana and hemp are Tetrahydrocannabinol (THC) and Cannabidiol (CBD), which are commonly reported on product information labels. However, there are many more cannabinoids present, and some states are increasing their requirements for the number that must be reported. Thereby, testing must be able to distinguish and provide accurate results for multiple cannabinoids.

In the case of marijuana and hemp flower, a common approach to cannabinoid testing incorporates a simple liquid extraction with methanol or ethanol, followed by HPLC-UV analysis. UV detector is preferred as it is easier and less expensive to operate than a mass spectral (MS) detector; however, it requires peaks to be separated chromatographically for proper identification and accurate quantitation.

Many HPLC methods for cannabinoid analysis utilize a gradient elution. But this often adds to the overall run time as an equilibration to initial conditions is required for every sample. Using an isocratic method eliminates the need for repeat equilibration, which may in turn allow more samples to be run per unit time. However, the tradeoff can be a loss in resolution during some portions of the run. In this work, we demonstrate both gradient and isocratic conditions capable of separating 17 cannabinoids on Ascentis^® Express HPLC columns. For each set of conditions, the overall elution time was …

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