By Rebecca Stevens

As mentioned in Part 1, the physiological effects of cannabis are mediated by a group of structurally related organic compounds known as cannabinoids. The cannabinoids are biosynthetically produced by a growing cannabis plant and Figure 1 details the biosynthetic pathways leading to some of the most important cannabinoids in plant material.

Potency figure 1 — Figure 1: The biosynthetic pathway of phytocannabinoid production in cannabis has been deeply studied through isotopic labeling experiments

The analytical measurement of cannabinoids is important to ensure the safety and quality of cannabis as well as its extracts and edible formulations. Total cannabinoid levels can vary significantly between different cultivars and batches, from about 5% up to 20% or more by dry weight. Information on cannabinoid profiles can be used to tailor cultivars for specific effects and allows end users to select an appropriate dose.

Routine Analysis vs. Cannabinomics

Several structurally analogous groups of cannabinoids exist. In total, structures have been assigned for more than 70 unique phytocannabinoids as of 2005 and the burgeoning field of cannabinomics seeks to comprehensively measure these compounds.¹

Considering practical potency analysis, the vast majority of cannabinoid content is accounted for by 10-12 compounds. These include Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabigerol (CBG), Δ9-tetrahydrocannabivarian (THCV), cannabidivarin (CBDV) and their respective carboxylic acid forms. The cannabinoids occur primarily as carboxylic acids in plant material. Decarboxylation occurs when heat is applied through smoking, vaporization or cooking thereby producing neutral cannabinoids which are more physiologically active.

Potency Analysis by HPLC and GC

Currently, HPLC and GC are the two most commonly used techniques for potency analysis. In the case of GC, the heat used to vaporize the injected sample causes decarboxylation of the native cannabinoid acids. Derivatization of the acids may help reduce decarboxylation but overall this adds another layer of complexity to the analysis² ³. HPLC is the method of choice for direct analysis of cannabinoid profiles and this technique will be discussed further.

A sample preparation method consisting of grinding/homogenization and alcohol extraction is commonly used for cannabis flower and extracts. It has been shown to provide good recovery and precision² ³. An aliquot of the resulting extract can then be diluted with an HPLC compatible solvent such as 25% water / 75% acetonitrile with 0.1% formic acid. The cannabinoids are not particularly water soluble and can precipitate if the aqueous percentage is too high.

To avoid peak distortion and shifting retention times the diluent and initial mobile phase composition should be reasonably well matched. Another approach is to make a smaller injection (1-2 µL) of a more dissimilar solvent. The addition of formic acid or ammonium formate buffer acidifies the mobile phase and keeps the cannabinoid acids protonated.

The protonated acids are neutral and thus well retained on a C18 type column, even at higher (~50% or greater) concentrations of organic solvent² ³.

Detection is most often done using UV absorbance. Two main types of UV detectors are available for HPLC, single wavelength and diode array. A diode array detector (DAD) measures absorbance across a range of wavelengths producing a spectrum at each point in a chromatogram while single wavelength detectors only monitor absorbance at a single user selected wavelength. The DAD is more expensive, but very useful for detecting coelutions and interferences.

References

Chemical Constituents of Marijuana: The Complex Mixture of Natural Cannabinoids. Life Sciences, 78, (2005), pp. 539
Development and Validation of a Reliable and Robust Method for the Analysis of Cannabinoids and Terpenes in Cannabis. Journal of AOAC International, 98, (2015), pp. 1503
Innovative Development and Validation of an HPLC/DAD Method for the Qualitative and Quantitative Determination of Major Cannabinoids in Cannabis Plant Material. Journal of Chromatography B, 877, (2009), pp. 4115

Rebecca is an Applications Scientist at Restek Corporation and is eager to field any questions or comments on cannabis analysis, she can be reached by e-mail, rebecca.stevens@restek.com or by phone at 814-353-1300 (ext. 2154)

November 30, 2016

The Practical Chemist

Appropriate Instrumentation for the Chemical Analysis of Cannabis and Derivative Products: Part 1

By Rebecca Stevens

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Election Day 2016 resulted in historic gains for state level cannabis prohibition reform. Voters in California, Maine, Massachusetts and Nevada chose to legalize adult use of Cannabis sp. and its extracts while even traditionally conservative states like Arkansas, Florida, Montana and North Dakota enacted policy allowing for medical use. More than half of the United States now allows for some form of legal cannabis use, highlighting the rapidly growing need for high quality analytical testing.

For the uninitiated, analytical instrumentation can be a confusing mix of abbreviations and hyphenation that provides little obvious information about an instrument’s capability, advantages and disadvantages. In this series of articles, my colleagues and I at Restek will break down and explain in practical terms what instruments are appropriate for a particular analysis and what to consider when choosing an instrumental technique.

Potency Analysis

Potency analysis refers to the quantitation of the major cannabinoids present in Cannabis sp. These compounds are known to provide the physiological effects of cannabis and their levels can vary dramatically based on cultivation practices, product storage conditions and extraction practices.

The primary technique is high performance liquid chromatography (HPLC) coupled to ultraviolet absorbance (UV) detection. Gas chromatography (GC) coupled to a flame ionization detector (FID) or mass spectrometry (MS) can provide potency information but suffers from issues that preclude its use for comprehensive analysis.

Pesticide Residue Analysis

Pesticide residue analysis is, by a wide margin, the most technically challenging testing that we will discuss here. Trace levels of pesticides incurred during cultivation can be transferred to the consumer both on dried plant material and in extracts prepared from the contaminated material. These compounds can be acutely toxic and are generally regulated at part per billion parts-per-billion levels (PPB).

Depending on the desired target pesticides and detection limits, HPLC and/or GC coupled with tandem mass spectrometry (MS/MS) or high resolution accurate mass spectrometry (HRAM) is strongly recommended. Tandem and HRAM mass spectrometry instrumentation is expensive, but in this case it is crucial and will save untold frustration during method development.

Residual Solvents Analysis

When extracts are produced from plant material using organic solvents such as butane, alcohols or supercritical carbon dioxide there is a potential for the solvent and any other contaminants present in it to become trapped in the extract. The goal of residual solvent analysis is to detect and quantify solvents that may remain in the finished extract.

Residual solvent analysis is best accomplished using GC coupled to a headspace sample introduction system (HS-GC) along with FID or MS detection. Solid phase microextraction (SPME) of the sample headspace with direct introduction to the GC is another option.

Terpene Profile Analysis

While terpene profiles are not a safety issue, they provide much of the smell and taste experience of cannabis and are postulated to synergize with the physiologically active components. Breeders of Cannabis sp. are often interested in producing strains with specific terpene profiles through selective breeding techniques.

Both GC and HPLC can be employed successfully for terpenes analysis. Mass spectrometry is suitable for detection as well as GC-FID and HPLC-UV.

Heavy Metals Analysis

Metals such as arsenic, lead, cadmium, chromium and mercury can be present in cannabis plant material due to uptake from the soil, fertilizers or hydroponic media by a growing plant. Rapidly growing plants like Cannabis sp. are particularly efficient at extracting and accumulating metals from their environment.

Several different types of instrumentation can be used for metals analysis, but the dominant technology is inductively coupled plasma mass spectrometry (ICP-MS). Other approaches can also be used including ICP coupled with optical emission spectroscopy (ICP-OES).