How is a cannabinoid analysis performed?

Have you ever wondered how the levels of CBD, THC and other cannabinoids in marijuana or CBD oils are determined? In this post we will explain the science behind the methods that allow cannabinoids to be quantified, such as gas chromatography, HPLC and mass spectrometry, and how these techniques are used together to analyze the components of cannabis.

Introduction to cannabinoid analysis

Cannabinoid analysis is used for a variety of purposes, both therapeutic and regulatory. Its primary purpose is to determine the potency and quality of cannabis products, but it can also be used to legally classify a sample as “hemp” or “marijuana” based on its THC content. It is thus a very useful tool for both consumers and professionals in the cannabis industry.

What are cannabinoids and how are they produced in the plant?

The cannabinoids are a class of chemical compounds found predominantly in the Cannabis sativa plant. Cannabis sativa plant . These are formed in the trichomes, very small glandular structures found mainly in the cannabis flowers of the female plants, better known as the marijuana buds. .

A particularity of cannabinoids is that they are produced by the plant in their acid form, expressed with an “A” at the end of the acronym. This is why some of the best known acidic compounds are Δ-9-tetrahydrocannabinol acid (THCA) and cannabidiolic acid (CBDA).

When cannabinoids are heated, they change to the neutral forms, the cannabinoids we are all familiar with or most popular, such as tetrahydrocannabinol (THC) y cannabidiol ( CBD ). This occurs with heating, as carbon dioxide (CO2) is lost in a process called decarboxylation. decarboxylation .

These acidic and neutral forms of cannabinoids can be measured in different ways, and in this paper I will explain some of them.

Understanding Chromatography: A Key Tool in Cannabinoid Analysis

What is chromatography?

One of the most common ways to measure cannabinoids in their acid and neutral forms is chromatography. Chromatography is a separation technique, performed in laboratories with specialized equipment, where the analytes or chemical compounds that make up a sample are isolated in order to identify them.

To understand it better, we can imagine chromatography as a race. The “track” is the stationary phase, which does not move and through which the mixture we want to analyze passes. The “runners” are the mobile phase, which moves along the track or stationary phase. Cannabinoid substances, like runners, move differently according to their physical properties: some adhere more to the stationary and move more slowly, separating from those that move faster(different retention time). The same would be true for the race simile: as the runners advance along the track, they will be separated according to their physical condition.

How chromatography works

  1. Sample preparation: All chromatography begins with a mixture, which will be the mobile phase. The latter is composed of a solvent fluid (which can be liquid or gaseous) and, in the case of marijuana, the mixture will be composed of cannabis flowers, the CBD oilThe cannabinoids in cannabinoids, which in this case are called analytes. These analytes are the components to be analyzed, those compounds that are separated and measured during chromatography. (The same technique is used to identify terpenes, flavonoids and other compounds, but this is a story for another writing).
  2. Injection: The sample is circulated under pressure through a column composed of a solid or semi-solid material(stationary phase).
  3. Separation: Each of the sample components differs in flow rate, size and polarity, leading to separation as they flow through the column (some adhere more than others).
  4. Detection: As the separated components exit the column, they pass through a detector that records the amount of each exiting. This is usually done by measuring the amount of ultraviolet light that each component absorbs.
  5. Data analysis: The separated components are detected and recorded on a computer, which generates a graph called a chromatogram, the interpretation of which allows accurate identification and quantification of the various cannabinoids present.
Gas chromatography, schematic
Gas chromatography, schematic

Gas chromatography: Analysis of cannabinoids in their neutral form.

Gas chromatography(GC) uses gas as the mobile phase and the separation occurs in a column, which can be a capillary tube. In this technique it is necessary to use heaters to have a uniform sample along the capillary column.

Gas chromatography can only measure cannabinoids in their neutral form.

As it is necessary to heat the sample, GC induces decarboxylation of the cannabinoids to their acid form. Therefore, when cannabinoids are evaluated under this technique, it can only be done with neutral cannabinoids, as heat decarboxylates the acidic forms. This may be one of the disadvantages of gas chromatography.

High Performance Liquid Chromatography (HPLC): Measurement of cannabinoids in acidic and neutral form

Liquid chromatography uses a liquid mobile phase, where a column can also be used. In the cannabis industry, one of the most widely used techniques is High Performance Liquid Chromatography (HPLC), where very small particles are packed at high pressure.

Unlike gas chromatography, liquid chromatography does not require heating of the mixture and can therefore be used to measure the concentration of different cannabinoids, such as CBD and THC, even in their acid form. This makes it the preferred method of analysis for cannabis products.

In HPLC, pumps are used to pass this pressurized liquid (solvent) containing the mixture to be analyzed through the HPLC column. Each of the sample components differs in its flow rate, leading to separation as it flows through the column.

Interpretation of cannabinoid HPLC analysis results

The following figure shows the result of a cannabinoid analysis using HPLC [1]. This type of chart, called chromatogramThe time plot shows the time on the X-axis and the detector signal strength on the Y-axis. Each peak on the graph represents a different component of the sample, and the area under the peak can be used to determine the amount of that component in the sample.

The X-axis (horizontal axis) shows the separation time of each analyte, therefore, the unit is minutes. As you can see, each of the analytes, which in this case are cannabinoids, differs in the time (the number of minutes) they take to pass through the liquid phase.

The Y-axis (vertical axis) represents the signal intensity, a measure of absorbance whose unit is the AU(Absorbance Unit ) and quantifies the amount of the analyte. This measurement is based on the amount of light absorption, i.e. how much light is absorbed by each of the analytes. The higher the peak on the y-axis, the higher the intensity, and therefore more of that analyte is present in the sample and its area under the curve is greater. In the figure below the unit on the Y-axis is mAU (milli-Absorbance Units), which is one thousandth of the Absorbance Unit.

Each of the cannabinoids has a particular retention time, which allows us to differentiate them in a chromatography. The higher the peak, the higher the concentration of this cannabinoid in the sample..

Then, the result of the chromatography tells us, at a given time (axis X), whether an analyte is (or is not) present at a particular intensity (X-axis). Y). In our case, each of the cannabinoids has a particular retention time, and the more of this particular cannabinoid in the sample, the higher the value on the Y-axis.

This is why, in order to differentiate each of the cannabinoids in the chromatogram, it is necessary to have a standard to know the specific retention time for each of the analytes (cannabinoids). The Y-axis is a unique fluorescence measurement for each of the cannabinoids and for this reason it is needed in standard, as a guide, to know which cannabinoid falls on that particular peak.

Example of a chromatogram of a cannabis sample. Cannabinoid analysis
Example of a chromatogram of a cannabis sample. Results of a cannabinoid analysis using HPLC. Figure taken from the article by Gul and collaborators [1] where the cannabinoids of a sample are exposed. Each of the analytes, which in this case are cannabinoids, differs in their retention time. It is for this reason that on the X-axis, which shows time and is measured in minutes, cannabinoids appear in different locations. The Y-axis showing the absorbance (milli-Absorbance Units) shows the intensity and therefore the amount of each of the analytes.

It took me quite some time to figure out what the first two peaks in the figure above represent and I had to inquire with colleagues who are experts in the field, who explained to me that the first peak, which has no name in the figure, is produced by the injection of the liquid phase and is the non-interacting molecules in the column, it is called the solvent front (solvent front).

The initial time before the peak of the solvent front is the time required for this mobile phase to pass through the column and is called the deadvolume.

The second peak, called “I.S” in the figure, is that of theinternal standard ( IS), which in the case of the article used 4-androstene-3,17-dione (4-androstene-3,17-dione). The peak of this chemical compound in the chromatogram serves as a reference to calculate the amount of cannabinoids present in the sample.

Mass Spectrometry (MS): Determination of the molecular weight of cannabinoids.

Mass spectrometry (MS) separates particles such as atoms and molecules by differentiating their charge-to-mass ratios. This technique is used to determine the weight of particles and is an analytical tool that measures the ratio between the mass and charge(m/z) of one or more molecules present in the sample.

These measurements can be used to calculate the exact molecular weight of the sample components. Mass spectrometry can identify unknown compounds by determining their molecular weight, and is also used to quantify known compounds and determine the structure of the components and molecules present.

Combined GC-MS: Improving accuracy in cannabinoid determination

One of the most widely used techniques for the determination of cannabinoids is the combination of gas chromatography-mass spectrometry (GC-MS).

Then, the ability of gas chromatography to separate the components of a sample and the ability of mass spectrometry to identify and quantify the individual components are combined. The result is very sensitive and accurate in measuring the amount and type of cannabinoids present in the sample.

In conclusion…

Well, here is some basic information about the different techniques currently used to measure cannabinoids in the cannabis industry. If there are any chemist readers out there, I would love to know what you think about this elementary chemistry information.

Referencias
  1. Gul, W., et al., Determination of 11 cannabinoids in biomass and extracts of different varieties of Cannabis using high-performance liquid chromatography. Journal of AOAC International, 2015. 98(6): p. 1523-1528.

Dra. Daniela Vergara
Investigadora y catedrática | Especialista en cultivos emergentes y consultora de cannabis

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