What’s IN Your Cannabis Analysis??
For many years, the only way that cannabis users could know the potency of the bud they bought was to burn one down, and that method was subjective at best. Medical marijuana has become commonplace in many US states, and with that came a new trend of potency testing cannabis.
It is not uncommon to walk into a store that sells marijuana and see a menu listing all of the strains available for purchase and the THC, CBD and other cannabinoid potencies. In some states, potency and health and safety testing are mandated by law.
This blog will help you understand the basics of cannabis testing and analysis, with some followup courses focused on specific state regulations where testing is required by law, like Colorado and Washington.
The most common cannabis analysis conducted on flowers concentrates and infused products is a potency test to determine the percentage of cannabinoids such as THC, THCA, CBD, etc.
It is important for medical and recreational users alike to know how strong the cannabis is and to understand the relative amounts of cannabinoids present. The quantity and ratio of cannabinoids can greatly impact how consuming cannabis will affect the user.
Because cannabis is a cultivated plant, there are some inherent health and safety risks. Was it treated with pesticides? Was it grown properly to prevent the formation of mold? How was it stored and handled?
Often there is a disconnect between the cultivators, retailers, and consumers of cannabis, so answering these questions directly can be difficult. Testing and cannabis analysis can provide many of these answers and make retailers and users more confident in the product they are selling and consuming.
Methods Of Cannabis Analysis
Methods are available to screen for pesticides, residual solvents, and chemicals used to make concentrates, mold, and mildew, microbial contaminants like bacteria and foreign matter such as incest parts or rodent droppings.
We will focus primarily on potency testing in cannabis analysis, but will also touch on the health and safety testing. This image shows the chromatographic separation of plant pigments on a paper strip.
In order to understand potency testing of cannabis, we must first understand the basics of chromatography which is the principle behind many modern methods of cannabis analysis in forensics and the food and drug industry.
Chromatography is a relatively new science and developed in the early 1900’s as a method to separate plant pigments such a chlorophyll.
The word chromatography means “color writing” and was derived because of the colorful separation of these plant pigments on paper strips.
Although early uses of chromatography involved separation of colored chemical components, the term is now used more broadly in separation science for a process that employs two phases of matter to separate chemical compounds: a solid stationary phase and a fluid (liquid or gas) mobile phase.
The fluid phase is used to carry the chemical compounds to be analyzed (the analytes) through some sort of solid material, usually packed into a column or tube or dispersed on a glass plate. The system is designed so the analytes have an attraction to the solid phase.
Different molecules will have a different level of attraction based on their chemical structure, so some of them will interact more with the solid phase and move slower through the column. Others will interact less with the solid phase and have a stronger interaction with the mobile phase and move through the column faster.
An analogy of chromatography that is often used is to imagine a group of people in a shopping mall. The individual people represent molecules in the fluid phase and the stores in the mall represent the solid phase.
If the group is made up of people who like shoes and the mall has mostly furniture stores, the group of people will move through quickly. If the mall is made up of many shoe stores, the group will move slower and people who really like shoes will take longer to leave the mall that those who do not.
The analogy is great because you can see that there are many different groups of people and stores possible that will affect how long individuals spend in the mall. There are many methods of chromatography (thin layer, liquid, gas and so on) and within each method many variations that allow for separation of specific classes of molecules.
Separation is only half of the chromatography though. In the early days, it was easy to see the separations because they were done using plant pigments which are colored. Most chemical compounds do not have colors in the visible spectrum, so even if there is a way to separate them, how do we know they are there?
This is the job of the detector. All chromatographic cannabis analysis methods employ some sort of detector after the separation step. Some detectors work in other regions of the electromagnetic spectrum, for example using ultraviolet light instead of visible light.
Others work by burning the molecules in a flame and detecting changes in the flame intensity and some work by detecting the molecular mass of the chemical compounds. The detector is not only capable of telling you that a particular compound is present, but it can also tell you how much there is.
We will learn more about quantitative cannabis analysis a little later.
Preparation For Cannabis Analysis
Before we get into technical details of the different types of chromatography equipment used to analyze cannabis, it is important to understand how the samples are prepared for cannabis analysis. Unfortunately, there is no method that can directly measure how much THC is in a marijuana flower.
In order to determine the potency of a flower or concentrate or infused edible product, it must be extracted into a solvent. This is most commonly done by soaking the material to be tested with isopropyl alcohol (or another solvent) to dissolve all of the cannabinoids (and other analytes).
It is important to develop a consistent and thorough extraction protocol to ensure that all analytes are dissolved every time so reliable potency results can be obtained. An example of a method to prepare flowers for cannabis analysis is as follows:
1) The flower is broken up by hand and a precise amount is weighed into a vial.
2) An exact amount of alcohol is then added to the vial and it is shaken vigorously.
The plant material is allowed to soak in the alcohol for several hours at room temperature with occasional mixing, and after about 5 hours all of the cannabinoids have been extracted. A TLC plate is developed in a chamber, notice how the solvent has moved partially up the plate.
A TLC plate with indicator dye illuminated under UV light, the four dark spots are THCA. The most basic method that can be used to analyze cannabis for potency is thin layer chromatography (TLC).
The mobile phase is a liquid solvent or mixture of solvents (commonly ethyl acetate and hexane blended at specific ratios) and the stationary phase is a coating of silica gel on a glass plate.
In this method, a small amount of the prepared sample is applied to the silica coating of the TLC plate and the plate is partially submerged in the liquid mobile phase. Capillary action causes the solvent to migrate up the plate and individual analytes are separated.
Because cannabinoids are not colored, secondary methods must be used to visualize them. Some scientists use reagent stains that change color when they contact cannabinoids and others use indicator plates with a UV dye to visualize the cannabinoids.
Either way, quantifying a number of cannabinoids present are challenging and is usually done with computer programs. The computer compares the size of the spot for the unknown sample with the size of the spot for a sample of known potency and uses the ratio to quantify the unknown sample potency.
TLC is a simple and inexpensive method of measuring cannabis potency, but it is not very accurate and sample preparation and cannabis analysis are time-consuming so it is not practical for large testing volumes.
Using Chromatography For Cannabis Analysis
Gas chromatography (GC) is another method that is often used for potency and is probably the most widely used cannabis analysis method. The principle behind GC is the same as other chromatographic methods, but instead of a liquid, a gas is used as the mobile phase.
The sample is prepared and then injected into the GC instrument, the analyte is carried by a gas, usually helium or hydrogen, and passes through a column that is often over 50 feet long. The inside of the column is coated with a stationary phase that interacts with the analytes and causes them to separate.
A detector is placed at the end of the column and is used to determine the presence of analytes. The most common detectors used for GC are flame ionization detectors (FID) or mass spectrometers (MS).
The FID detector contains a flame that burns the analytes coming off of the column, so any organic material (like terpenes and cannabinoids) will be combusted and generate a detector response. On the other hand, GCMS uses complex machinery to detect the molecular weight of the analytes and is a very powerful tool for characterizing unknown analytes.
The data that comes out of a GC looks much different than TLC but is in fact very similar. A chromatogram is a plot of detector response versus time, where a flat horizontal line indicates a zero response and peaks are due to analytes being detected.
The retention time of each peak is unique and corresponds to a different analyte and allows for qualitative cannabis analysis. Quantitative cannabis analysis is possible to determine the amount of each analyte present using a calibration standard.
The size of each peak in the chromatogram corresponds to the amount of that substance present, so if you inject a known amount of analyte as a standard you can use the relative peak sizes to determine how much is in an unknown sample. GC is fast, accurate and can be affordable with basic instruments starting around $12,000 (more advanced GCMS models can cost over $100,000).
The biggest limitation of GC is that it cannot accurately measure THCA. Because THCA is heat sensitive and the GC method involves heating the sample, it will cause the THCA to decarboxylate into THC.
Best Method For Cannabis Analysis
It combines the speed and sensitivity of GC with the principles of TLC, and because it does not heat the sample HPLC can be used to measure THCA.
The prepared sample is injected into the HPLC and a liquid solvent mixture carries the analytes through the column where they separate.
After leaving the column, the analytes reach the detector which for HPLC is most often an ultraviolet (UV) detector or mass spectrometer like the one used for GC.
The UV detector works by irradiating the analytes with ultraviolet light and measuring how much is absorbed so that any chemical that absorbs UV is detected. Cannabinoids absorb strongly in the UV region of the spectrum, so this detector works well for routine potency cannabis analysis.
However, many terpenes and other molecules do not respond to UV light so this detector is not as useful for this kind of cannabis analysis and mass spec should be used instead. HPLC is very accurate, rapid and allows for the detection of THCA.
However, HPLC equipment is expensive, sensitive and can be difficult to maintain. There are other methods that can be used to test cannabis for potency that does not use chromatography, however, they are not nearly as common.
One method uses a technique called nuclear magnetic resonance (NMR), but the equipment is very expensive and difficult to operate and maintain. NMR is primarily used in university and government labs, but there are compact and inexpensive instruments that are available on the market that may become competitive options for cannabis analysis.
There are other methods that use the electromagnetic spectrum (ultraviolet, visible and infrared light) that are becoming more popular because of their simple methodology and inexpensive instrumentation. There is at least one company that produces an instrument that uses near-infrared light to measure THC and other cannabinoid potencies.
As the testing and analysis market expands, we will see more and more rapid and inexpensive methods of measuring cannabis analysis and potency. There are other tests that are conducted on cannabis analysis beyond just potency.
Most consumers and retailers are only aware of potency testing because this applies directly to the product and can be used as a marketing and sales tool. However, there are several other cannabis analysis that is becoming more and more common and is required by law in states like Colorado and Washington.
Much of the testing here is focused on health and safety and is intended to look for dangerous contaminants in marijuana.
Because of the prohibition of cannabis for many years and the thriving black market, regulators in states with legal marijuana want to control all aspects of production and ensure that the public is safe as this stigmatized plant becomes more and more common.
Cannabis analysis tests with GC or HPLC for chemical contaminants like pesticides and harmful chemicals to ensure that it is being cultivated in a responsible and safe manner. These same analysis methods can also be used to test for chemical adulterants and illegal drugs that may have been intentionally added, although this is not a problem with legal marijuana but has been an issue with black market cannabis.
GC has also used cannabis analysis with concentrates for residual solvents (such as butane) used to make hash and other concentrates. Chemical contaminants like pesticides and residual solvents are only part of the health and safety testing.
Microbial contamination is also a major concern because mold and fungus grow readily on cannabis under the right conditions. E. coli and other microorganisms have also been detected in cannabis and may appear due to improper handling and fertilization.
These types of microbial contaminants are of particular concern for medical marijuana patients, some of whom may have compromised immune systems and have a high risk of infection from these types of contaminants.
Microbial contaminants can be detected visually in some cases, but there are more reliable methods such as culturing the sample in a petri dish, incubating and counting the microbes using a microscope. This method is very time and labor intensive but is inexpensive and accurate when done properly.
Another method called qPCR amplifies and quantifies the DNA of microbes like e. coli and is quite expensive, but is very accurate, sensitive, easy to automate and fast. Additionally, visual inspection of the cannabis flowers is often conducted to look for foreign contaminants such as hair, rodent excrement, and insects.
For more information on cannabis analysis and testing, we highly recommend the American Herbal Pharmacopoeia’s booklet titled Cannabis Inflorescence. This short book goes into great detail about the different methods that can be used to cannabis analysis and is a great primer for those looking to learn more about this new and exciting topic.
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