Proper analytical testing is vital to the hemp industry. In order to qualify as “industrial hemp” under the 2014 Farm Act (or as “hemp” under the 2018 Farm Bill), cannabis must be tested for delta 9 (∆9) tetrahydrocannabinol (THC) concentrations to ensure that it does not exceed three tenths of one percent (0.3 percent) on a dry weigh basis.
Analytical testing raises a number of issues, including, but certainly not limited to, the specific testing methods that are (and should be) used, at what point in the growth cycle of the cannabis plant testing should occur, what part(s) of the plant should be tested, from which part(s) of the field plant samples should be acquired, how the plant material should be combined to form a test sample, whether the acid form of THC should be decarboxylated as part of the testing process, and the scope of the test (i.e., the degree to which other compounds are measured, if at all, in a particular test).
This article, by Asheville-based cannabis attorney Rod Kight, addresses the primary types of tests currently used in the industry and the ones most appropriate for hemp.
[Image of a North Carolina hemp plant by Evan Anderson, 2018.]
Part 1- Different Tests Produce Different Results
A cannabis plant sample can be subjected to several different types of scientific tests to determine its ∆9-THC concentrations.
I was initially surprised to learn that different tests produce different results. In fact, since testing requires both separating plant molecules and then measuring them, there are a number of different combinations of tests, all of which can generate different outcomes. In a very real and practical way, using one testing method can result in a different legal outcome than a using a different method.
This article addresses the primary types of tests currently used in the industry and the ones most appropriate for hemp.
Part 2- Evaluation of Commonly Used Testing Methods
The cannabis plant has a complicated biological profile. It includes more than 400 compounds, including cannabinoids, terpenoids, alkaloids, flavonoids, fatty acids, etc. Teasing out and measuring one of those compounds, ∆9-THC, can be done using several different methods.
I argue that high performance liquid chromatography (HPLC) combined with mass spectrometry (MS) is the most accurate and reliable testing protocol for measuring ∆9-THC concentrations in hemp.
a. Separation techniques
All cannabis testing utilizes chromatographic techniques, which are chemical tests that determine whether the characteristic chemical constituents of the plant (in our case, ∆9-THC) are present.
On a fundamental level, this requires separating out the various compounds. Separation is typically done by using chromatography, which is a laboratory technique for separating a mixture. The mixture, a cannabis sample, is dissolved in a fluid or a gas called the mobile phase, which carries it through a structure holding another material, a solid or liquid supported by a solid, called the stationary phase. The various constituents of the mixture travel through the stationary phase at different speeds causing them to separate. In other words, ∆9-THC travels through the stationary phase at a different rate than the other cannabis compounds. It is separated from them by virtue of its unique speed during the test.
[Mobile readers: Article continues after the videos.]
[Video: “Introduction to Ultra High Performance Liquid Chromatography Whiteboard Video” by Chromatography & Mass Spectrometry Solutions via YouTube.]
[Video: “Mass Spectrometry” by Professor Dave Explains via YouTube. From the video description:
Who wants to smash molecules into little bits? A mass spectrometer does, that’s who. This is a good technique for corroborating molecular structure, because we can make sure the puzzle pieces fit the right way.]
“… ∆9-THC travels through the stationary phase at a different rate than the other cannabis compounds. It is separated from them by virtue of its unique speed during the test.”
The most common chromatographic procedures are thin-layer chromatography, gas chromatography, and high performance liquid chromatography. Each operates differently.
i. Thin Layer Chromatography (TLC)
The simplest, and least used, chromatography method is TLC. This technique is performed on a sheet of glass, plastic, or aluminum foil that is coated with a thin layer of absorbent material, such as silica gel, as the stationary phase. The mobile phase is a liquid. The primary advantages of TLC are that it is relatively simple and inexpensive, that it requires less time than other techniques, and that it uses fewer amounts of substances. Its primary disadvantages are that its detection limit is relatively high (ie, it is not very sensitive) and that it is an “open system”, which means factors such as humidity and temperature will affect the result. TLC is not appropriate to hemp because of these limitations.
ii. Gas Chromatography (GC)
A commonly used chromatograph technique is GC, in which the mobile phase is a gas (vapor) that passes through a column which is packed with a solid stationary phase (usually silica). With GC the sample must be heated in order to vaporize it. This allows it to pass through the column with a carrier gas. GC is a very good technique for separating volatile organic compounds, such as terpenes, which easily become vapors or gases.
However, the fact that GC requires heating the sample is particularly problematic for separating ∆9-THC. GC heats the cannabis sample to a temperature that changes its molecular structure in such a way that higher concentrations of the molecule being measured (ie, ∆9-THC) are created. This can cause a sample which initially contains ∆9-THC concentrations within the statutory limit to test above the limit. In other words, the GC technique can, and often does, give a “false positive” result. This transformation is called decarboxylation.
[Mobile readers: Article continues after these videos.]
[Video: Learn about how chemicals can be separated based on polarity through thin layer chromatography (TLC). By Angela Guerrero for the Khan Academy, which is dedicated to making education available to all, via YouTube.]
[Video: Understand how to separate and purify chemicals through gas chromatography and how to interpret a gas chromatogram. Created by Angela Guerrero for the Khan Academy via YouTube.]
Using (gas chromatography) to separate ∆9-THC for measurement is somewhat akin to a highway patrol officer using a radar gun that speeds up the vehicle it is measuring.
On a molecular level, all cannabinoids contained within cannabis trichomes have an extra carboxyl ring or group (COOH) attached to their chain. Decarboxylation is a chemical reaction that removes a carboxyl group and releases carbon. It converts a cannabinoid, such as THC-A (the “A” stands for acid), from its acid form to its neutral form. The conversion is caused by heat. THC-A is the precursor to ∆9-THC, which is a neutral form of the THC cannabinoid. THC-A converts to ∆9-THC when heated. The heat used in GC is sufficient to decarboxylate THC-A and convert it to ∆9-THC.
In other words, GC literally creates higher levels of the very compound being measured. Using GC to separate ∆9-THC for measurement is somewhat akin to a highway patrol officer using a radar gun that speeds up the vehicle it is measuring. The mere act of testing increases the quantity (or, in the radar gun analogy, the speed) of the thing being tested. For this reason, GC is an inappropriate and unreliable technique for separating ∆9-THC from a cannabis sample.
Given that GC is an inappropriate chromatograph technique for hemp, it is reasonable to ask why is it used at all, much less as the de facto test in much of the United States. Although there is no single answer, its popularity in the marijuana industry is a likely reason.
GC is a popular analytical technique in adult-use (“recreational”) marijuana states. Generally speaking, the average consumer who purchases cannabis in a recreational state wants as much ∆9-THC “bang for the buck” as possible. Cannabis companies know this and understandably focus on marketing the ∆9-THC concentrations in their products. Using GC as part of a testing protocol necessarily involves increasing (some would say “inflating”) the reported ∆9-THC levels of a given cannabis plant. If we assume the average consumer wants more ∆9-THC, then this testing method results in higher sales.
Although at first glance this may appear to be somewhat deceptive from a marketing standpoint, the fact is consumers of flower products in adult-use states almost always decarboxylate their cannabis before or during ingestion by smoking, vaping, or heating it as part of an edibles recipe.
So, while the GC technique is technically inaccurate when it comes to separating the actual ∆9-THC in a plant, it is accurate in its practical application since most people in adult-use states heat the cannabis flowers they purchase and decarboxylate the THC-A in them. In this way, GC is a somewhat appropriate method to determine the total “available ∆9-THC” in a marijuana sample. For cannabis testing in recreational states, at least for cannabis destined to be heated by the consumer, this is fine and perhaps even the most appropriate measure. Additionally, since the legal status of a cannabis crop in an adult-use state is not determined by whether or not it exceeds a statutorily defined ∆9-THC limit, the consequences of an inaccurate result are not usually significant.
However, for hemp states an inaccurate result can result in the loss of an entire crop. Finally, there are a number of different uses for hemp flower and biomass, most of which do not involve heat and decarboxylation. GC is a decidedly inappropriate and unreliable technique in these situations.
… for hemp states an inaccurate result can result in the loss of an entire crop.
As an aside, I have heard and evaluated the argument that a mathematical step can be inserted into the GC protocol to account for the effect of decarboxylation and determine the true ∆9-THC in a cannabis plant sample. The idea is that while GC increases the ∆9-THC present in a sample, a mathematical step can fix this issue in the final result.
Although relatively easy in theory, this does not work well in practice. In order to obtain a true measure of the ∆9-THC concentrations in a given plant you must have a method to determine the concentrations present in the initial sample. Adding a mathematical step to GC necessarily makes an assumption of the amount of ∆9-THC and the amount of other molecules which can convert to ∆9-THC originally present. Although somewhat “circular”, this can be done; however, doing so introduces a new opportunity for uncertainty (ie, by making an assumption of the original amount of ∆9-THC present) and also adds an additional source for potential error.
iii. High performance liquid chromatography (HPLC)
In HPLC a liquid mobile phase is pumped at high pressure through a porous solid, such as glass, silica, or aluminum that is packed into a glass or metal tube or that constitutes the walls of an open-tube capillary.
HPLC is the most reliable separation method for ∆9-THC because it does not involve high temperatures and leaves all relevant molecules intact. This means it is highly accurate and does not require the addition of a mathematical step or assumptions about the concentrations of the very thing that it is separating.
Aside from not decarboxylating the sample, another advantage to using HPLC is speed. Due to the pressure exerted on the mobile phase, HPLC also produces the fastest results. Its disadvantages include that it can cause coelution, in which two compounds with similar structure and polarities exit the HPLC device at the same time. This makes measuring the relative quantities of the respective compounds difficult or even impossible. Fortunately, coelution of cannabinoids is rare.
HPLC is also generally more expensive than GC, though not significantly.
b. Measurement techniques
Once the ∆9-THC has been separated from other compounds in a cannabis sample by using chromatography, the actual amounts must be detected and measured. This can be done by using several different devices, the most common of which are a flame ionization detector, an ultraviolet detector, and a mass spectrometer.
i. Flame Ionization Detector (FID)
A commonly used detector for GC is FID. It is a “mass sensitive instrument” because it measures ion mass per unit of time. FID operates by collecting and measuring the amount of analyte (ie, the substance being measured) in a gas stream. The fact that it operates in the context of a vapor makes it particularly useful for GC; however, for this reason it is not used with HPLC, which does not emit a gas. This is its primary disadvantage for use with hemp. Given that GC is inappropriate for separating ∆9-THC for the reasons discussed above, FID is inappropriate by virtue of the fact that it operates with GC. Additionally, of all of the detection and measuring methods described in this article, it is the least specific. (As an aside, it is also the detection method most utilized by the DEA.)
ii. Ultraviolet Detector (UV)
This detection device operates on the principal that chemicals absorb UV light at specific wavelengths. The principal at issue, Beer’s Law, states that the concentration of a chemical is directly proportional to the absorbance of a solution. In UV, the concentration of ∆9-THC can be determined by the amount of UV absorption that occurs. Although UV is generally an accurate detecting technique, it can generate a false positive result when UV absorbing molecules in addition to ∆9-THC are present during the detection stage.
iii. Mass Spectrometer (MS)
MS detects various molecules based on their distinctive mass and mass fragmentation pattern. It does this in three stages. The first stage, ionization, vaporizes molecules in a sample and then bombards them with an electron beam, which causes them to become charged particles (ie, ions). The ions are then sorted by two processes, acceleration and deflection, and measured. MS only measures the mass of ions. Neutral molecules are not detected. The result is a spectrum. A high quality mass spectrometer detects and measures a fragmentation of each molecule. This removes much uncertainty and generates a very accurate result. For this reason, MS is the most reliable method to detect and measure ∆9-THC in a cannabis sample.
Part 3- Conclusion
Testing protocols may seem abstract. Teasing out their differences can be tedious. However, the testing protocol used to measure ∆9-THC concentrations can mean the difference between a lawful hemp harvest and a “hot” crop. This can translate to having a profitable crop or a total loss.
Although HPLC in conjunction with MS is the most reliable protocol for testing ∆9-THC concentrations in a sample, it is not the most prevalent. Anecdotally, it appears that GC-FID is the most common protocol, despite the fact that GC increases ∆9-THC in the sample being tested, and FID is the least specific detector of the available options. The reason for the prevalence of this testing protocol is unknown. One of the most likely reasons is that GC-FID is commonly used in adult-use marijuana states, where higher ∆9-THC levels often mean higher sales and where measuring “available ∆9-THC” makes sense because most consumers will smoke, vape, or heat it to make edibles. However, the same protocol is inappropriate and unreliable for hemp.
Federal law explicitly states that the operative issue for determining whether a particular cannabis crop is lawful hemp or unlawful marijuana is its ∆9-THC concentration. Any reliable testing method must, at a minimum, measure the actual concentrations of ∆9-THC in order to comply with the statute. Attempting to quantify the total “available THC” using a testing method that distorts the result by creating the very molecule it is measuring is contrary to law.
Additionally, most hemp plants are not destined to be heated (ie, by smoking, vaping, or cooking in a recipe). The THC-A molecules in these plants will never be decarboxylated. For this reason, and aside from conflicting with the statute’s requirements, testing total “available ∆9-THC” makes improper assumptions that do not always, or even often, apply to hemp. The only consistently reliable testing protocol is HPLC-MS.
As the hemp industry continues to consolidate standards it must promote and adopt reliable testing methods. Since the threshold issue in determining a cannabis plant’s legal status is its ∆9-THC concentrations, an error caused by an unreliable method is intolerable and contrary to law. The most reliable testing protocol is HPLC-MS, which should be adopted as the de facto test for measuring ∆9-THC in hemp.
Since the threshold issue in determining a cannabis plant’s legal status is its ∆9-THC concentrations, an error caused by an unreliable method is intolerable and contrary to law.
Cannabis Attorney, Contributor
Rod Kight is an attorney who represents lawful cannabis businesses. He speaks at cannabis conferences across the country, drafts and presents cannabis legislation to foreign governments, is regularly quoted on cannabis matters in the media and maintains the Kight on Cannabis legal blog, where he discusses legal issues affecting the cannabis industry. You can contact him here.
His articles are republished on CarolinaCannabisNews.com with permission. They originally appear on his blog Kight on Cannabis.