Hemp is a naturally variable, inhomogeneous material. Analyzing one or a few cuttings from a grow will not give data representative of a field. Here I propose a scientifically based representative sampling plan for hemp grows detailing how to collect composite samples, how many to collect, and how to analyze them. I want this industry to commit itself to the goal of analyzing one plant per 1000. Technologies available to handle the resultant increased sample load are reviewed.

With passage of the 2018 Farm Bill and promulgation of the United States Department of Agriculture’s (USDA) interim final rule in October 2019, it is now legal to grow hemp provided the total tetrahydrocannabinol (THC) level in dried plant material does not exceed 0.3% dry weight (1–3). To enforce this law, extensive testing of hemp crops is being performed around the country by state departments of agriculture. The USDA has issued a sampling guidelines document (3). The USDA sampling document defines a “lot” of hemp plants as a (3) “…contiguous area in a field, greenhouse, or indoor growing structure containing the same variety or strain of cannabis throughout.”

My concerns with this sampling plan are noted below, along with my proposal for an improved hemp representative sampling plan. First however, we must address the topic of sampling error, something a good representative sampling plan tries to minimize.

The entirety of anything that we wish to sample, be it all the people in the US or all the plants in a hemp grow, is called a 

 (4). In a perfect world, we would measure the quantity of interest in the entire population. For example, asking the opinion of everyone in the US for a poll, or analyzing every hemp plant in a field. However, this is impractical given the enormity of the task. Instead, we choose a subset of the population from which to collect data, for example, we could choose to ask 1000 Americans their opinion for a poll or choose to analyze one plant per 1000 in a hemp grow. Sampling error rears its ugly head because there is always a chance the population subset we choose to analyze will not give us the same result as if we had analyzed the entire population.

Sampling error is different from the random error discussed in a previous column (5). Random error is caused by things we have no control over such as instrumental and environmental fluctuations. Random error is minimized by more and better testing (5). When testing a hemp field then for cannabinoid content, the amount of error in the final measurement is a combination of random error and sampling error (assuming systematic error is zero [5]).

I am not a statistician but a chemist, so I refer you to these references if you desire a detailed mathematical treatment of margin of error and population subset size (6,7). However, everything I have read on the subject points to the same conclusion: the way to minimize sampling error is to maximize the size of the population subset that is analyzed. Intuitively this hopefully makes sense. There are more than 300 million people in the United States. When running an opinion poll, the more people you talk to the better the chances you will gather data that is representative of the nation. When it comes to hemp fields, when taking one or a few cuttings there is a high chance that the plants sampled are not representative of an entire grow. The way around this is to analyze as many hemp plants in a field as is practicable.

The limiting factor in performing representative sampling for hemp field testing is time and expense. Therefore, we must find a happy medium between minimizing sampling error while not generating an inordinate number of samples for testing. I believe the best compromise here is achieved if we test one hemp plant per 1000 in a field. I want our industry to commit itself to this testing goal.

The USDA hemp sampling document (3) presents equations for calculating the minimum number of cuttings to collect in a lot. Example calculations are given and are summarized in tables showing the minimum number of cuttings to collect based on the acreage of hemp plants. In the examples given, one cutting per acre or less is collected per lot. The document calls for compositing all cuttings into a single sample. Typically, one aliquot of this composite sample is tested (8–10).

The following sections summarize my concerns about this approach.

The calculations for the minimum number of cuttings to collect are based on this assumption (3): “…the proportion of hemp plants having THC content greater than the acceptable hemp THC level…are based on past experience in the same or similar growing areas.”

The example calculations assume the proportion of hot hemp plants is 0.01 (3).

One problem with this assumption is that in some areas of the country hemp is being planted for the first time this year, so the needed historical data does not exist. Additionally, since it only became legal nationwide to plant hemp as of January 2019 (1), there is little historical data for the rest of the country upon which to base an assumption of the proportion of hot plants. Lastly, even if we had enough historical data, given the vicissitudes of the weather, every growing season is going to be different in every area. Absent any peer-reviewed data to the contrary, I do not believe it is safe to assume that any given growing season will be predictive of any future growing season. Rather than basing a sampling scheme assuming we know the proportion of hot plants, would it not make more sense to measure that proportion of hot plants instead?

My concern here is that, on average, the USDA sampling document calls for collecting one cutting per acre or less (3). The problem is that hemp farmers plant at different densities. For example, Farmer x may grow 2000 plants/acre, while Farmer y may grow 4000 plants/acre. Remember that the population being sampled is the hemp plants, not the area of the field they occupy. Basing the number of samples to collect based on acreage is like choosing the number of people to include in an opinion poll based on a state’s area. Using this logic, you would talk to more people in Alaska rather than New Jersey for a poll, even though New Jersey has 12 times as many people as Alaska (11).

Our goal should be to achieve the same level of sampling error for all lots analyzed. To do this, the number of samples to collect needs to be determined by the number of plants in a plot, not their acreage. In our examples then, and following the goal of sampling one plant per 1000, Farmer x’s plot should be sampled at a rate of 2 plants/acre, while Farmer y’s plot should be sampled at a rate of 4 plants/acre. Only by basing the sample size on the number of plants in a plot can we assure the best representative sample is obtained.

Where on the plant should we collect cuttings? The USDA sampling document states that cuttings are to be taken from the top 1/3 of the plant (3). Work by myself and others has clearly shown that cuttings from the same hemp plant vary in cannabinoid content (12) as seen in 

The top of a hemp plant normally has the highest cannabinoid content, and hence only collecting plant tops means a sample will be collected that will be biased towards containing more THC than the field in general. This makes it more likely that its total THC content will be above 0.3% and that the farmer will lose his crop.

Ultimately, I believe that the plant parts sampled need to reflect how the crop will be harvested. For smokable hemp, the buds are the article of commerce, which means that harvesting the top 1/3 of the plant may be appropriate for this type of harvest. However, when growing hemp for extraction, the entire plant may be ground up and utilized. In this case cuttings from the top, middle, and bottom would be appropriate.

Composite Samples Are Not Necessarily Uniform

The USDA’s sampling document calls for compositing all the cuttings from a lot into one sample (3).

There are two problems with this. First, the regulations assume that every composite sample is homogeneous. Where is the peer-reviewed science on this? A simple visual inspection of ground hemp samples will show they are not uniform. In my own experience, I have found that composite samples are not uniform. I have taken composite hemp samples and had their cannabinoid content measured in triplicate at an ISO licensed, state certified laboratory (13). I found cannabinoid concentration variation across the samples well above the margin of error as reported by the laboratory, which means these composite samples were not homogeneous, and that sampling error exists for composite samples.

The second problem with composite samples is only testing them once. This assumes the sample is uniform. Wouldn’t it be better to test multiple aliquots and reduce the sampling error rather than only testing one aliquot and assuming the sampling error is zero? When it comes to science, I strongly feel that actual measurements are always better than assumptions. At minimum three aliquots of composite samples should be tested and an average and a standard deviation calculated to get a handle on the sampling error.

Another concern I have is that different states are sampling in different ways (8-10). Some states only collect a specific number of cuttings regardless of grow size. Some states are using sampling based on acreage and not number of plants. Some states give specific details on how to walk a field and collect samples, but there is no agreement on what pattern to walk. Lastly, some states are testing using liquid chromatography (8,9), while others are using gas chromatography (10). Until there is nationwide standardization on how to collect and analyze hemp samples, different states will obtain different results on the same samples.

A Proposed Hemp Representative Sampling Plan

Patricia Atkins and I have written in this journal on the importance of representative sampling in cannabis testing (14,15). I offer here my representative sampling proposal for hemp. This proposed plan does not necessarily conform with regulations promulgated by the USDA or any state’s department of agriculture. My proposal is based on my understanding of statistics, my 30+ years experience in chemical analysis, and my PhD in physical chemistry.

The gist is this. Rather than assuming we know the proportion of hot plants, why not use statistical sampling techniques to generate enough data to get an excellent estimate of the average total THC value for a hemp lot? Rather than only collecting one composite sample per lot, assuming that sample is uniform, and then only testing it once, why not collect multiple composite samples from a lot, test each of them multiple times, and maximize the amount of data we have on a lot minimizing the sampling error?

The number of samples to collect from any given lot needs to be based on the number of plants in that lot as the plants are the population we are testing and care about. I want our industry to commit itself to sampling one hemp plant per 1000 planted.

My own instinct on this is that sampling locations should be chosen randomly. This is to make sure that location does not bias the sample. For our 10,000 plant example plot this means 10 locations should be chosen at random. However, there is work indicating that collecting samples according to a pattern is a good idea as well (15). I am persuadable on what might be the best approach here.

What Parts of the Plant Should Be Sampled?

Research has shown that the cannabinoid content from different parts of the same plant varies appreciably (Figure 1). As stated above, the portion of the plant tested should correspond to how the plant will be harvested. If only plant tops are to be harvested, then only plant tops should be tested. If the entire plant is to be harvested then cuttings from the top, middle, and bottom of plants should be collected and tested.

Since cannabinoid content varies from plant to plant, taking a cutting from only one plant at a given location might not be representative of that spot. To overcome this problem, cuttings from three plants at a given location should be collected to obtain a more representative sample. If harvesting just plant tops, the upper 1/3 of nine plants from a given location should be sampled. If harvesting the entire plant, then a cutting from the top, middle, and bottom on each of three plants for nine cuttings total should be collected. The cuttings from a given location should be combined to comprise a composite sample representative of that spot. For a 10,000 plant hemp lot then, 10 composite samples should be collected from 10 locations.

The law clearly states that dried hemp is the material to be tested. In my own work I dry hemp cuttings at 140 °F in a standard food dehydrator overnight. This gets the material down to about 5% moisture. I grind about 5 g of dried cuttings in a coffee grinder for 1 min to homogenize the sample. There are other ways of drying and grinding hemp. I welcome input on this. The most important thing is that we, as an industry, standardize how we prepare hemp samples for analysis (16).

How Many Aliquots of Each Sample Should Be Tested?

The problem with compositing all the cuttings from a lot and testing only one aliquot were discussed above. At minimum, any composite sample should be tested three times and the results averaged to obtain a more representative picture of the composite. Using our example then, each of the 10 composite samples should be tested three times for a total of 30 data points.

For a given hemp lot, the average total THC value for a lot should be calculated. For our example hemp plot, this would mean calculating the average of 30 values. The standard deviation (17) of these values should be calculated as well. This number is a measure of the variance in the data, and it is important because it takes into account sampling error.

The “acceptable hemp THC level” as defined by the USDA is calculated by adding the margin of error for total THC for a given method to 0.3 wt. % total THC (2). For example, if the margin of error is 0.04 wt. % for an analyzer (18), the acceptable hemp THC level for this analyzer would be 0.34%. If the average total THC value for a given lot is within a 95% confidence interval (17) of the acceptable hemp THC level for an analyzer, it should be considered legal. For hemp with total THC values above this level it should be considered illegal.

For compliance purposes the law states that a hemp grow should be sampled within 15 days of harvest (1,2). However, for the purposes of managing a hemp grow to maintain compliance, I recommend farmers sample and test their crops more often. One approach is to begin testing about halfway through the growing season, and to test a lot once per week. I highly recommend tracking your results over time to spot trends. As harvest approaches, sampling a couple of times a week is a good idea.

For our 10,000 plant plot, I recommended collecting 10 composite samples and performing 30 analyses total. Sending these samples out to a third party laboratory is not practical because it can take weeks to get results and may cost thousands of dollars to analyze one weeks’ worth of samples. This means that hemp farmers need to perform their own testing to manage their grows to maintain compliance.

In a previous column, I compared different technologies for cannabis potency testing (19) using the criteria of speed, accuracy, cost, and the ability to perform representative sampling. By necessity, any technique that is going to be used for representative sampling must be fast, easy, and inexpensive to make handling the representative sampling analysis load feasible.

I showed (19) that chromatography can accurately analyze a given sample but it is slow and expensive to use. As a result, it is not amenable to representative sampling. Another problem with chromatography is its difficulty of use, which is why some states require degreed scientists to run these instruments (19). I believe these problems with chromatography are why we are not performing proper representative sampling in this industry.

Spectroscopy is fast and inexpensive, but because it is a secondary technique it is not necessarily as accurate as chromatography (20). There exist spectroscopic analyzers capable of an accuracy or ±0.04 wt.% accuracy for the determination of total THC in dried, ground hemp (18). Since this is several times lower than the legal total THC limit of 0.3%, these analyzers are of sufficient accuracy to measure total THC in hemp. This technology is fast enough, inexpensive enough, and accurate enough to handle the sample load required to allow true representative sampling of hemp lots for farmers and regulators.

For state departments of agriculture, I recommend a two-tier approach to efficiently handle the sample load necessitated by proper representative sampling. Infrared spectroscopy could be used as an initial screen of samples. If a lot tests as being legal that is the end of it. If a lot tests hot, samples should be subsequently tested by chromatography to confirm the analysis. The advantage of this approach means that time and money are not wasted analyzing legal samples by chromatography.

There is natural variability amongst hemp plants, hence their grows need to be sampled in a representative manner. There are issues with how hemp fields are currently sampled. Here I propose that one plant per every 1000 should be analyzed. This is a reasonable compromise between minimizing sampling error and not generating an overwhelming number of samples to test. How to collect, prepare, analyze the samples, and report the data was discussed. Technologies available to handle the increased sample load resulting from representative sampling were covered.

115th United States Congress, Senate Bill S.2667,” Hemp Farming Act of 2018.

https://www.federalregister.gov/documents/2019/10/31/2019-23749/establishment-of-a-domestic-hemp-production-program

https://www.ams.usda.gov/sites/default/files/media/SamplingGuidelinesforHemp.pdf

https://en.wikipedia.org/wiki/Sampling_error

https://en.wikipedia.org/wiki/Sample_size_determination

https://agriculture.delaware.gov/plant-industries/hemp-program

Massachusetts State Hemp Plane, Massachusetts Department of Agricultural Resources, Submitted to USDA March 9, 2020.

Kentucky Department of Agriculture Standard Operating Procedure KDA-HEMP-20190204-1, issued Feb. 4, 2019.

https://en.wikipedia.org/wiki/List_of_states_and_territories_of_the_United_States_by_population

M. Giese, M. Lewis, L. Giese, and K. Smith, 

https://en.wikipedia.org/wiki/Confidence_interval

California Bureau of Cannabis Control Regulations, Section 5719.

, is Founder, CEO, and Chief Technical Officer of Big Sur Scientific. He is the inventor of the BSS series of patented mid-infrared based cannabis analyzers. Dr. Smith has done pioneering research and published numerous peer-reviewed papers on the application of mid-infrared spectroscopy to cannabis analysis, and sits on the editorial board of 

. He has worked as a lab director for a cannabis extractor, as an analytical chemist for Waters Associates and PerkinElmer, and as an analytical instrument salesperson. He has more than 30 years of experience in chemical analysis and has written three books on the subject. Dr. Smith earned his PhD on physical chemistry from Dartmouth College.

Articles

Cannabis Analysis 

Here a scientifically based representative sampling plan for hemp grows is presented, detailing how to collect composite samples, how many to collect, and how to analyze them.

A Proposed Representative Sampling Plan for Hemp Grows

This article reviews the chemical makeup of cannabis extracts and how they affect the human body. From terpenes to cannabinoids, this article takes a deep dive into the chemistry of cannabis extracts. It also demonstrates the opportunity for discovery and what we have yet to uncover.

Cannabis is widely known for tetrahydrocannabinol (THC) and cannabidiol (CBD), two prominent compounds found in relatively high concentrations in the flowers of 

 (1). As the legal cannabis industry develops, product offerings have expanded exponentially. Smoking cannabis flower is no longer the only option available to consumers, though it does maintain its position as the majority of product sales (

Cannabis extraction has emerged as the gateway to product development. Various solvents are used to produce a number of different cannabis extracts, which can be sold as inhalable products or used to infuse ingestible and topical products. The chemical profile of the extract is dependent on the solvent used during the extraction, the parameters used during the extraction, as well as the input material.

 (2) is optimal for efficient cannabinoid extraction. The solubility of ethanol allows for all grades of cannabis plant matter to be used in the extraction, with nearly 100% of cannabinoids able to be extracted within 20 min. However, by optimizing for cannabinoids this extraction method sacrifices terpenoids, flavonoids, and other light compounds within cannabis that are lost in the process.

Warm or Room Temperature Ethanol Extracts

Warm or room temperature ethanol extracts contain:

⦁ Cannabinoids (THC, CBD, cannabigerol [CBG], and so on)

⦁ Plant pigments such as chlorophyll and carotenoids

Because of the plant pigments and waxes, further refinement is necessary to remove these undesirable by-products. In turn, there is more loss of the delicate flavonoids and terpenoids leaving a highly concentrated extract full of phytocannabinoids. Some higher temperature ethanol extraction simultaneously decarboxylates the tetrahydrocannabinolic acid (THCA) turning it into THC. While we know the aforementioned compounds are found in the extract, we are still unsure of about 20–40% of the chemical makeup of cannabis extract depending on the cannabinoid potency. It is thought that warm or room temperature ethanol extracts are full of medically beneficial compounds and are best used in tinctures, capsules, and sublingual products.

The cold temperatures of cryogenic ethanol extraction allow for efficient extraction of cannabinoids without the by-products such as pigments or waxes. The result is a high potency cannabinoid fraction that is not decarboxylated. Advanced cold extraction techniques are able to produce inhalable products called 

, which is a hard-candy consistency product used as an inhalable. It is made up of 80–90% THCA crystals.

Ethanol extracts are growing in popularity because of their efficiency and potency, but lack in versatility. These extracts are often used to make high potency distillates as well as a variety of distillate infused ingestible products (

Hydrocarbon extraction started as a low-tech but innovative extraction method in early medical and illicit markets. Its products, known as “concentrates” or “dabs,” have been a popular choice for many experienced cannabis users because of the amplified flavor and higher concentration of cannabinoids.

Hydrocarbon solvents are used under cold temperatures and under relatively low pressures. This is fast and captures the desirable components of the cannabis plant.

⦁Terpenoids, especially mono-terpenes and mono-terpenoids

Because of the higher potency of cannabinoids and terpenoids, hydrocarbon extracts often have potencies over 75% cannabinoids (

While hydrocarbons quickly extract terpenes, aromatic hydrocarbons found in the plant, and cannabinoids extensive post-processing is required to remove the solvent from the extract. This requires more time and explosion-proof extraction and solvent removal equipment. The gravity of scalability and safety of this extraction method is significant.

Carbon dioxide is used in a number of other industries including hops oil extraction. Because of the nature of the cannabis plant, and the familiar similarities to hops, it was clear that 

 would be a good fit for cannabis (4). CO

 is easily manipulated into different phases using pressure and temperature. Naturally, this makes CO

 one of the most versatile solvents in cannabis extraction.

Despite requiring a longer extraction time, 

 from the plant because of its various phase properties (5). Subcritical and Supercritical CO

 are the two most popular phases for cannabis extraction. Supercritical fluids maintain the properties of both liquid and gas. This gives CO

 the ability to freely flow through the plant material, like a gas, while penetrating the trichomes and surface of the plant material, like a liquid. Subcritical CO

 is operated at a lower temperature becoming a less efficient solvent overall, but focusing primarily on the volatile aromatic compounds such as terpenoids and terpenes. CO

 is more efficient with extracting the full terpene profile of the plant including monoterpenes, sesquiterpenes, and a wide array of terpenoids, as demonstrated in a terpene fraction (

 pressures can be ramped up to the supercritical phase to increase the efficiency of cannabinoid extraction. With that, the extraction time is dramatically reduced but more undesirable components such as waxes and pigments are also extracted. This reduces the crude potency of the extract, but a wax removal process known as “winterization” can be utilized to remove the lipids. This increases the potency of the extract. The terpene fraction and cannabinoid fractions are blended together to create high quality extract for vapes (

In addition to the extraction solvent and parameters, the starting plant material also dictates the end result of the extract’s chemical profile. If starting with material that is low in terpenes but high in cannabinoids, the end result will be a low terpene high cannabinoid extract. If decarboxylated plant material is extracted with hydrocarbons, a THCA crystalline product will not be possible to produce as the THCA has already been converted to THC. There are many variables to the process that must be considered prior to extraction.

While testing methods have advanced significantly since the dawn of the modern era of cannabis, there remains a wide frontier of opportunity to discover and learn. Even in 2020, 10–30% of the total mass of a cannabis extract remains unidentified on a typical certificate of analysis (COA). This demonstrates that we are unable to identify a relatively large portion of the extract’s chemical makeup, or that it is not economically viable to do so. Generally, it is known that there are compounds such as esters, flavonoids, various pigments, alkaloids, and so on within cannabis extract. Despite the potential to influence experiences with cannabis, these compounds have yet to capture public interest or significant scientific inquiry with respect to product profiles and consumption.

Over time, discoveries have been made which demonstrate that each compound affects the human experience with cannabis. THC can enable euphoria, CBD reduces inflammation, limonene is energizing, 

 (7), linalool is relaxing—yet none of these compounds work the same together as in isolation. Each compound influences the behavior of the entire network to provide synergistic effects. Through studying cannabis interactions with the human body, scientists have unveiled entire networks of systems within the human body, namely the endocannabinoid system (ECS). Our understanding of cannabis chemistry remains limited, and this new frontier provides ample opportunity for research and insights that can enable people to get more from this plant.

https://en.wikipedia.org/wiki/Cannabis_sativa

https://www.edenlabs.com/coldfinger/ethanol-extraction-process/

https://www.edenlabs.com/co2-extraction/co2-extraction-process/

https://www.heylocannabis.com/post/what-is-rawx-why-does-heylo-cannabis-follow-this-method

https://www.heylocannabis.com/post/cbg-cannabigerol

 is the founder, CEO, and Chief Extractor of Heylo. With a background in chemistry and clinical research, Lo was inspired to explore cannabis as medicine and to enter the emerging industry. She joined Eden Labs, a leading CO

 extraction equipment manufacturer to support and expand a Research and Development department. There she managed the development of their latest and greatest CO

 extraction system. In 2017, after working with Eden Labs and another cannabis processor, Lo launched Heylo with a mission to help people get more out of life with cannabis.

Extraction Science

A review of the chemical makeup of cannabis extracts and how they affect the human body. From terpenes to cannabinoids, this article takes a deep dive into the chemistry of cannabis extracts.

Exploring the Chemical Makeup of Cannabis Extract by Method

Presentations and panel discussions with the Knox Family are always the highlight of every cannabis science event. For this installment of “Cannabis Crossroads,” I interviewed these true pioneers and leaders of endocannabinology—the Knox Family. This amazing family of four doctors are key opinion leaders in cannabis therapeutics and cannabinoid medicine. Collectively, they have counseled thousands of patients and dedicate time to educating many more by way of talks, seminars, blogs, and videos. 

It is amazing to me that the Knox family not only has four medical doctors, but that all of you are deeply involved with cannabis medicine. How did this come to be?

 Dr. Janice had been a board-certified anesthesiologist for 32 years before retiring from the operating room. She was invited to work for a medical marijuana clinic to sign authorizations for patients to access their cannabis legally, and was open to trying that. Subsequently, after hearing her experiences with the patients in the clinic, Dr. David also had the opportunity to begin seeing patients in the same clinics, while still a full time emergency physician. Superficially, it was easy to sign the authorizations, as patients clearly had conditions on the state approved list of qualifying conditions, but this introduction to cannabis medicine was so eye-opening for us that we had to actively pursue further knowledge.

To start, the patients we were seeing defied any expectation we may have initially had. Our prior exposure to cannabis in the conventional medical space was that it was simply a drug of abuse. This was quickly disproved, as these were patients from all walks of life, with complex medical histories, who were using cannabis therapeutically for many reasons, but were having better results than any conventional therapies had given them. After a few thousand people give you the same story about cannabis, you have to listen to what they are saying.

This was the impetus for us to pursue the research and science about cannabis and the discovery of the endocannabinoid system (ECS). At that time, Drs. Rachel and Jessica were completing their residency training in family medicine and preventative medicine, respectively. Dr. Janice, especially, talked with them extensively about her experiences with cannabis patients, and piqued their interest in the field. As they were always interested in a more holistic care model than that of conventional medicine, they quickly got on board with learning about the ECS and cannabis therapeutics.

It has frequently been stated that students training to become medical doctors have learned almost nothing about the ECS in the past. In a previous column I wrote about the University of Maryland School of Pharmacy offering a master's degree in medical cannabis science and therapeutics. Is endocannabinology being positively embraced by the medical community? What challenges remain in educating more physicians about cannabinoid medicine?

Very few medical training institutions are yet teaching about the ECS, especially as to the fully understood extent and complexities of the system (and the endocannabinoidome). A recent count was that only 13% of medical schools in the US mention the ECS somewhere in their curricula. This is beginning to change as attitudes evolve, even though the legality at the national level has not. Several schools are now offering more extensive courses such as the University of Maryland, but notably these are more likely to be associated with schools of pharmacy rather than medical schools. Younger physicians are, however, more open to accepting the science and institutions are opening up to doing more studies, but we don’t think this is to the point of the mainstream medical community embracing endocannabinology. The challenge is still to do away with a lot of antiquated attitudes (stigma) about cannabis, and to let the science of the ECS and pharmacology of the plant be heard. In some ways, when considering all of the complexities of the ECS and the endocannabinoidome, and the equally convoluting variables in cannabinoid profiles, the average physician just hasn’t had time to figure out what to do with it yet, let alone the institutions figuring out how to teach it. This doesn’t excuse academic institutions of the responsibility to teach their students about the ECS, however. Arguably, it’s not only their due diligence, but a moral obligation to do so given that 100% of patients have one, that all bodily systems are regulated by it, and that cannabis use is increasing significantly across the US population. Failing to teach the ECS is failing to prepare students for the real world.

Q: You must be approached frequently by physicians or students that would like to get more involved in cannabinoid medicine. What advice do you give them?

 We teach them how fundamental learning the science of the ECS is, and recommend that they begin their education there. Applying the science and pharmacology of cannabis comes more easily with that foundation in place. Understanding and practicing cannabinoid medicine is about so much more than just cannabis. The underlying physiology of the ECS explains why and in what ways cannabis is effective (or sometimes ineffective), but that is only part of the story.

"Ancient Healing" and "Revolutionary Care" are two of the three tenants listed on your website, DoctorsKnow.com. As medical technology advances, do cultures preserve their ancient knowledge, or is much of it lost as attention turns to the development of new drugs and treatments? Have we preserved our ancient healing knowledge or has much of it been lost over time?

 Our modern approach to medicine, unfortunately, has not retained a lot of ancient wisdom that still exists in what are now looked upon as “alternative” medicine fields, including naturopathy, ayurvedic medicine, Chinese medicine, and acupuncture—and probably by design. We’ve even coined them “alternative,” when the real alter (counter-to) native (natural) medicine is conventional, Western medicine. So there is some acknowledgment of those areas, but always as inferior to the power of conventional medicine.

Cannabis itself was displaced from a level of prominent use until less than 100 years ago, by the introduction of more specific monomolecular pharmaceuticals as well as by the political machinations to outlaw the plant. This deliberate act to remove cannabis from the pharmacopoeia and severely limit any study on the plant certainly resulted in a loss of knowledge, and a loss of at least two generations of research that could have been so helpful to so many. Much has been lost in terms of the broad education of medical practitioners in general, although the knowledge can still be found in smaller arenas. The understanding of the ECS is helping to support why many ancient and alternative practices have efficacy, and we hope will contribute to a new awakening of integrating all methods toward better health for all.

Q: What is "integrative cannabinology"? What is most important for our readers to know about this field?

Functional and integrative endocannabinology is the application of functional and integrative medicine practices to correct endocannabinoid dysfunctions (and thus disease) at their root cause. Several common and difficult to treat conditions—such as migraines, fibromyalgia, post-traumatic stress disorder (PTSD), epilepsy, and obesity among others—have been associated with ECS dysfunction. The functional piece of endocannabinology means that rather than treating only the symptoms of these conditions, we seek to correct the underlying endocannabinoid dysfunction—the root cause. The integrative piece means we address the symptoms and root cause with a combination of treatment modalities, including not only cannabis, but also nutrition, cannabimimetic substances and practices (for example, herbs, spices, terpenes, mind-body therapies, exercise), and even conventional therapies when appropriate. This is a burgeoning field of medicine that we believe all clinicians should know something about, and that we hope will become a board-certified specialty or subspecialty in its own right in the near future.

Q: In very recent history we have witnessed an opiate crisis that has had a devastating impact on our families and communities. The very people seeking freedom from chronic pain were suddenly snared in a deadly addiction trap that robbed everything from their life. How important is cannabis and other natural medicines in allowing people to break their opiate addiction and what has the medical community learned from this disaster?

 The current opioid catastrophe resulted when big pharma seeking big money pushed their pharmaceuticals to expand their market. The push to define pain as the “5th vital sign” and the declaration that pain was not being adequately treated, plus the questionable studies that opioids were not addicting for true pain patients, allowed for the huge increase of opioids available to everyone. It can be extremely difficult to distinguish between a patient with legitimate pain, especially chronic pain, and those simply needing the opiate to stave off withdrawal. The current push to limit opioids for everyone now results in denial of relief for those truly needing opioids for pain management. In many cases, this results in practitioners treating the policies rather than the patient. I’m not sure the medical community as a whole has learned much, rather that this is just the swing of the pendulum we operate under.

Cannabis and other integrative modalities, on the other hand, do have a lot to offer for pain management and opiate addiction. The anecdotal attestation from thousands of our patients that they were able to reduce or eliminate the need for opioids for their chronic pain conditions is astoundingly important, even if some controlled studies that have been published do not prove this with a statistically significant difference. Many patients do get pain relief from cannabis alone, and studies show the synergism of cannabis with opioids. This means that with cannabis use the patients get better pain relief from a lower dose of opioids, and do not develop the same increasing tolerance and need for a higher opiate dose over time. This lowers the risk associated with treatment with opioids such as respiratory depression and even death. The fact is that pain relief from cannabis does not require any level of intoxication from THC. We can’t count the number of patients who state emphatically that cannabis saved their lives from opioids.

Unfortunately, in the national forums on the opiate crisis, there have been many subjects discussed but minimal, if any, mention of cannabis or of other natural medicines, such as kratom. This is likely due to no US Food and Drug Administration (FDA) approval of any of these other modalities.

Q: Pharmaceutical companies have embraced biotherapeutics, but there still seems to be a heavy focus on isolating a single, bioactive component and developing this single component drug to market. Medical cannabis and whole plant medicine is the antithesis of this approach and patients benefit from the synergistic action of many plant compounds (cannabinoids, terpenes, bioflavonoids). Is the isolation of a single component by pharma companies a failed strategy? How do we bridge the gap between these pharma approaches and nutraceuticals?

It is hard to call it a completely failed strategy, as the FDA approval of Epidiolex (essentially a CBD isolate) resulted from this approach by GW Pharmaceuticals. This does highlight the difficulties of studying the effects of a complex polypharmaceutical represented by the whole plant versus a single molecule. This is like multivariable calculus versus linear addition or subtraction, and does not lend itself amenable to the double blinded controlled studies the FDA and pharmaceutical developers require for approval. That system is designed to work with isolates, but as we know, the entourage effect is much more powerful. Epidiolex works, with a significant benefit in reducing seizures, but often requires precise and higher doses with more risk of side effects; whole plant extracts with the full entourage work better, at a much lower dose of CBD. The entire interplay of all the cannabinoids, terpenes, and other components carries a lower risk of side effects. Studying complex plant medicines would require a change in the fundamental approach taken by the FDA to conduct research. The agency would need to broaden its focus on isolates as pharmaceutical drugs to include whole plant, nutraceutical research.

When you look at the history and spectrum of cannabis use, with its remarkable safety profile compared to most pharmaceuticals, there is no reason that whole plant cannabis needs to be subject to that same level of controlled study. While inexact for actual dosing, the approach of time and titration of the cannabis profile (CBD:THC amounts and ratios, other cannabinoids, terpenes, and flavonoids present) has been effective for multitudes of people for millennia. We hope that this approach can be refined to be more exact for any individual patient, but including cannabis as part of one’s approach to health and lifestyle is very low risk, and the FDA needs to realize this.

There seems to be a shift in healthcare—a moving away from reactive treatment towards more proactive health and lifestyle management. Is medicine moving from disease treatment to disease prevention?

 On one hand, much of our modern medical approach to treatment is in reaction to illness or injury. This is necessary to help patients recover and regain their life, but has not been very effective in resolving or controlling many chronic disease processes. Medicine has always had a niche for preventive medicine, but it does not yet seem to be a big movement in the medical establishment. Many people are realizing this, and that they individually need to be much more proactive in managing all those elements in their lifestyle that contribute to the risk of developing or managing those chronic medical conditions. This includes everything from diet and nutrition, exercise and activity, family and relationships, to chemical and toxic exposures. Our current Western approach really is poorly designed for that, as overall health is so dependent on the entire environment we are living in 24/7/365. You see your doctor for 10 minutes, who may refer you out for dietary counseling, physical therapy or exercise, other programs such as smoking cessation and so forth, but these are piecemeal approaches in many cases. Where you live can be a huge factor if you have limited access to clean water, healthy food choices, and the ability to exercise. We talk about health equity, but that cannot be separated from systemic, total equity, something we call “health equity.”

We are learning so much more about cannabis. What are you most excited about regarding cannabinoid medicine?

 We believe there will be a renaissance in the clinical studies needed to bring cannabinoid medicine into the forefront. The science of endocannabinology will continue to develop and explore the workings of the complex physiology of the endocannabinoidome. The lack of well-designed clinical studies (the reasons why we’ve already discussed) on the use of cannabinoid therapeutics has been the limiting factor for most physicians to ignore or downplay any role for incorporating cannabinoids into their practice. With the increased acceptance of cannabis, we hope the path will be cleared and this research can move ahead. Already there are proposals to study the use of CBD or other cannabinoids in the treatment of COVID-19, especially those patients suffering a cytokine storm, the hyperinflammatory reaction that causes severe illness. High profile studies such as that may be the impetus to allow for even more clinical studies on the myriad of conditions we know cannabinoids can help

 received her bachelor’s degree from Harvard University before going on to earn her medical and business degrees from Tufts University. She completed her postgraduate training in Preventive Medicine at the University of California San Diego while earning her public health degree from San Diego State University. Dr. Jessica believes that using all-natural remedies to fight disease and promote well-being should be the first-line approach in medical care. With healthcare costs soaring, and access to medical care as limited as ever, she believes the application of lifestyle and natural medicine is key to adequately addressing the current state of public health in our communities and country as a whole. It was out of this belief that Dr. Jessica’s interest in cannabis therapeutics blossomed.

 has more than 35 years in the practice of medicine. She is a graduate of the University of California at Berkeley and the University of Washington School of Medicine. She is a wife, mother, best selling author, and board certified anesthesiologist. Over her years in the operating room, Dr. Janice witnessed the increased use of life threatening pain medications and unnecessary medical interventions that yield low success rates while patients continued to get sicker. This awareness motivated Dr. Janice to become an expert in cannabis and the fields of cannabis therapeutics and endocannabinology.

 is a graduate of the University of Washington and University of Washington School of Medicine. After 37 years of clinical experience in emergency medicine and counting, he has expanded his expertise to include cannabis therapeutics. Dr. David has consulted with more than 5000 patients across the Pacific Northwest within the past year, noting the degree of results cannabis has had on a variety of maladies, but perhaps none more common than chronic pain, a condition which plagues emergency rooms nationwide.

 received her medical and business degrees from Tufts University after completing her undergraduate studies at Duke. She trained in family and integrative medicine before pursuing additional study in the areas of functional, cannabinoid, and cannabis medicine. Though her formal training in family and integrative medicine provides her with the know-how to treat acute illness and chronic disease, she is passionate about teaching her patients how to avoid and even reverse them altogether through natural means.

 is the columnist and editor of “Cannabis Crossroads” and a contributing editor to 

 magazine. Crossney is also the president and CEO of CSC Events. Direct correspondence to: 

Cannabis Crossroads

An interview with an amazing family of four doctors—the Knox Docs—who are key opinion leaders in cannabis therapeutics, endocannabinology, and cannabinoid medicine.

The First Family of Cannabinoid Medicine: The Knox Docs

Federal law defines legal hemp as cannabis plant material that contains not more than 0.3% dry weight total tetrahydrocannabinol (THC). Because of confusion over the law, a number of false arrests and seizures of legal hemp have occurred. Additionally, the United States Department of Agriculture recommends about one sample per acre of hemp should be tested. This means regulatory agencies may be swamped with samples come harvest season and might have difficulty keeping up with the demand. There is a need then for law enforcement and regulatory agencies to measure total THC in cannabis plant material to discriminate between legal hemp and illegal marijuana accurately, quickly, and easily to alleviate these problems. We have developed a novel mid-infrared spectrometer that measures total THC in dried, ground cannabis plant material in 2 min. By applying a unique total THC hierarchical classification algorithm to the data, high total THC cannabis is sorted from low THC cannabis with a success rate of 99.4% based on a challenge set of 491 samples. The algorithm distinguishes low total THC marijuana from hemp with a success rate of 95.1% based on a challenge set of 284 samples. The applications of this analyzer for law enforcement, forensic laboratories, and regulatory agencies are discussed.

For the purposes of this paper these terms will have the following meanings.

: The dried, ground buds, flowers, stems, stalks, leaves, and seeds of genus 

: Cannabis plant material containing equal to or more than 5% dry weight percent total THC.

: Cannabis plant material containing less than 5% dry weight percent total THC.

: Cannabis plant material containing more than 0.3% dry weight percent total THC as defined by Federal law (1,2).

: Cannabis plant material containing 0.3% or less dry weight percent total THC as defined by Federal law (1,2).

 The weight percent of an analyte in a sample.

The 2018 United States Farm Bill as passed by the United States Congress (1) and a subsequent Interim Final Rule as promulgated by the United States Department of Agriculture (USDA) on October 29, 2019 (2) makes it legal to grow and possess hemp in the United States so long as the total THC content by dry weight does not exceed 0.3%. Undried or “wet” hemp is not analyzed because the law clearly states that dry hemp must be analyzed (1,2). Additionally, drying hemp changes cannabinoid concentrations, and we are not aware of any peer-reviewed literature that correlates the composition of wet hemp with dried hemp. Until this happens, the industry should continue to test dry hemp only.

Since passage of the Farm Bill, several legal hemp shipments have been seized by law enforcement around the country under the mistaken assumption that the material was illegal marijuana (3–7). Places where this has happened include Idaho (3), Oklahoma (4), New York City (5), Texas (6), and Colorado (7). The latter is surprising since medicinal and recreational marijuana are legal in Colorado. However, one must still possess the proper paperwork and licenses to transport hemp and marijuana in that state (7). As a result of these seizures, innocent people have spent time in jail, thousands of dollars of valuable hemp have been seized, bad press for law enforcement has ensued, and lawsuits have been filed (3). Another outcome of the change in the law is that forensic laboratories are now being swamped with cannabis plant material samples, many of which are legal hemp, tying up resources and spending money that is better spent elsewhere.

The law and interim final rule task state departments of agriculture with developing programs for hemp testing (1,2). The interim final rule contains sample calculations that recommend that about one cutting per acre of hemp be tested (2). This means come harvest season each state must test thousands of samples during a short harvest season. We fear that many states may not have the testing capacity to perform the Federally recommended amount of testing.

There is a need then for law enforcement to analyze seized cannabis plant material in the field to prevent false arrests, for forensic laboratories to weed out nonprosecutable cases to help streamline their operations, and for state departments of agriculture to increase their cannabis testing capacity so hemp farmers receive results in a timely fashion. Some current testing technologies are nonquantitative and hence not congruent with the law since they do not produce a total THC value. Other technologies produce a total THC value, but require expensive consumables, significant sample preparation, skilled operators, and are slow with a high cost per analysis. Ideally, then there would exist a method for distinguishing hemp from marijuana that is fast, easy-to-use, portable, inexpensive, and accurate. Our progress in using mid-infrared (mid-IR) spectroscopy and a unique hierarchical total THC classification algorithm towards this goal is discussed.

Criteria for Evaluating Chemical Analysis Methods

 (click to enlarge) shows the “Golden Rectangle of Chemical Analysis,” four figures of merit that should be used when evaluating any analytical chemical method.

The criteria listed are accuracy, speed, cost, and the ability to perform representative sampling. 

 is a measure of how far away an analysis is from its true value (8). Speed includes the time to prepare a sample and how long it takes for an instrument to analyze it. Cost includes the initial cost of the instrument, the cost to analyze each sample, and the cost of maintenance and upkeep of the system. The representative sampling criterion comes into play when analyzing inhomogeneous substances such as cannabis plant material. The proper way to analyze these materials is to collect many random samples, analyze them, and report the average result with an appropriate margin of error (9,10). This criterion dovetails off the others since for representative sampling a technique must be fast enough and inexpensive enough to allow the requisite number of samples to be analyzed at a realistic cost in a reasonable amount of time.

In a perfect world, an analytical method would be accurate, fast, low cost, and allow for representative sampling. The reality is that there are trade-offs between these criteria. For example, the most accurate methods, such as typical laboratory analyses, can be slow and expensive. Field analyzers, while perhaps not as accurate, may be faster, cheaper, and more readily allow representative sampling than laboratory methods. Keeping these criteria in mind, we will review the technologies currently available for distinguishing hemp from marijuana.

Technologies for Distinguishing Hemp and Marijuana

For cannabis plant material the presence of THC is legal so long as it contains less than 0.3% total THC (1,2). This means that for a chemical analysis technique to be used by regulatory and law enforcement officials to distinguish between hemp and marijuana it must report a weight percent total THC value

We draw the analogy to drunk driving laws. These laws typically state a blood alcohol value below which it is legal to drive and above which it is illegal to drive. An analyzer that only tests for the presence of alcohol in one’s body without generating a blood alcohol level would be pointless because it cannot distinguish between legal and illegal drivers. Similarly, a cannabis plant material total THC test that only tests for the presence of THC and does not generate a total THC value is pointless because it cannot distinguish between legal hemp and illegal marijuana as defined by Federal law. There are several methods in place that claim to be able to distinguish between hemp and marijuana that do not generate a total THC value. These include organoleptic analysis, color analysis, genetic analysis, and Raman spectroscopy.

Organoleptic analysis is the use of the human senses to analyze a material (11). For cannabis there are people, particularly botanists, who are trained to distinguish cannabis from other plants by look and smell. Before the legalization of marijuana anywhere in the United States, this would have been probable cause for arrest. However, now that cannabis plant material with upwards of 0.3% total THC is legal this is no longer sufficient because legal cannabis plant material may contain some THC. We believe some of the false arrests documented above were caused by failure of law enforcement to understand this problem. We have seen no peer-reviewed data showing a human can organoleptically determine total THC levels. Hence this method should be avoided.

There exist color tests that disclose the presence of THC in a sample (12). These tests do not produce a weight percent total THC result so are not compliant with what the law requires. Also, practically every cannabis sample contains THC, so this technology would not be useful in distinguishing different types of cannabis plant materials from each other because of the large number of positives from legal hemp samples.

A recent paper in this journal disclosed the use of genetic testing to distinguish hemp from marijuana (13). Marijuana cultivars are bred for high THC values, whereas hemp cultivars are bred for low THC values. It makes sense then that these strains would have different genotypes. However, DNA testing is not compliant with the law since it does not measure a total THC value. Additionally, a cannabis plant’s genotype is not the only predictor of its total THC concentration; growing conditions play a role as well. It is the combination of genotype and growing conditions that determines the cannabinoid profile of a cannabis grow. Thus, genetic testing may be useful to classify cannabis plants into strains, but it does not give us the total THC measurement needed to determine legality.

In a recent paper, claims were made that a Raman spectrometer successfully distinguished between hemp and marijuana 100% of the time (14,15). However, their reports contain no total THC measurement data on the samples analyzed, and no science supporting the ability of their spectrometer to quantitate total THC in cannabis plant material. Thus, we must conclude like the other techniques listed here, that Raman spectroscopy is not congruent with the law and should not be used to distinguish hemp from marijuana.

In summary then nonquantitative methods may be fast, inexpensive, and allow a high sample throughput enabling representative sampling, but are inaccurate since they do not provide a total THC value.

For the quantitative determination of total THC in cannabis plant material chromatography and spectroscopy have been used (16–19).

In chromatography, cannabis plant material is prepared by weighing, grinding, extracting, filtering, and diluting to obtain a sample of purified cannabinoids dissolved in solvent (16). In high performance liquid chromatography (HPLC) (17,18), the sample is injected into a flowing stream of liquid that passes through a column containing a bed of separating medium (16). Different molecules interact with the medium with different strengths and leave the column at different times (17,18). These purified materials are then quantitated. For the determination of cannabinoids by HPLC, a common detection scheme is diode array ultraviolet-visible spectroscopy (17,18). In gas chromatography (GC), the sample is vaporized in a heated injection port, swept into a bed of separating medium by a flow of gas, this bed is heated, cannabinoids vaporize based in part on their boiling points, and are swept towards a detector for quantitation. Flame ionization is one type of GC detection scheme that has been used to analyze cannabis (19).

Chromatography is certainly accurate, but it is slow (16-19). Between the sample preparation steps mentioned above and running the instrument it can take 20 min or more to analyze a single sample (17–19). Chromatography is also expensive. Each analysis requires expensive consumables such as solvents, vials, filters, and syringes. The required chemical operations and the use of a chromatograph is difficult enough that state regulations require cannabis laboratory technicians using these devices be degreed and highly trained (20). These people do not work cheap, driving up the cost per analysis. There is also the cost of legally disposing of the solvents used in HPLC. In total, between the expense of the instrument, consumables, time, labor, and waste disposal it can cost $30 or more per sample to analyze cannabis samples (16). The slowness and expense of using chromatographs prevents the analysis of the large number of samples needed for appropriate representative sampling (9,10). Thus, when considering the Golden Rectangle of Chemical Analysis chromatography is certainly accurate but suffers when considering cost, speed, and throughput.

Infrared spectroscopy is the study of the interaction in infrared light, otherwise known as heat, with matter (21,22). Two different wavelength regions, the near infrared (NIR) and the mid-infrared (mid-IR) are commonly used to analyze samples. NIR wavelengths are shorter than mid-IR wavelengths (21). NIR is used to determine analytes such as fat, protein, and moisture in agricultural crops including hay, forage, and grain (27,28). Given the history of NIR spectroscopy’s use on agricultural crops, its extension to cannabis analysis seems logical. NIR has been used to determine cannabinoid content in hemp (24).

However, NIR suffers from some problems. Firstly, NIR is not selective, it can typically only see chemical functional groups that contain O-H, N-H, and C-H bonds (27,28). This is fine for gross measurements, such as total protein, but NIR struggles to distinguish cannabinoids of similar structure such as THCA and THC. Another problem NIR suffers from is lack of sensitivity. Because of the physics of the situation, NIR absorbances are 100 times weaker than mid-IR absorbances (23,28). This means NIR can struggle with accurately determining total THC at low levels in cannabis plant material.

Mid-IR is used throughout the food, forensic, pharmaceutical, and many other industries (21). Mid-IR has been used to determine cannabinoids in marijuana (25), hemp (26), cannabis distillates (29), extracts (30), and to study the rate and mechanism of potency degradation in cannabis oils (31). Mid-IR has excellent selectivity since it can see hundreds of different kinds of chemical bonds (22). Therefore, it can determine total THC and it can quantitate both THC and THCA in hemp and marijuana (25,26). Since mid-IR absorbances are 100 times more intense than NIR absorbances (22,27,28), this allows mid-IR to accurately determine low levels of total THC in hemp (26). Additionally, mid-IR requires no complex sample preparation or consumables so the cost per analysis is $0, a single cannabis plant material sample can be analyzed in 2 min (26), and both of these advantages lend themselves well for handling the large number of samples needed for true representative sampling. For these reasons, we chose to study the utility of mid-IR spectroscopy for distinguishing hemp from marijuana. We will show that mid-IR is sufficiently accurate for reliably distinguishing hemp from marijuana while maintaining the advantages of speed and low cost needed for representative Sampling.

Dried cannabis plant material samples (<10% moisture) were prepared for analysis by taking ~5 g aliquots and grinding them for 1 min in a standard coffee grinder. Samples were scanned using a BSS 3000 Cannabis Analyzer from Big Sur Scientific. It is a general purpose quantitative mid-IR spectrometer and can be used in other applications besides cannabis (32).

The analyzer is 4-in. x 6-in. x 8-in. and weighs 2 lbs so it is compact, lightweight, and portable. Using its carrying case the unit can be taken into the field and used almost anywhere including laboratories, offices, greenhouses, barns, trade shows, and the bed of pickup trucks. It can be powered by a battery giving portable power. The optics are pinned in place at the factory yielding a rugged design that is insensitive to vibrations. The analyzer is precalibrated and validated and works out of the box with a few minutes of setup and testing. So long as the device is kept dry, has a power source, and a horizontal surface to sit upon it will work well.

The spectrometer design has been disclosed in detail before (32); a brief description will be provided here. 

 (click to enlarge) shows a schematic layout of the spectrometer.

In the figure, the number 1 denotes a solid state mid-IR source that is pulsed electronically to discriminate between IR light coming from the source and ambient heat. Number 2 is a transfer optic that directs the infrared beam to number 3, a zinc selenide (ZnSe) triple bounce attenuated total reflectance (ATR) crystal (21). The top surface of the ATR crystal is where the sample, number 5, is placed, and hence is called the sampling surface. The infrared beam, which is internal to the ZnSe crystal, will internally reflect from a spot on the sampling surface (21,33,34). At this spot, some infrared amplitude will appear above the crystal surface forming an “evanescent wave” (21,33,34). Samples placed on the sampling surface can absorb the evanescent wave, and the infrared beam is then analyzed to determine the sample’s spectrum (21).

To analyze liquids by ATR, they are spread on the sampling surface and their spectra are scanned (21). To ensure reproducible interaction between solid samples and the evanescent wave, a clamp, 4 in Figure 2, is used to secure these samples to the sampling surface. 

 (click to enlarge) shows a sample of ground cannabis plant material being applied to the sampling surface of the analyzer prior to clamping.

The clamp features a slip-clutch mechanism that will slip when a specific pressure is reached, and the user is notified of this by an audible click. This ensures all solid samples have the same amount of pressure applied to them, which is important for reproducible sample-window contact and hence quantitative analysis (21,23). The ATR method was chosen because it requires little sample preparation, is fast, easy-to-use, and has been shown to be quantitative when applied to cannabis plant material (25,26). Additionally, the technique is nondestructive, which is good if the sample is particularly valuable, or needs to be preserved as evidence.

After the infrared beam interacts with the sample it leaves the ATR crystal and impinges upon transfer optic (number 6),which directs the beam to the spectrometer-on-a-chip (number 7). The spectrometer is a Fabry-Perot interferometer (FPI), which consists of an optical cavity with two reflective surfaces (mirrors) (32). A scan is performed by moving one of the mirrors away from the other. At any given mirror position only a fixed bandwidth of wavelengths will pass through the spectrometer and onto the detector, number 8, and changing the mirror position changes the wavelengths that impinge on the detector. Scanning a spectrum is a matter of moving the mirror through a given set of positions and measuring the amount of light hitting the detector at each position. The detector material used was lithium tantalate. The spectrometer and detector comprise a monolithic unit about the diameter of a dime. We call the spectrometer a Fabry-Perot Interferometer-Attenuated Total Reflectance spectrometer or FPI-ATR for short. It has been the tradition in spectroscopy to name spectrometer designs after their inventors. For example, the well-known Czerny-Turner spectrometer is named after its inventors (35). Hence, we will use the terms “FPI-ATR” spectrometer and “Smith spectrometer” interchangeably for this device.

A Microsoft Surface Pro computer (number 9) running the Windows 10 Pro operating system was used. Big Sur Scientific’s Cannabis Analyzer software controlled and scanned the spectrometer, measured spectra, applied calibration models to the spectra to predict concentrations, and displayed results. Communications between the analyzer and computer are via a USB cable.

Mid-IR spectra were measured from 1250 to 960 cm

 instrumental resolution. This spectral region was chosen because THC and other cannabinoids absorb strongly in this wavenumber range, enhancing sensitivity. A background spectrum with no sample on the sampling window is measured prior to the analysis of each sample and takes 1 min. After cannabis plant material is clamped to the sampling window, the sample scan takes an additional 1 min. It thus takes 2 min to analyze a single sample. The sampling window is cleaned before and after each analysis with a paper towel wetted with a small amount of alcohol.

After each analysis the results are presented to the user on the computer’s screen. The user has the option to print the results, copy and paste the results into a different application, or generate a certificate of analysis in Adobe Acrobat PDF format. Each sample spectrum measured is automatically saved in an industry standard spectroscopy file format (Thermo GRAMS *.SPC format), and each set of results is automatically saved as a text file. The unit comes with a system check sample that is run daily to ensure the analyzer is functioning properly.

HPLC reference data from a state licensed, ISO registered laboratory (36) were used to calibrate the Smith spectrometer. The laboratory did not correct the measured weight percent cannabinoid values for variations in moisture content of the cannabis plant material. Pure cannabinoid standards were not used for calibrating the mid-IR analyzer because this presents a calibration applicability problem (23). For chromatographic cannabis potency analyses the final sample is a solution of cannabinoids (17–19), and hence it is appropriate to use solutions of cannabinoids as standards. However, in practice the mid-IR spectrometer analyzes dried, ground cannabis plant material not solutions of cannabinoids. For the spectroscopic models to be applicable to cannabis plant samples, the spectra and weight percent cannabinoids as determined by HPLC of standard cannabis plant samples were used to build calibrations.

The partial least squares algorithm (PLS1) was used to build the calibration models used (23). An advantage of this algorithm over traditional single peak Beer’s Law analyses is that it works well even if spectral peaks contain overlaps from multiple analytes (23). Thus, there is no need for each analyte to have a spectrally resolved peak for quantitation to be achieved (23).

The Hierarchical Classification algorithm employed involves using spectroscopic calibrations to measure the total THC in a cannabis sample, and then sorting the sample into an appropriate category based on this result. Here is how it works. In an initial step, a broad concentration range calibration is used to determine the total THC in cannabis plant material, and the samples are sorted into high and low total THC cannabis categories. In a second step, a narrower range calibration is applied to the low total THC cannabis plant material and this number is used to sort samples into legal hemp or illegal marijuana.

The quality of the total THC spectroscopic calibrations was measured by analyzing a validation set by mid-IR and applying a calibration that did not include spectra of the validation samples. The validation samples were analyzed in triplicate by mid-IR; the predicted values reported here are the average of these values. The predicted total THC values for the validation set were compared to the known values for these samples as measured by HPLC (36). The accuracies of the spectroscopic calibrations were determined as a standard error of prediction (SEP) (23). The SEP is the best measure of calibration accuracy since it determines how well a calibration performs on samples it has not seen before, which is how real-world calibrations work (23). The ability of the Hierarchical Algorithm to distinguish between hemp and marijuana correctly was measured with a set of challenge samples that were known to be either hemp or marijuana. The percentage of samples correctly sorted is reported as the success rate.

Previous work (17,18) has shown that for cannabis plant material much of the contribution to the total THC value is from THCA rather than THC. The mid-IR spectrum of pure THCA is seen in 

(click to enlarge). The portion of the spectrum from 1250 to 960 cm

 is shown, the region scanned with Smith spectrometer used in this study.

 here because it is the shoulder of a larger peak that tops out at 1256 cm

, beyond the range scanned. Peak B is at 1190 cm

. In going from hemp with low total THC values to recreational marijuana with high THC values for quantitation to be possible there should be mid-IR spectral features whose size changes with total THC content.

The same three mid-IR features present in the spectrum of pure THCA as seen in Figure 4 are clearly present in the spectra of cannabis plant material seen in 

 (click to enlarge) and are labeled the same in both figures. As the total THC content goes from 0.1%, to 14%, to 30% in Figure 5, the mid-IR features of THCA clearly get larger. This correlation between peak size and total THC value means Beer’s Law (23) can be used as the basis for using mid-IR to quantitate total THC in cannabis plant material.

Obtaining accurate HPLC reference data for calibrating the spectrometer proved difficult. The problem of inter-laboratory error, different cannabis laboratories obtaining varying results on the same samples, has been researched and is well documented (29,37–42). Causes of this problem include lack of standard reference materials, laboratories using different sample extraction methods, and lack of adequate training (29,41–43).

At first, we attempted to use reference data from multiple laboratories in our spectroscopic calibrations. However, the inter-laboratory error problem caused the resultant calibrations to be of poor quality. When we built calibrations using data from a single laboratory, calibration quality improved markedly. This meant we had to choose a laboratory whose reference data we would use and trust. We literally roamed the country visiting many cannabis analysis laboratories, and vetted them by interviewing their laboratory directors, reviewing their standard operating procedures, and watching them perform analyses. Additionally, at some laboratories we ran the same cannabis plant samples by mid-IR spectroscopy and HPLC to provide an orthogonal test of the quality of the chromatographic results. We took the correlation between mid-IR and HPLC data as a measure of chromatographic quality, assuming that mid-IR is an unbiased and independent method. Based on our work we chose a state licensed, ISO certified laboratory (36), and used their weight percent cannabinoid values to calibrate the mid-IR analyzer.

The Search for Cannabis Standard Reference Materials

In the United States, the National Institutes of Standards and Technology (NIST) is the Federal agency tasked with generating standard reference materials (SRMs) for industry (43). SRMs are materials of known and agreed upon composition that can be used in round robin studies to evaluate the accuracy of different laboratories, the quality of different methods, and to evaluate instrumentation. For example, NIST supports the green tea industry with a standard reference material (44). This means that NIST knows how to create SRMs for agricultural crops. It would make sense then that NIST should create SRMs for the cannabis industry.

However, recall that in the United States at the Federal level all cannabis plant material with a total THC value of greater than 0.3% is considered marijuana and is illegal (1,2). Thus, it would be against Federal law for NIST to develop SRMs for marijuana analysis. But there is nothing preventing NIST from developing SRMs for legal hemp. NIST is in fact developing such materials (45). However, it may be some time until these SRMs are ready.

Fortunately, the University of Kentucky (UKY) has developed their own set of hemp standard reference materials as part of their Hemp Proficiency Program (HPP) (46). This sample set consists of four legal hemp samples, each of which has been analyzed by more than 50 cannabis analysis laboratories. The certificates of analysis for these samples contain the average total THC, total CBD, and other cannabinoid weight percents as determined by these laboratories. Barring the introduction of any NIST cannabis SRMs, we believe the UKY samples are the best validation set now available in the cannabis industry, and we encourage all cannabis analysis laboratories to use these samples to vet their methods and to participate in the HPP.

 A novel mid-infrared spectrometer that measures total THC in dried, ground cannabis plant material in 2 min is discussed as well as the applications of this analyzer for law enforcement, forensic laboratories, and regulatory agencies.

July/August 2020

A Proposed Representative Sampling Plan for Hemp Grows

Exploring the Chemical Makeup of Cannabis Extract by Method

The First Family of Cannabinoid Medicine: The Knox Docs

Distinguishing Hemp from Marijuana by Mid-Infrared Spectroscopy

America’s Heartland Tackles Hemp Cultivation with Education

The Role of Cannabinoids in the Global Fight Against Antibiotic Resistance

Setting the Standard for Cannabis Labeling: Introducing the Universal Cannabis Product Symbol and the Universal Cannabis Information Label

From the Editor: Behind Our Redesign