As mentioned in Part 1, the physiological effects of cannabis are mediated by a group of structurally related organic compounds known as cannabinoids. The cannabinoids are biosynthetically produced by a growing cannabis plant and Figure 1 details the biosynthetic pathways leading to some of the most important cannabinoids in plant material.
The analytical measurement of cannabinoids is important to ensure the safety and quality of cannabis as well as its extracts and edible formulations. Total cannabinoid levels can vary significantly between different cultivars and batches, from about 5% up to 20% or more by dry weight. Information on cannabinoid profiles can be used to tailor cultivars for specific effects and allows end users to select an appropriate dose.
Routine Analysisvs. Cannabinomics
Several structurally analogous groups of cannabinoids exist. In total, structures have been assigned for more than 70 unique phytocannabinoids as of 2005 and the burgeoning field of cannabinomics seeks to comprehensively measure these compounds.¹
Considering practical potency analysis, the vast majority of cannabinoid content is accounted for by 10-12 compounds. These include Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabigerol (CBG), Δ9-tetrahydrocannabivarian (THCV), cannabidivarin (CBDV) and their respective carboxylic acid forms. The cannabinoids occur primarily as carboxylic acids in plant material. Decarboxylation occurs when heat is applied through smoking, vaporization or cooking thereby producing neutral cannabinoids which are more physiologically active.
Potency Analysis by HPLC and GC
Currently, HPLC and GC are the two most commonly used techniques for potency analysis. In the case of GC, the heat used to vaporize the injected sample causes decarboxylation of the native cannabinoid acids. Derivatization of the acids may help reduce decarboxylation but overall this adds another layer of complexity to the analysis² ³. HPLC is the method of choice for direct analysis of cannabinoid profiles and this technique will be discussed further.
A sample preparation method consisting of grinding/homogenization and alcohol extraction is commonly used for cannabis flower and extracts. It has been shown to provide good recovery and precision² ³. An aliquot of the resulting extract can then be diluted with an HPLC compatible solvent such as 25% water / 75% acetonitrile with 0.1% formic acid. The cannabinoids are not particularly water soluble and can precipitate if the aqueous percentage is too high.
To avoid peak distortion and shifting retention times the diluent and initial mobile phase composition should be reasonably well matched. Another approach is to make a smaller injection (1-2 µL) of a more dissimilar solvent. The addition of formic acid or ammonium formate buffer acidifies the mobile phase and keeps the cannabinoid acids protonated.
The protonated acids are neutral and thus well retained on a C18 type column, even at higher (~50% or greater) concentrations of organic solvent² ³.
Detection is most often done using UV absorbance. Two main types of UV detectors are available for HPLC, single wavelength and diode array. A diode array detector (DAD) measures absorbance across a range of wavelengths producing a spectrum at each point in a chromatogram while single wavelength detectors only monitor absorbance at a single user selected wavelength. The DAD is more expensive, but very useful for detecting coelutions and interferences.
Chemical Constituents of Marijuana: The Complex Mixture of Natural Cannabinoids. Life Sciences, 78, (2005), pp. 539
Development and Validation of a Reliable and Robust Method for the Analysis of Cannabinoids and Terpenes in Cannabis. Journal of AOAC International, 98, (2015), pp. 1503
Innovative Development and Validation of an HPLC/DAD Method for the Qualitative and Quantitative Determination of Major Cannabinoids in Cannabis Plant Material. Journal of Chromatography B, 877, (2009), pp. 4115
Rebecca is an Applications Scientist at Restek Corporation and is eager to field any questions or comments on cannabis analysis, she can be reached by e-mail, firstname.lastname@example.org or by phone at 814-353-1300 (ext. 2154)
When a cannabis sample is submitted to a lab for testing there is a four-step process that occurs before it is tested in the instrumentation on site:
It is ground at a low temperature into a fine powder;
A solution is added to the ground powder;
An extraction is repeated 6 times to ensure all cannabinoids are transferred into a common solution to be used in testing instrumentation.
Once the cannabinoid solution is extracted from the plant matter, it is analyzed using High Pressure Liquid Chromatograph (HPLC). HPLC is the key piece of instrumentation in cannabis potency testing procedures.
While there are many ways to test cannabis potency, HPLC is the most widely accepted and recognized testing instrumentation. Other instrument techniques include gas chromatography (GC) and thin layer chromatography (TLC). HPLC is preferred over GC because it does not apply heat in the testing process and cannabinoids can then be measured in their naturally occurring forms. Using a GC, heat is applied as part of the testing process and cannabinoids such as THCA or CBDA can change form, depending on the level of heat applied. CBDA and THCA have been observed to change form at as low as 40-50C. GC uses anywhere between 150-200C for its processes, and if using a GC, a change of compound form can occur. Using HPLC free of any high-heat environments, acidic (CBDA & THCA) and neutral cannabinoids (CBD, THC, CBG, CBN and others) can be differentiated in a sample for quantification purposes.
Near infrared (NIR) has been used with cannabis for rapid identification of active pharmaceutical ingredients by measuring how much light different substances reflect. Cannabis is typically composed of 5-30% cannabinoids (mainly THC and CBD) and 5-15% water. Cannabinoid content can vary by over 5% (e.g. 13-18%) on a single plant, and even more if grown indoors. Multiple NIR measurements can be cost effective for R&D purposes. NIR does not use solvents and has a speed advantage of at least 50 times over traditional methods.
The main downfall of NIR techniques is that they are generally less accurate than HPLC or GC for potency analyses. NIR can be programmed to detect different compounds. To obtain accuracy in its detection methods, samples must be tested by HPLC on ongoing basis. 100 samples or more will provide enough information to improve an NIR software’s accuracy if it is programmed by the manufacturer or user using chemometrics. Chemometrics sorts through the often complex and broad overlapping NIR absorption.
Bands from the chemical, physical, and structural properties of all species present in a sample that influences the measured spectra. Any variation however of a strain tested or water quantity observed can affect the received results. Consistency is the key to obtaining precision with NIR equipment programming. The downfall of the NIR technique is that it must constantly be compared to HPLC data to ensure accuracy.
At Eurofins Experchem , our company works with bothHPLC and NIR equipment simultaneously for different cannabis testing purposes. Running both equipment simultaneously means we are able to continually monitor the accuracy of our NIR equipment as compared to our HPLC. If a company is using NIR alone however, it can be more difficult to maintain the equipment’s accuracy without on-going monitoring.
What about Terpenes?
Terpenes are the primary aromatic constituents of cannabis resin and essential oils. Terpene compounds vary in type and concentration among different genetic lineages of cannabis and have been shown to modulate and modify the therapeutic and psychoactive effects of cannabinoids. Terpenes can be analyzed using different methods including separation by GC or HPLC and identification by Mass Spectrometry. The high-heat environment for GC analysis can again cause problems in accuracy and interpretation of results for terpenes; high-heat environments can degrade terpenes and make them difficult to find in accurate form. We find HPLC is the best instrument to test for terpenes and can now test for six of the key terpene profiles including a-Pinene, Caryophyllene, Limonene, Myrcene, B-Pinene and Terpineol.
Quality systems between different labs are never one and the same. Some labs are testing cannabis under good manufacturing practices (GMP), others follow ISO accreditation and some labs have no accreditation at all.
From a quality systems’ perspective some labs have zero or only one quality system employee(s). In a GMP lab, to meet the requirements of Health Canada and the FDA, our operations are staffed in a 1:4 quality assurance to analyst ratio. GMP labs have stringent quality standards that set them apart from other labs testing cannabis. Quality standards we work with include, but are not limited to: monthly internal blind audits, extensive GMP training, yearly exams and ongoing tests demonstrating competencies.
Maintaining and adhering to strict quality standards necessary for a Drug Establishment License for pharmaceutical testing ensures accuracy of results in cannabis testing otherwise difficult to find in the testing marketplace.
Important things to know about testing
HPLC is the most recommended instrument used for product release in a regulated environment.
NIR is the best instrument to use for monitoring growth and curing processes for R&D purposes, only if validated with an HPLC on an ongoing basis.
Quality Systems between labs are different. Regardless of instrumentation used, if quality systems are not in place and maintained, integrity of results may be compromised.
GMPs comprise 25% of our labour costs to our quality department. Quality systems necessary for a GMP environment include internal audits, out of specification investigations, qualification and maintenance of instruments, systems controls and stringent data integrity standards.
Dr. Zacariah Hildenbrand, chief scientific officer and partner at C4 Laboratories, is currently researching some of the lesser-known molecules in cannabis, and he’s on to something. His research focuses on discovering new molecules, determining their therapeutic effects and expanding our understanding of the constituents of cannabis.
Dr. Hildenbrand received his Ph.D. from the University of Texas at El Paso where he researched the molecular architecture involved in hormone-dependent cancers. At the University of Texas Southwestern Medical Center in Dallas, his post-doctoral research contributed to the development of a novel therapy for the treatment of chronic myeloid leukemia, a blood-borne cancer that afflicts small children. He has published over 25 peer-reviewed scientific journal articles and hopes to do the same with his research in cannabis.
After a career of scientific consulting, Dr. Hildenbrand met Ryan Treacy, founder and chief executive officer of C4 Laboratories, in 2015 when Treacy launched the company. In June of 2015, the laboratory began operations, providing Dr. Hildenbrand the opportunity to embark on a new and exciting field of research- cannabis.
They currently collaborate with Dr. Kevin Schug of the Shimadzu Center for Advanced Analytical Chemistry (SCAAC) at the University of Texas, Arlington and together Drs. Schug and Hildenbrand are pursuing a DEA license to expand their current cannabis research. The SCAAC is a $10.0+ million analytical laboratory with instrumentation that only a handful of people in the world has access to.
C4 Laboratories, based in Mesa, Arizona, currently offers a range of services for cannabis analysis including terpene and cannabinoid analytics, microbial, pesticide, fungicide and insecticide testing. In addition to the standard gamut of tests, they also specialize in cultivation analytics like mold and mildew culture testing, viral detection with sentinel plants and comprehensive analysis of environmental conditions.
What makes their company unique is their multidisciplinary effort to characterize the therapeutic compounds found in cannabis, the C4 Cannabinomics Collaborative. We sit down with Dr. Zac Hildenbrand to talk cannabis science, his research and what they hope to accomplish with the C4 Cannabinomics Collaborative.
CannabisIndustryJournal: What is the C4 Cannabinomics Collaborative?
Dr. Zacariah Hildenbrand: The C4 Cannabinomics Collaborative is an open collaboration between growers and scientists to discover new molecules in cannabis and to have a better characterization of individual cannabis strains based on the active constituents found in each sample. We are facilitating the collaboration of some of the world’s best cannabis growers with world-class scientists to find new information about the plant.
What we want to accomplish in this work is identifying novel molecules. Because of the [federal government’s] restrictions in researching cannabis, there is very little peer-reviewed literature on many of the compounds found in cannabis. We want to secondarily find out what those molecules do in the human body and thus make recommendations for strains targeting specific conditions.
We also want to understand the strains currently out there by determining the most established cannabinoids and terpenes via chemotyping. You hear a lot of people talking about the effects of an Indica or Sativa and making recommendations based on that. We want to find chemical signatures based on cannabinoids and terpenes and make recommendations based on that. There are a lot of problems at hand when discussing strain names scientifically. There are nomenclature issues- people calling the same strain different names, people giving multiple names to the same strain to make it appear that their strain portfolios are more diverse.
We can identify the chemical signatures in strains based on the major cannabinoids and terpenes. Based on the terpenes and chemical profile we can determine more accurate recommendations for patients as well as in recreational applications. All of this, again, discovering the new molecules, identifying the current strains, is so we can make more informed decisions regarding cannabis use. It is not a panacea but it is a very robust plant. There are a lot of terpenes with anti-inflammatory responses. Other molecules help with blood flow, sleep, regulating blood glucose, and we all know the cases of CBD helping children with convulsions and epilepsy. We want people to make sure they have the most up-to-date information.
CIJ: How is your collaboration with the SCAAC at UT Arlington contributing to this work?
Dr. Hildenbrand: One of the instruments we use there is a supercritical-fluid-extraction supercritical-fluid-chromatography mass-spectrometer (SFE-SFC-MS). With that instrument, we can do the extraction on the machine with an extreme level of sensitivity. It is ideal for drug discovery and identifying molecules in the parts-per-quadrillion range. This particular instrument allows us to detect molecules with an extreme level of sensitivity without volatizing them during the sample extraction process.
We want to acquire samples of unique cannabis from growers that will work with us to discover new cannabis constituents. We are in the process of getting a DEA license so that we can send products across state lines to the center at UT Arlington to perform the advanced characterization. They have instrumentation that only a handful of people in the world have access to, which gives us the best opportunity to explore the unknown. When we discover new molecules, find out what they do on the molecular level, we can then isolate these compounds and ultimately use this newfound knowledge for the development of effective nutraceuticals.
CIJ: What molecules are you researching right now?
Dr. Hildenbrand: Some of the low-hanging fruit in our research looks at identifying compounds similar to the better-studied compounds such as THC and CBD. THCV has a very similar structure to THC, but has a shorter acyl carbon chain (3 carbons vs. 5).
THCV doesn’t induce a psychoactive response (like THC), but it does improve fat utilization, so it has remarkable potential for medicine. We are looking at what conditions are required for it to occur naturally. Cannabis doesn’t produce THCV in a high amount. 0.7% by weight is the most we have seen in Arizona. In Oregon, where craft cannabis has been refined to a much higher degree, we have heard rumblings of some strains containing up to 3% THCV. We want to find out if this is a possible weight loss tool. Our research in CBDV is very much the same.
CBL is the breakdown product of CBC when it is treated with ultraviolet light. We know absolutely nothing about what CBL does. If we find a strain that produces high amounts of CBC, we can then treat it with UV light and force the conversion to CBL, and then ultimately determine what it does. This is a good example of low-hanging fruit and the versatility of cannabis. Based on the biogenesis of the cannabinoids, we can alter the profile of cannabis products using a series of biochemical reactions.
For example, we have been helping clients in Arizona look for a quality sleep aid in cannabis. Certainly, Indica strains will help, but the molecule CBN helps specifically with sleep abnormalities. As CBN is formed as a byproduct when CBD or THC are oxidized, we see some producers using liquid nitrogen to oxidize CBD, leading to higher CBN levels. I would like to think we are in the age of understanding CBD, THC and the major terpenes,but there are a whole milieu of compounds that require our attention and THCV, CBDV and CBL are just a few that we want to devote our efforts to right away.
CIJ: What are your plans in the immediate future?
Dr. Hildenbrand: We are in the process of finalizing the documents to bring a C4 laboratory into Oregon where we can do quite a bit of research and where we’ll have access to some very unique cannabis. We will offer full compliance testing per ORELAP and OLCC regulations, but we also want to acquire samples (free of charge) from growers that want to collaborate with us to discover new molecules. We’ve been lucky enough to start working with growers like Adam Jacques and Chris West in Eugene, but we also want to be available to other growers who want to contribute to this research.
CIJ: What are your long-term goals with this project?
Dr. Hildenbrand: At a basic level, we hope to expand the current understanding of the cannabis plant. There is a lot of “bro science” and anecdotal claims out there. There is so much that we don’t know about cannabis that we cannot simply rely on anecdotal claims for each strain. We want to bring cannabis into the same light as any pharmaceutical-grade or biomedical research.
We need to be characterizing this plant with the same level of detail as other pertinent molecular therapies. In doing so there are a lot of potential discoveries to be made and we might be able to unlock the future of medicine. A drug like Marinol, for example, has been met with mixed reviews because its only one dimensional. Furthermore, we find that the terpene molecules are tremendously beneficial and this interplay between cannabinoids and terpenes is something that we want to explore further. All and all we wish to further illustrate the therapeutic capacities of cannabis within the contexts of specific ailments and medical conditions, while discovering the medicine of the future.
David Goldstein, co-founder and chief executive officer of PotBotics, launched a medical cannabis recommendation engine called PotBot with the goal to better inform patients to target their conditions with more accurate recommendations based on scientific research. “This is a tool to help move the market away from the thousands of strain names that are mainly just marketing or branding indicators,” says Goldstein. The medical application is designed to inform patients on peer-reviewed data, research on the treatment of their ailments with cannabis and the specific cannabinoids that are necessary for treating their condition. They began development on PotBot in October of 2014, launching the beta version to 400 users in November of 2015. On April 20th, 2016, Goldstein launched officially in the Apple Store, and the program will be available on Android in July.
Rather than focusing on strain names, PotBot focuses on the cannabinoid values to help patients gain an understanding of the correlation between which compounds might best target their condition. “This is a great tool for patients trying to familiarize themselves with what strains might work best,” says Goldstein. “For example, insomnia patients generally need cannabis with higher CBN levels, so we first educate the patient on cannabinoid ranges to shoot for and what strains might help. PotBot would recommend the strain Purple Urple because it is an indica found to have higher CBN values,” adds Goldstein. The program goes into great detail with the patient’s preferences including everything down to consumption methods so they know why it might recommend certain strains.
The recommendation tool is accessible via kiosks at dispensaries, on a desktop version for the computer as well as on the Apple Store for iPads and iPhones. “I do not see it as a way of replacing budtenders, rather supplementing them with knowledge,” says Goldstein. PotBot is designed as a tool to supplement the budtender’s understanding of cannabis, so the budtender does not need to know everything off the top of their head or recommend strains based on anecdotal information, according to Goldstein.
Goldstein’s team at PotBotics performed extensive research prior to launching PotBot, spending two years doing strain testing to develop the program. “There is currently no regulatory body [for strain classification] so we took it upon ourselves to work with the best testing laboratories for truly robust analyses and properly vetted growers to get the most valid data,” says Goldstein. “The current strain classification system and nomenclature is rather unscientific so we focus on cannabinoid values and soon we will be able to incorporate terpene profiles in the recommendation.” Moving away from the common focus on taste, smell and other qualitative values, they focus on medical attributes of cannabinoid profiles because they have the most peer-reviewed research available today.
As an OEM, the company designed the tool to work with each dispensary’s inventory, to provide recommendations for strains that a patient can access on site, however anyone can access the recommendation tool for free at PotBot.com. Goldstein’s company and their mission represent an important development in the cannabis industry; this could begin a key transition from thousands of understudied strain names to a more scientific and calculated method to treating patients’ conditions with cannabis.