California was the first state to step up to defend consumers from false marketing claims that ozone generators are safe, effective air purifiers. In reality, ozone is a lung irritant, especially harmful to allergy and asthma sufferers. In 2009, California became the first state in the nation to ban ozone generators. The Air Resources Board of the California Environmental Protection Agency states:
Not all air-cleaning devices are appropriate for use — some can be harmful to human health. The ARB recommends that ozone generators, air cleaners that intentionally produce ozone, not be used in the home or anywhere else humans are present. Ozone is a gas that can cause health problems, including respiratory tract irritation and breathing difficulty.
The regulation took effect in 2009 along with a ban on the sale of air purifiers that emit more than 0.05 parts per million of ozone. The ARB says that anything beyond this is enough to harm human health; however, some experts say that there is no safe level of ozone.
The National Institute for Occupational Safety and Health recommends an exposure limit to ozone of 0.1 ppm and considers levels of 5 ppm or higher “immediately dangerous to life or health.”
If you’re shopping for an air purifier, it’s best to avoid ozone generators, especially if you have a respiratory condition. Ozone generators, and ionic air cleaners that emit ozone, can cause asthma attacks in humans while doing little to nothing to clean the air.
O3 is a free radical, an oxidizer; when it meets any organic molecule floating around it bonds to it and destroys it. In a grow room, organic molecules include the essential oils in cannabis which produce the fragrance. When using ozone within your grow room, too much will not only all but eliminate the smell of your flowers but with prolonged exposure, it begins to actually degrade the cell walls of trichomes and destroy the structure of the glands.
Despite the claims of some manufacturers, ozone does not have an anti-microbial effect in air unless levels far exceed the maximums of the regulation and is therefore harmful humans.
Keeping the grow room clean of mold and bacteria is important, but ozone is not the technology you want to employ to satisfy this goal. Looking into a combination of UVC and Filtration will better meet the goal while keeping both your plants and staff healthy.
Editor’s Note: The following is based on research and studies performed in their Santa Cruz Lab, with contributions from Mikhail Gadomski, Lab Manager, Ryan Maus, Technical Services Analyst, Dr. Laurie Post, Director of Food Safety & Compliance, Andy Sechler, Lab Director, Toby Astill, Senior Business Development Leader at Perkin Elmer and Charles Deibel, President of Deibel Cannabis Labs.
Pesticides represent the leading cause of batch failures in the cannabis industry. They are also the hardest tests to run in the laboratory, even one equipped with state-of-the-art equipment. The best instruments on the market are HPLC and GC dual mass spectrometer detectors, called “HPLC-qqq”, “GC-qqq,” or just triple quads.
As non-lab people, we envision a laboratory that can take a cannabis sample, inject it into a triple quad and have the machine quickly and effortlessly print out a report of pesticide values. Unfortunately, this is far from reality. The process is much more hands on and complex.In the current chemistry lab, trained analysts have to first program the triple quads to look for the pesticides of concern; in cannabis pesticide testing, this is done by programming the first of two mass spectrometers to identify a single (precursor) mass that is characteristic of the pesticide in question. For BCC requirements in California, this has to be done for all 66 pesticides, one at a time.
Next, these precursor ions are degraded into secondary chemicals called the “product” ions, also called transition ions. The second of the two mass spectrometers is used to analyze these transition ions. This process is graphed and the resulting spectrum is analyzed by trained chemists in the lab, pesticide by pesticide, for all the samples processed that day. If the lab analyzes 10 samples, that translates to 660 spectra to analyze (66 pesticides x 10 samples). When looking at the spectra for each pesticide, the analysts must compare the ratios of the precursor ions to the product ions.
If these spectra indicate a given pesticide may be present, the chemists must then compare the ratios between the precursor and the products. If these ratios are not what is expected, then the analyst must perform confirmation testing to prove the precursor mass either is or is not the pesticide of concern. If the ratios are not what is expected, it means the molecule is similar to the pesticide in question, but may not be that pesticide. This confirmatory testing is key to producing accurate results and not failing batches when dealing with closely related chemicals. This process of analyzing spectra is done in all labs that are performing pesticide testing. In this fledgling industry, there are few published cannabis pesticide methods.
The need for this type of confirmation testing doesn’t happen all of the time, but when it does, it will take longer than our targeted three-day turn-around time. In the picture above, one precursor mass is ionized into several product masses; but only two are large enough to be used for comparison. In this hypothetical situation, two product masses are produced for every one precursor, the expected ion abundance ratio should be less than 30%. When performing any confirmatory testing, if the ion abundance ratio is >30%, it means the original precursor molecule was not the pesticide of concern. For example, if the ion abundance ratio was 50%, then the original molecule broke down into too many parts; it was not the pesticide we were looking for. This ion abundance ratio threshold was established by FANCO, the international organization that sets guidelines for all pesticide testing.
Methodology: In this fledgling industry, there are few published cannabis pesticide methods. The identification of the precursor mass and product ions are not always published, leaving labs to research which ions should be used. This adds to the potential for differences between lab results. Once selected, labs should validate their research, through a series of experiments to ensure the correct precursor and transition (product) ions are being used in the method.
Sample Preparation: Beyond the time-consuming work that is required to develop sound pesticide methods, the extraction step is absolutely critical for credible results. If the pesticides aren’t fully extracted from the cannabis product, then the results will be lower than expected. Sample preparations are often not standardized between labs, so unless a given extraction technique is validated for accuracy, there is the possibility for differences between labs.
Getting a Representative Sample
The current California recommended amount of sample is one gram of product per batch. Batch sizes can vary greatly and it is entirely likely that two different one gram samples can have two different results for pesticides. Has the entire plant been evenly coated with exactly the same amount of pesticide onto every square inch of its leaves? No, probably not. That is why it is imperative to take a “random” sample, by taking several smaller samples from different areas of the entire batch.
Sampling Plans: We can learn a lot from the manufacturing and sampling best practices developed by the food industry through the years. If a food manufacturer is concerned with the possibility of having a bacteria pathogen, like Salmonella, in their finished product, they test the samples coming off their production lines at a statistically relevant level. This practice (theory) is called the sampling plan and it can easily be adapted to the cannabis industry. The basic premise is that the more you test, the higher your likelihood of catching a contaminate. Envision a rectangular swimming pool, but instead of water, it’s filled with jello. In this gelatinous small pool, 100 pennies are suspended at varying levels. The pennies represent the contaminates.
Is the pool homogenized? Is jello evenly represented in the entire pool? Yes.
Is your concentrate evenly distributed in the extraction vessel? Yes. The question is, where are the pennies in that extraction vessel? The heavy metals, the microbial impurities and the pesticides should be evenly distributed in the extraction vessel but they may not be evenly represented in each sample that is collected. Unfortunately, this is the bane of the manufacturing industry and it’s the unfortunate reality in the food industry. If you take one random cup of jello, will you find the penny? Probably not. But it you take numerous 1 cup samples from random areas within the batch, you increase your chances of finding the contaminate. This is the best approach for sampling any cannabis product.
The best way to approve a batch of cannabis product is to take several random samples and composite them. But you may need to run several samples from this composite to truly understand what is in the batch. In the swimming pool example, if you take one teaspoon scoop, will you find one of the pennies? The best way to find one of the pennies is to take numerous random samples, composite them and increase the number of tests you perform at the lab. This should be done on any new vendor/cultivator you work with, in order to help establish the safety of the product.
The combination of gas chromatography and infrared spectroscopy (GC/IR) is a powerful tool for the characterization of compounds in complex mixtures. (1-5) Gas chromatography with mass spectroscopy detection (GC/MS) is a similar technique, but GC/MS is a destructive technique that tears apart the sample molecules during the ionization process and then these fragments are used to characterize the molecule. In GC/IR the molecules are not destroyed but the IR light produced by molecular vibrations are used to characterize the molecule. IR spectrum yields information about the whole molecule which allows the characterization of specific isomers and functional groups. GC/IR is complementary to GC/MS and the combination results in a powerful tool for the analytical chemist.
A good example of the utility of GC/IR vs GC/MS is the characterization of stereo isomers. Stereo isomers are mirror images such as a left hand and a right hand. In nature, stereo isomers are very important as one isomers will be more active then its mirror image. Stereo isomers are critical to medicinal application of cannabis and also a factor in the flavor components of cannabis.
GC/MS is good at identifying basic structure, where GC/IR can identify subtle differences in structure. GC/MS could identify a hand, GC/IR could tell you if it is a left hand or right hand. GC/MS can identify a general class of compounds, GC/IR can identify the specific isomer present.
Gas chromatography interfaced with infrared detection (GC/IR), combines the separation ability of GC and the structural information from IR spectroscopy. GC/IR gives the analyst the ability to obtain information complementary to GC/MS. GC/IR gives the analyst the power to perform functional group detection and differentiate between similar molecular isomers that is difficult with GC/MS. Isomer specificity can be very important in flavor and medical applications.
Gas chromatography with mass spectrometry detection (GC/MS) is the state-of-the-art method for the identification of unknown compounds. GC/MS, however, is not infallible and many compounds are difficult to identify with 100 % certainty. The problem with GC/MS is that it is a destructive method that tears apart a molecule. In infrared spectrometry (IR), molecular identification is based upon the IR absorptions of the whole molecule. This technique allows differentiation among isomers and yields information about functional groups and the position of such groups in a molecule. GC/IR complements the information obtained by GC/MS.
Initial attempts to couple GC with IR were made using high capacity GC columns and stopped flow techniques. As GC columns and IR technology advanced, the GC/IR method became more applicable. The advent of fused silica capillary GC columns and the availability of Fourier transform infrared spectrometry made GC/IR available commercially in several forms. GC/IR using a flow cell to capture the IR spectrum in real time is known as the “Light Pipe”. This is the most common form of GC/IR and the easiest to use. GC/IR can also be done by capturing or “trapping” the analytes of interest eluting from a GC and then measuring the IR spectrum. This can be done by cryogenically trapping the analyte in the solid phase. A third possibility is to trap the analyte in a matrix of inert material causing “Matrix Isolation” of the analyte followed by measuring the IR spectrum.
The physical state of the sample has a large effect upon the IR spectrum produced. Molecular interactions (especially hydrogen bonding) broadens absorption peaks. Solid and liquid samples produce IR spectra with broadened peaks that loses much of the potential information obtained in the spectra. Surrounding the sample molecule with gas molecules or in an inert matrix greatly sharpens the peaks in the spectrum, revealing more of the information and producing a “cleaner” spectrum. These spectra lend themselves better to computer searches of spectral libraries similar to the computer searching done in mass spectroscopy. IR spectral computer searching requires the standard spectra in the library be of the same physical state as the sample. So, a spectrum taken in a gaseous state should be searched against a library of spectra of standards in the gaseous state.
Gas Phase – Lack of molecular interactions sharpen absorption peaks.
Matrix Isolation – Lack of molecular interactions sharpen absorption peaks.
GC/IR yields chromatograms of infrared absorbance over time. These can be total infrared absorbance which is similar to the total ion chromatogram (TIC) in GC/MS or the infrared absorbance over a narrow band or bands analogous to selected ion chromatogram. This is a very powerful ability, because it gives the user the ability to focus on selected functional groups in a mixture of compounds.
Gas chromatography with infrared detection is a powerful tool for the elucidation of the structure of organic compounds in a mixture. It is complementary to GC/MS and is used to identify specific isomers and congeners of organic compounds. This method is greatly needed in the Cannabis industry to monitor the compounds that determine the flavor and the medicinal value of its products.
GC–MS and GC–IR Analyses of the Methoxy-1-n-pentyl-3-(1-naphthoyl)-Indoles: Regioisomeric Designer Cannabinoids, Amber Thaxton-Weissenfluh, Tarek S. Belal, Jack DeRuiter, Forrest Smith, Younis Abiedalla, Logan Neel, Karim M. Abdel-Hay, and C. Randall Clark, Journal of Chromatographic Science, 56: 779-788, 2018
Simultaneous Orthogonal Drug Detection Using Fully Integrated Gas Chromatography with Fourier Transform Infrared Detection and Mass Spectrometric Detection , Adam Lanzarotta, Travis Falconer, Heather McCauley, Lisa Lorenz, Douglas Albright, John Crowe, and JaCinta Batson, Applied Spectroscopy Vol. 71, 5, pp. 1050-1059, 2017
High Resolution Gas Chromatography/Matrix Isolation Infrared Spectrometry, Gerald T. Reedy, Deon G. Ettinger, John F. Schneider, and Sid Bourne, Analytical Chemistry, 57: 1602-1609, 1985
GC/Matrix Isolation/FTIR Applications: Analysis of PCBs, John F. Schneider, Gerald T. Reedy, and Deon G. Ettinger, Journal of Chromatographic Science, 23: 49-53, 1985
A Comparison of GC/IR Interfaces: The Light Pipe Vs. Matrix Isolation, John F. Schneider, Jack C. Demirgian, and Joseph C. Stickler, Journal of Chromatographic Science, 24: 330- 335, 1986
Gas Chromatography/Infrared Spectroscopy, Jean ‐ Luc Le Qu é r é , Encyclopedia of Analytical Chemistry, John Wiley & Sons, 2006
Imagine this: you are taking medication for cancer pain. One day, it works perfectly. The next, you feel no relief. On some days, you need to take three doses just to get the same effect as one. Your doctor can’t be completely positive how much active ingredient each dose contains, so you decide for yourself how much medication to take.
Doesn’t seem safe, right? It is crucial that doctors know exactly what they are prescribing to their patients. They must know that their patients are receiving the exact same dose of medication in their prescription each time they take it, and that their medication contains only the intended ingredients.
consistency is key to creating products that are safe for consumers.In the cannabis industry, lack of certainty on these important factors is a major problem for drug manufacturers as they attempt to incorporate cannabidiol (CBD), a compound found in cannabis that has no psychoactive effects but many medical benefits, into pharmaceutical drugs.
When using these compounds as medications, purity is essential. Cannabis contains a wide variety of compounds. Delta-9 tetrahydrocannabinol (THC) is the most well-known compound and its main psychoactive one1. Safety regulations dictate that consumers know exactly what they are getting when they take a medication. For example, their CBD-based medications should not contain traces of THC.
The cannabis industry greatly needs a tool to ensure the consistent extraction and isolation of compounds. In 2017, the cannabis industry was worth nearly $10 billion, and it is expected to grow $57 billion more in the next decade2. As legalization of medical cannabis expands, interest in CBD pharmaceuticals is likely to grow.
If compounds such as CBD are going to be used in pharmaceutical drugs, consistency is key to creating products that are safe for consumers.
CBD is a non-psychoactive compound that makes up 40 percent of cannabis extracts1. It is great for medical applications because it does not interfere with motor or psychological function. Researchers have found it particularly effective for managing cancer pain, spasticity in multiple sclerosis, and specific forms of epilepsy3.
Other compounds derived from cannabis, such as cannabichromene (CBC) and cannabigerol (CBG), may also be beneficial compounds with medical applications. CBC is known to block pain and inflammation, and CBG is known for its use as a potential anti-cancer agent1.
Along with these compounds that provide medical benefits, there are psychoactive compounds that are used recreationally, such as THC.
“It will definitely be an advantage to have cannabis-based medications with clearly defined and constant contents of cannabinoids,” says Kirsten Müller-Vahl, a neurologist and psychiatrist at Hannover Medical School in Germany.
Creating a Standard Through Centrifugal Partition Chromatography
To obtain purified compounds from cannabis, researchers need to use technology that will extract the compounds from the plant.
Many manufacturers use some sort of chromatography technique to isolate compounds. Two popular methods are high performance liquid chromatography (HPLC) and flash chromatography. These methods have their places in the field, but they cannot be effectively and cost-efficiently scaled to isolate compounds. Instead, HPLC and flash chromatography may be better suited as analytical tools for studying the characteristics of the plant or extract. As cannabis has more than 400 chemical entities4, compound isolation is an important application.
This method is highly effective for achieving both high purity and recovery.Although molecules such as CBD can be synthesized in the lab, many companies would rather extract the compounds directly from the plant. Synthesized molecules do not result in a completely pure compound. The result, “is still a mixture of whatever cannabinoids are coming from a particular marijuana strain, which is highly variable,” says Brian Reid, chief scientific officer of ebbu, a company in Colorado that specializes in cannabis purification.
Currently, there is only one method available to researchers that completely allows them to isolate individual compounds: centrifugal partition chromatography (CPC).
The principle of CPC is similar to other liquid chromatography methods. It separates the chemical substances as the compounds in the mobile phase flow through and differentially interact with the stationary phase.
Where CPC and standard liquid chromatography differs is the nature of the stationary phase. In traditional chromatography methods, the stationary phase is made of silica or other solid particles, and the mobile phase is made of liquid. During CPC, the stationary phase is a liquid that is spun around or centrifuged to stay in place while the other liquid (mobile phase) moves through the disc. The two liquid phases, like oil and water, don’t mix. This method is highly effective for achieving both high purity and recovery. Chemists can isolate chemical components at 99 percent or higher purity with a 95 percent recovery rate5.
“CPC is ideal for ripping a single active ingredient out of a pretty complex mixture,” says Reid. “It’s the only chromatographic technique that does that well.”
The Need for Pure Compounds
High levels of purity and isolation are necessary for cannabis to be of true value in the pharmaceutical industry. Imagine relying on a medication to decrease your seizures, and it has a different effect every time. Sometimes there may be traces of psychoactive compounds. Sometimes there are too much or too little of the compound that halts your seizures. This is not a safe practice for consumers who rely on medications.“It’s hard to do studies on things you can’t control very well.”
Researchers working with cannabis desperately need a technology that can extract compounds with high purity rates. It is hard to run a study without knowing the precise amounts of compounds used. Reid uses a Gilson CPC 1000 system at ebbu for his cannabinoid research. With this technology, he can purify cannabinoids for his research and create reliable formulations. “Now that we have this methodology dialed in we can make various formulations —whether they’re water-soluble, sublingual, inhaled, you name it —with very precise ratios of cannabinoids and precise amounts of cannabinoids at the milligram level,” says Reid.
Kyle Geary, an internist at the University of Illinois at Chicago, is currently running a placebo-controlled trial of CBD capsules for Crohn’s disease. This consistent isolation is helpful for his research, as well. “Ideally, the perfect study would use something that is 100 percent CBD,” says Geary. “It’s hard to do studies on things you can’t control very well.”
The State of the Industry
While CBD is not considered a safe drug compound under federal law in the United States6, 17 states have recently passed laws that allow people to consume CBD for medical reasons7. Half of medicinal CBD users solely use the substance for treatment, a recent survey found8. As the industry quickly grows, it is crucial that consumer safety protocol keeps pace.
In June, the US Food and Drug Administration (FDA) approved the first drug that contains a purified drug substance from cannabis, Epidiolex9. Made from CBD, it is designed to treat Dravet Syndrome and Lennox-Gastaut syndrome, two rare forms of epilepsy. FDA Commissioner Scott Gottlieb said in the news release that although the FDA will work to support the development of high-quality cannabis-based products moving forward, “We are prepared to take action when we see the illegal marketing of CBD-containing products with serious, unproven medical claims. Marketing unapproved products, with uncertain dosages and formulations can keep patients from accessing appropriate, recognized therapies to treat serious and even fatal diseases.”
The industry should be prepared to implement protocols to ensure the quality of their CBD-based products. The FDA has issued warnings in recent years that some cannabinoid products it has tested do not contain the CBD levels their makers claim, and consumers should be wary of such products10. It’s hard to know when or if the FDA will begin regulating CBD-based pharmaceuticals. However, for pharma companies serious about their reputation, there is only one isolation method that creates reliable product quality: CPC.
National Institute on Drug Abuse. (2015, June 24). The Biology and Potential Therapeutic Effects of Cannabidiol. Retrieved from https://www.drugabuse.gov/about-nida/legislative-activities/testimony-to-congress/2016/biology-potential-therapeutic-effects-cannabidiol
Atakan, Z. (2012). Cannabis, a complex plant: Different compounds and different effects on individuals. Therapeutic Advances in Psychopharmacology,2(6), 241-254. doi:10.1177/2045125312457586
Gilson. (n.d.). Centrifugal Partition Chromatography (CPC) Systems. Retrieved from http://www.gilson.com/en/AI/Products/80.320#.WzVB2lMvyMI
Mead, A. (2017). The legal status of cannabis (marijuana) and cannabidiol (CBD) under US law. Epilepsy & Behavior, 70, 288-291.
ProCon.org. (2018, May 8). 17 States with Laws Specifically about Legal Cannabidiol (CBD) – Medical Marijuana – ProCon.org. Retrieved from https://medicalmarijuana.procon.org/view.resource.php?resourceID=006473
Borchardt, D. (2017, August 03). Survey: Nearly Half Of People Who Use Cannabidiol Products Stop Taking Traditional Medicines. Retrieved from https://www.forbes.com/sites/debraborchardt/2017/08/02/people-who-use-cannabis-cbd-products-stop-taking-traditional-medicines/#43889c942817
U.S. Food & Drug Administration. (2017). Public Health Focus – Warning Letters and Test Results for Cannabidiol-Related Products. Retrieved from https://www.fda.gov/newsevents/publichealthfocus/ucm484109.htm
By Dr. Zacariah Hildenbrand, Allegra Leghissa, Dr. Kevin A. Schug 2 Comments
Have you ever wondered why all beers have that strong, characteristic smell? Or why you could tell the smell of cannabis apart from any other plant? The answer is simple – terpenes.
These 55,000 different molecules are responsible for a majority of the odors and fragrances around us, from a pine forest, to the air diffuser in your house 1–3. They all share the same precursor, isoprene, and because of that, they are all related and have similar molecular structures. Unfortunately, it is this uncanny similarity that makes their analysis so challenging; we still lack a complete list of which terpenes expected to be found in each given plant species 1,2.
Many different methods have been developed in an effort to provide a time-optimized and straightforward analysis. Gas chromatography (GC) is usually center stage due to the volatility of the terpenes. Therefore, there is significant concern with the type of GC detector used 2.
The flame ionization detector (FID) is a good quantitative detector for GC, but qualitatively it does not provide any information, except for retention time; the differentiation between terpene species is achieved solely by use of retention indices (RI), which are based on elution times from a particular GC stationary phase. The best part of the FID is its low cost, reliability, and relatively easy interface, which make it an effective tool for quality control (QC) but less so with respect to research and discovery 2.
The primary choice for a research setting is the mass spectrometer (MS) detector. It is more expensive and complicated than FID, but importantly, it provides both good quantitative capabilities, and it provides mass spectra for each species that elutes from the chromatograph. However, for terpene analysis, it may still not be the best detector choice. Since terpene class molecules share many structural and functional similarities, even their fragmentation and sub-sequential identification by MS may lead to inconsistent results, which need to be confirmed by use of RI. Still, MS is a better qualitative analysis tool than the FID, especially for distinguishing non-isobaric terpenes 2.
Recently, new technology based on vacuum ultraviolet spectroscopy (VUV) has been developed as a new GC detector. The VUV detector enables analysis of virtually all molecules; virtually all chemical compounds absorb light in the range in the 125-240 nm wavelength range probed by the detector, making it an essentially universal detector 4–11. Previously, spectroscopic absorption detectors for GC have lacked sufficient energy to measure absorption of most GC-amenable species. The VUV detector fills a niche, which is complementary to MS detection in terms of the qualitative information it provides.
With the VUV detector, each compound exhibits its own unique absorbance spectrum. Even isomers and isobars, which are prevalent in terpene mixtures and can be difficult to distinguish different species by their electron ionization mass spectra, can be well differentiated based on their VUV spectra 6,9,10. Nevertheless, because analytes exhibit different spectra, it is not required to achieve a perfect chromatographic separation of the mixture components. Co-eluting peaks can be separated post-run through the use of library spectra and software inherent to the instrument 4,10. This ability is called “deconvolution”, and it is based on the fact that two co-eluting terpenes will give a peak with an absorbance spectrum equal to the sum of the two single absorbance spectra 4. Figure 1 shows the deconvolution process for two co-eluting terpenes, camphor and (-)-isopulegol. Due to their different absorbance spectra (Figure 1C), it is possible to fully separate the two peaks in post-run, obtaining sharp peaks for both analytes 6.
The deconvolution process has been shown to yield precise and accurate results. Thus, chromatographic resolution can be sacrificed in favor of spectroscopic resolution; this enables the development of methods with faster run times. With the ability to deconvolve unresolved peaks, a long temperature ramp to chromatographically separate all isomeric terpenes is not required 6. Additionally, the presence of coeluting components, which might normally go undetected with some GC detectors, can be easily judged based on comparison of the measured spectra with pure reference spectra contained in the VUV spectral library.
The other issue in terpenes analysis is the extraction process. Terpenes can be extracted with the use of solvents (e.g., methanol, ethanol, hexane, and cyclohexane, among others), but the process is usually time-consuming, costly and not so environmentally-friendly 2. The plant needs to be manually crushed and then aliquots of solvent are used to extract components from the plant, ideally at least 3 times and combined to achieve acceptable results. The problem is that some terpenes may respond better to a certain solvent, making their extraction easier and more optimized than for others 2. The choice of solvent can cause discrimination against the extraction some terpenes, which limits the comprehensiveness of analysis.
Headspace is another technique that can be used for the sample preparation of terpenes. Headspace sampling is based on heating the solid or liquid sample inside a sealed vial, and then analyzing the air above it after sufficient equilibration. In this way, only volatile analytes are extracted from the solid/liquid sample into the gas phase; this allows relatively interference-free sampling 12–14.
How do we know whether our extraction analysis methods are correct and comprehensive for a certain plant sample? Unfortunately, there is not a complete list of available molecules for each plant species, and even if two specimens may smell really similar to our nose, their terpenes profiles may be notably different. When working with a new plant material, it is difficult to predict the extraction efficiency for the vast array of terpenes that may be present. We can only perform it with different extraction and detection methods, and compare the results.
The route for a comprehensive and fast analysis of terpenes is therefore still long; however, their intoxicating aromas and inherent medicinal value has provided a growing impetus for researchers around the world. Considering the evolving importance of Cannabis and the growing body of evidence on the synergistic effects between terpenes and cannabinoids, it is likely that newly improved extraction and analysis methods will be developed, paving the way for a more complete list of terpene species that can be found in different cultivars. The use of new analytical technologies, such as the VUV detector for GC, should aid considerably in this endeavor.
 Breitmaier E., Terpenes: Flavors, Fragrances, Pharmaca, Pheromones. John Wiley & Sons 2006.
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By Dr. Zacariah Hildenbrand, Dr. Kevin A. Schug No Comments
Much has been made about the plummeting market value of cannabis grown outdoors in Oregon. This certainly isn’t a reflection of the product quality within the marketplace, but more closely attributable to the oversaturation of producers in this space. This phenomenon has similarities to that of ‘Tulip Mania’ within the Dutch Golden Age, whereby tulip bulbs were highly coveted assets one day, and almost worthless the next. During times like these, it is very easy for industry professionals to become disheartened; however, from a scientific perspective, this current era in Oregon represents a tremendous opportunity for discovery and fundamental research.
As we have mentioned in previous presentations and commentaries, our research group is interested in exploring the breadth of chemical constituents expressed in cannabis to discover novel molecules, to ultimately develop targeted therapies for a wide range of illnesses. Intrinsically, this research has significant societal implications, in addition to the potential financial benefits that can result from scientific discovery and the development of intellectual property. While conducting our experiments out of Arlington, Texas, where the study of cannabis is highly restricted, we have resorted to the closet genetic relative of cannabis, hops (Humulus lupulus), as a surrogate model of many of our experiments (Leghissa et al., 2018a). In doing so, we have developed a number of unique methods for the characterization of various cannabinoids and their metabolites (Leghissa et al., 2018b; Leghissa et al., 2018c). These experiments have been interesting and insightful; however, they pale in comparison to the research that could be done if we had unimpeded access to diverse strains of cannabis, as are present in Oregon. For example, gas chromatography-vacuum ultraviolet spectroscopy (GC-VUV) is a relatively new tool that has recently been proven to be an analytical powerhouse for the differentiation of various classes of terpene molecules (Qiu et al., 2017). In Arlington, TX, we have three such GC-VUV instruments at our disposal, more than any other research institution in the world, but we do not have access to appropriate samples for application of this technology. Similarly, on-line supercritical fluid extraction – supercritical fluid chromatography – mass spectrometry (SFE-SFC-MS) is another capability currently almost unique to our research group. Such an instrument exhibits extreme sensitivity, supports in situ extraction and analysis, and has a wide application range for potential determination of terpenes, cannabinoids, pesticides and other chemical compounds of interest on a single analytical platform. Efforts are needed to explore the power and use of this technology, but they are impeded based on current regulations.
Circling back, let’s consider the opportunities that lie within the abundance of available outdoor-grown cannabis in Oregon. Cannabis is extremely responsive to environmental conditions (i.e., lighting, water quality, nutrients, exposure to pest, etc.) with respect to cannabinoid and terpene expression. As such, outdoor-grown cannabis, despite the reduced market value, is incredibly unique from indoor-grown cannabis in terms of the spectrum of light to which it is exposed. Indoor lighting technologies have come a long way; full-spectrum LED systems can closely emulate the spectral distribution of photon usage in plants, also known as the McCree curve. Nonetheless, this is emulation and nothing is ever quite like the real thing (i.e., the Sun). This is to say that indoor lighting can certainly produce highly potent cannabis, which exhibits an incredibly robust cannabinoid/terpene profile; however, one also has to imagine that such lighting technologies are still missing numerous spectral wavelengths that, in a nascent field of study, could be triggering the expression of unknown molecules with unknown physiological functions in the human body. Herein lies the opportunity. If we can tap into the inherently collaborative nature of the cannabis industry, we can start analyzing unique plants, having been grown in unique environments, using unique instruments in a facilitative setting, to ultimately discover the medicine of the future. Who is with us?
Leghissa A, Hildenbrand ZL, Foss FW, Schug KA. Determination of cannabinoids from a surrogate hops matrix using multiple reaction monitoring gas chromatography with triple quadrupole mass spectrometry. J Sep Sci 2018a; 41: 459-468.
Leghissa A, Hildenbrand ZL, Schug KA. Determination of the metabolites of Δ9-Tetrahydrocannabinol using multiple reaction monitoring gas chromatography – triple quadrapole – mass spectrometry. Separation Science Plus 2018b; 1: 43-47.
Leghissa A, Smuts J, Changling Q, Hildenbrand ZL, Schug KA. Detection of cannabinoids and cannabinoid metabolites using gas chromatography-vacuum ultraviolet spectroscopy. Separation Science Plus 2018c; 1: 37-42.
Qiu C, Smuts J, Schug KA. Analysis of terpenes and turpentines using gas chromatography with vacuum ultraviolet detection. J Sep Sci 2017; 40: 869-877.
I’m not much of an oenophile but I recently came across a very interesting set of documentaries about sommeliers, which are experts on the science of wine and, most importantly, how wines are to be paired with food. What struck me as the most fascinating topic pertained to how mistakes made in the vineyard could be concealed by the barrel in which the wine is stored. For example, if the weather conditions throughout the season had been particularly tumultuous, and you end with sub-optimal grapes that are lacking complexity, then you can compensate for this by aging the wine in a variety of different oak barrels to enhance the flavor. To me, this is synonymous with the way that I’ve seen cannabis concentrates being handled, particularly with respect to terpenes. More specifically, it has recently become somewhat fashionable to supplement cannabis extracts with commercially available terpenes to reestablish an aroma profile that is most representative of the original stock material. Taken one step further, I have even heard of hemp extracts being supplemented with terpenes to achieve a particular strain phenotype, which I cannot imagine pans out very well. In my opinion, this is a very bad idea for two reasons:
One, cannabis is incredibly complex and can contain over 100 different terpene molecules, which can collectively act as anti-inflammatories (Chen et al., 2014), anti- microbial agents (Russo, 2011), sleep aids (Silva et al., 2007), bronchodilators (Falk et al., 1990), and even insulin regulators (Kim et al., 2014). So let’s say that you get your stock material tested and the laboratory screens the product for the top 25 most-prevalent terpenes: alpha- and beta-pinenes, linalool, limonene, beta-myrcene, etc. At that point you utilize this information to supplement your extraction product with these terpenes. However, you still may be missing information about other important molecules such as trans-2-pinanol, alpha-bisabolene and alloaromadendrene that are produced at extremely low, yet therapeutically relevant concentrations in the plant. So essentially with the limited information of the terpenes actually present in your stock material, you would be trying to rebuild a puzzle with only a small fraction of the pieces. Even Ben Affleck’s character in the movie ‘The Accountant’ can’t effectively pull this off.
Secondarily, not all commercially available terpenes are created equal. I’ll be the first to admit that I don’t have decades of experience vetting the quality of terpenes currently on the market; however, the several times that I have thrown samples into the GC-FID (Gas Chromatograph equipped with a Flame Ionization Detector) I have been unpleasantly surprised. Expecting beta-caryophyllene and detecting caryophyllene oxide is frustrating and in my opinion, such inaccuracies are wrong and should not be accepted as colloquialisms.
The moral of the story here is that in order to produce premium cannabis extracts/concentrates, the stock material needs to be handled with extreme care in order to retain the bouquet of terpenes in their natural ratios. This is incredibly important given the volatile nature of terpenes and their seemingly ephemeral, yet vital, nature in cannabis. Thankfully in this bourgeoning industry there are a number of extraction professionals who are delicately navigating the balance between art and science to produce premium products that are incredibly terpene-rich. However, for every alchemyst there is also someone trying to circumvent nature and while as a scientist I am inherently in favor of experimentation, I am also an admirer of natural processes.
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.
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