Two major active components of cannabis, cannabidiol (CBD) and tetrahydrocannabinol (THC), as well as minor cannabinoids and associated terpenes, are very poorly soluble in water. This property of cannabis presents major problems for product design and formulation that are difficult to overcome. One of the strategies for effective solubilization is based on using cyclodextrins (CD) that are cyclic polysaccharides capable of forming inclusion complexes with hydrophobic molecules. Cyclodextrins are considered generally recognized as safe (GRAS) by the U.S. Food and Drug Administration (FDA) and have been widely used in food, personal care, and pharmaceutical applications. This article reviews state-of-the-art approaches to produce CD-phytocannabinoid complexes, related processing and analytical techniques, and implications for cannabis product development. Continued interest for improved water solubility has been dictated by both highly desirable improvements in low and variable bioavailability of phytocannabinoids as well as development of better end user experience (such as taste, ease and convenience of use, better shelf product stability, and so on). Thus, the supply of liquid ingredients will be critically important for the rapidly expanding scope of novel consumer products for the recreational use, cannabis-enriched food, food additives, and personal care markets.

Solubility of tetrahydrocannabinol (THC) in water is so low that traditional direct methods were found unusable for accurate measurements (1). THC solubility estimates were derived from the more soluble water–ethanol mixtures with variable ethanol content, by the linear extrapolation of the 240 nm ultraviolet (UV) band for 5–15% ethanol solution to 100% aqueous. This classical experiment produced an often-cited THC solubility value of 2.8 µg/mL at 23 °C that gets further reduced (almost to a quarter of that number) by desalting effects, to 0.77 µg/mL, in 0.15 M NaCl. The water solubility of CBD falls in the same low range and has been reported at 0.1 µg/mL (2).


Historical recreational use of phytocannabinoids has been almost exclusively limited to inhalation of the cannabis-derived smoke or concentrate’s vapors. So the first phytocannabinoids solubilization approaches came mostly from pharmaceutical development and relied on using alcohol or oil mixtures (solubility enhancement enabling concentrations >100 mg/mL). These found use in Marinol softgel capsules of THC (glycerol and sesame oil), oromucosal Sativex THC–cannabidiol (CBD) spray (ethanol, propylene glycol, and peppermint oil), and recently the U.S. Food and Drug Administration (FDA)-approved orally administered CBD liquid dose of Epidiolex (ethanol and sesame oil; recommended doses as high as >5 mg/kg).

Complexation with cyclodextrins (CDs) has several important benefits when compared to other solubilization approaches. Cyclodextrins keep the active molecules solubilized at high dilutions without precipitation and helps to avoid using organic solvents, surfactants, emulsifiers, and other polymeric additives. This process therefore reduces toxicological concerns and offers simple production options (3).

Cyclodextrins form a toroid (truncated conical) configuration with multiple hydroxyl groups at each side and a lipophilic inner cavity. Their interactions with hydrophobic substrates are often described within a concept of the noncovalent “host–guest” complexes, optimization of the hydrophobic interactions, and the best spatial fit of the substrate in the available binding pocket of the ligand. The cyclodextrin solubility in water at 25 °C differs considerably, with γ-cyclodextrin being the most soluble (0.249 g/mL) followed by α-cyclodextrin (0.184 g/mL) and β-cyclodextrin (0.12 g/mL) (4). Typical cyclodextrins contain six to eight glucose monomer units. The rings are cone-shaped with a cavity depth of ~0.7–0.8 nm and cavity diameter of ~0.5–0.8 nm. Molecules with larger rings have also been made, but so far remain primarily in the realm of academic research. As an example, Figure 1 shows the chemical and crystallographic structures of β-cyclodextrins (5). (See upper right for Figure 1, click to enlarge.) Solvent molecules are easily drawn into the inner cavity and bind outside via hydrogen bonding giving rise to various clathrate structures, which are particularly plentiful with water.

CDs are commercially manufactured from starch, by its treatment with two key enzymes, amylase and cyclodextrin glucosyltransferase. The use of CDs has continued to grow across the industries to an estimated >400,000 metric tons in 2016, with food grade materials costing $50–400/kg. The most common CD derivatives are shown in Figure 2. (See upper right for Figure 2, click to enlarge.)

Post-processing chemical modifications (usually, alkylation or acylation of the hydroxy groups, with variable numbers of molar substitutions per sugar unit, 0.5–2) provide access to adjusting the solubility profile of cyclodextrins. One of the biggest suppliers of high quality materials (including pharma grades) worldwide is Wacker Chemie that distributes and supports the products in the U.S. market via Ashland Group under the trademarks CAVITRON, CAVAMAX, and CAVASOL.

As an example of an inclusion complex with higher bioavailability, Wacker sells a dietary supplement product CAVACURMIN that is a 1:1 complex of curcumin with γ-CD. This material showed a 40-fold improved bioavailability compared to unformulated curcumin in small human clinical trials (6). Structurally similar 2-hydroxypropyl modified (2-HP-β-CD) and native beta-cyclodextrins are offered by the French agro conglomerate Roquette under the brand name KLEPTOSE. CAPTISOL brand by Cedex Pharmaceuticals is sulfobutylether (SBE) anionic derivative of β-CD with enhanced (>50x) solubility in water compared to native material.

Both European and United States Pharmacopoeias include three parent CDs and two synthetic derivatives, 2-HP-β-CD and SBE-β-CD-Na+ that are more water-soluble variants. Two other CD derivatives are approved as excipients in human pharmaceutical products, larger ring 2-HP-γ-CD and more hydrophobic randomly methylated-β-CD (Me-β-CD). About 35 pharmaceutical formulations that contain cyclodextrins on the ingredients list are currently in the marketplace with β-CD and 2-HP-β-CD at the top of the list (3).

Characterization and Optimization of the CD Complexes

Formation of the CD complexes has been typically studied by constructing related substrate-ligand phase solubility Higuchi-Connors diagrams (7) (more accessible analyses may be found in many contemporary surveys, such as one found in the literature [8]) that would plot substrate solubility versus CD concentration (Figure 3). (See upper right for Figure 3, click to enlarge.) Such experiments include adding an excess amount of substrate to aqueous CD solution of variable concentration, incubation with shaking for 1–2 h, followed by filtration of the unbound substrate. The stability constant 

 and complexation efficiency (CE) of the inclusion complex are then calculated from the slope of the phase solubility diagrams as follows:

where S0 is the equilibrium substrate solubility in water without CD. CE represents a concentration ratio between cyclodextrin in a complex and free cyclodextrin.

In most cases, the results of solubility studies are interpreted as formation of either 1:1 or 2:1 CD-to-substrate complexes. In recent years, however, this simplified picture has been complemented with growing evidence of existing larger assemblies including homo- and hetero-aggregates, micellar structures, non-inclusion complexes, and so forth (8). For all practical purposes, initial solubility screening studies that test a standard panel of commercially available CDs appear to be a good paradigm to begin product development. Besides solubility testing explored by ultraviolet-visible (UV-vis) spectroscopy or high performance liquid chromatography (HPLC), formation of the CD complexes could be conclusively confirmed by differential scanning calorimetry (DSC) and qualitatively characterized by optical microscopy as well as other methods (9). Nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy (Job’s titration plots) could sometimes be helpful but may be harder to interpret quantitatively because of signal overlaps and only minor shifts upon complexation.

A number of practical tips on the rapid assessment of solubility regarding effective experimental designs have been reported by Loftsson and colleagues (10). An important observation derived from their comprehensive commercial drug screening program revealed that for poorly soluble active pharmaceutical ingredients (<30 µg/mL), there are substantial negative deviations of the extrapolated (to the nil CD concentration) solubility value St from dissolution diagrams compared to the intrinsic solubility S0. The origins of this phenomenon are still poorly understood. But the slope-only dependent parameter CE, rather than a stability constant 

 calculated from S0, now appears as a much more practically relevant estimate. So we should be putting more emphasis on CE when looking at phytocannabinoids.

Most of the information we have about cyclodextrin complexes of phytocannabinoids were collected in a few investigations conducted in the mid-2000s. This work identified optimal cyclodextrins with better binding properties and provided some proof-of-principle animal data to illustrate possible utility compared to unbound materials. Among the later work, US patent WO 2017/183011 describes the formation of β-CD and 2-HP-β-CD complexes of THC and CBD that were obtained by the melt/no-solvent method and with addition of propylene glycol (11). It claimed improved water solubility of the resulting products. However, the patent provided little characterization of the CD-encapsulated cannabinoids.

Widely quoted publications by Mannila and colleagues (2,12) reported several cyclodextrin complexes of THC-9 and CBD, including 2-HP-β-CD, Me-β-CD, and β-CD. The related binding constants and complexation efficiencies are presented in Table I. (See upper right for Table I, click to enlarge.) Me-β-CD and β-CD emerged as far better ligands compared to 2-HP-β-CD. Comparable results have been obtained in the studies reported by Hazekamp and colleagues (13) that investigated α-, β-, γ-, and Me-β-CD complexation by THC-9. A THC-9 concentration of about 14 mg/mL was reached by using 24% (~0.2 M) Me-β-CD solution, which amounts to an increase in solubility of four orders of magnitude. The deviation from linearity and realization of the AP type of the phase solubility diagrams has been interpreted as possible formation of the high order complexes, however, one group proposed a 1:2 THC-to-CD stoichiometry (12), whereas other investigators (13) favored 2:1 ratio with an out-of-cavity binding mode of the second cannabinoid molecule. The 1:2 THC-to-CD complex has been isolated by the precipitation method. Future X-ray crystallography studies may help to resolve the question and establish an exact binding mode for both THC and CBD ligands. Complexation preferences of THC-8 and binding constants were found to be analogous to THC-9 (14) though the total solubility was lower (Table I).

Production Methods and Process Economics

Cyclodextrin inclusion complexes can be prepared by several methods such as kneading, common solvent evaporation, coprecipitation, and freeze-drying methods (3). Most of the published cannabinoid pilot work used freeze-drying methods in which the alcohol solution of the substrate was added to the concentrated aqueous CD solutions and the solvents were removed by freeze-drying. However, it may be not an optimal scale up production method. From an energy saving perspective, kneading and precipitation appear to be superior methods. As this point, the reports on the commercially relevant phytocannabinoid encapsulation technologies are still lacking.

Dissolution experiments are a strong predictor of the drugs performance in the living systems. Faster dissolution kinetics have been strongly correlated with better bioaviability for the highly lipophilic and poorly soluble drugs. Though a potential of the phytocannabinoid CD complexes has never been explored in human clinical trials, available in vitro and animal data may support improved delivery. The idealized phytocannabinoid in vitro dissolution curves are presented in Figure 4. (See upper right for Figure 4, click to enlarge.) Apparently, complexation with CDs allowed for reducing the dissolution time to just a few minutes. Consequently, pharmacologically relevant superior outcomes have been obtained in several studies. The rabbit in-vivo model showed comparable area-under-the curve (AUC) numbers achieved between sublingual ethanolic CBD solutions and sublingual CBD-Me-β-CD complex, whereas oral administration of CBD revealed no quantifiable parent molecules in plasma at any time point (2). In an ex-vivo study, a transcorneal permeation of THC-8 could only be achieved with an addition of cyclodextrins to the formulations (14).

Complexation with CD was universally found to stabilize phytocannabinoids. The early data (15) were fully supported by subsequent studies. THC-9 complexes with Me-β-CD displayed no degradation in water solutions in the range of pH 5–9 for 8 weeks at ambient temperature (13). In similar conditions, including more detrimental acidic, THC-8 aqueous solutions containing 5% ethanol and 5% cyclodextrin showed no degradation with an addition of Me-β-CD and SPE-β-CD and significantly slowed degradation for 2-HP-β-CD (14).

There are some proof-of-principle published data indicating efficient encapsulation of phytocannabinoids into cyclodextrin complexes with enhanced solubility. A small range of in vivo animal data support a notion that such complexes may have better pharmacological properties than unbound materials. Successful development of multiple pharmaceutical and nutritional products already in the market place leads us to believe that using CD complexation of phytocannabinoids may be beneficial.

Possible improvements in stability and taste control or masking are the areas that have not been investigated in much detail so far, but clearly represent a growing industry need. Cyclodextrin complexes may also be useful in helping to capture more elusive “entourage effects” by virtue of concurrent complexation of the contributing terpenes, polyphenols, and other minor components in natural cannabis and hemp isolates (16).

Further structural and process optimization along with development of the viable and economical production scale up methods seems to be logical next steps to consider for cannabis technologists. If taken, it would enable a strong feedback loop to the extended animal and human testing and new product prototype development both for the consumer markets and medical applications.

J. Mannila, T. Järvinen, K. Järvinen, and P. Jarho, 

E. Sabadini, T. Cosgrove, and C. and Fdo Egídio, 

M. Purpura, R.P. Lowery, J.M. Wilson, H. Mannan, G. Münch, and V. Razmovski Naumovski, 

T. Loftsson, P. Jarho, M. Másson, and T. Järvinen, 

T. Loftsson, D. Hreinsdottir, and M. Másson, 

U.S. Patent, WO 2017/183011 Al (2017), “Water-soluble cannabinoid inclusion complexes.”

J. Mannila, T. Järvinen, K. Järvinen, M. Tarvainen, and P. Jarho, 

K. Hippalgaonkar, W. Gul, M.A. ElSohly, M.A. Repka, and S. Majumdar, 

Y. Shoyama, S. Morimoto, and I. Nishioka, 

J. Lou, Z. Teng, L. Zhang, J. Yang, L. Ma, F. Wang, X. Tian, R. An, M. Yang, Q. Zhang, L. Xu, and Z. Dong, 

 is an Adjunct Professor at the Center for Drug Discovery at Northeastern University in Boston, Massachusetts where his interests are focused on the translational research related to new drugs targeting the endocannabinoid system. Alex is a principal in several biotech and phytocannabinoid startup companies and an expert consultant to the cannabis industry in the area of drug formulation and drug delivery. Direct correspondence to: 

Articles

This article reviews state-of-the-art approaches to produce cyclodextrinphytocannabinoid complexes, related processing and analytical techniques, and implications for cannabis product development.

Solubilization of Phytocannabinoids Using Cyclodextrins

As the cannabis industry matures, terpene isolation from cannabis plant material has become a primary issue. So far, there have only been two practical applications: steam distillation, which is time consuming and smaller in scale, or CO

 extraction, which requires expensive equipment and an experienced operator. Solvent-free microwave-assisted extraction solves these problems with a cost-effective, efficient solution. The process requires nothing other than water, takes roughly 1 h per run, and yields tetrahydrocannabinol (THC) free, strain-specific terpenes from a variety of starting materials. Throughout this article, I discuss the capabilities of microwave-assisted extraction and how the cannabis industry can utilize it.

Percy Spencer invented the microwave in 1945 (1) and since that time it has revolutionized the way food has been cooked and prepared. However, microwaves have numerous uses outside of the kitchen. By the mid-1970s, chemists began using them for a wide variety of applications in inorganic and organic sample preparation (2) with materials ranging from soils and food to electronic boards (3). This instrumentation has been an asset to the scientific community over the last 40 years and will continue to be. In this article, we are going to discuss the capabilities of this technology in the isolation of strain-specific terpenes from the cannabis plant.

Harvesting terpenes from cannabis is generally completed in two ways. One utilizes steam distillation in a Clevenger apparatus or similar set-up to facilitate terpene removal. The other process utilizes CO

 as the extraction solvent, collecting the terpenes as an initial fraction within the closed loop system. While both processes work adequately, they each have drawbacks. Steam distillation requires a significant amount of heat under a long dwell time to achieve a full extraction. The length of time under heat can have an effect on product quality. It is also difficult to scale in comparison to CO

 and microwave-assisted extraction techniques. While subcritical CO

 is an efficient way to remove terpenes from plant material, the technique lacks the ability to efficiently process wet or fresh frozen material. This is because of the interaction between water and carbon dioxide forming carbonic acid (CO

) (4), which can cause an acidic pH change in the final product affecting the overall flavor. The implementation of a commercial supercritical CO

 extraction system is also a large initial capital investment that requires an experienced operator to achieve optimal results.

Microwave-assisted extraction solves these problems with the introduction of an affordable and efficient system that requires minimal experience to operate. Utilizing a set of magnetrons, the system channels microwaves throughout the sample cavity and uses them as a form of energy to excite polar molecules. In the cannabis application, that polar molecule is water. As the microwaves pass through the sample matrix, every change in the electric field of the wavelength causes a dipole rotation of the water molecules. This rotation generates friction, causing a tremendous amount of heat very rapidly (5).

This heat eventually causes the water to change from its liquid phase to steam. The increase in heat and pressure caused from this steam releases the terpenes from the plant material (6). As the steam begins to exit the microwave cavity into the distillation head, it carries these newly released terpenes with it. The microwave process is more efficient than standard steam distillation because instead of using convection heat, the microwaves heat evenly throughout the sample matrix.

The development work was done using an Ethos X microwave system with a 5 L vessel and glass fragrance extraction kit manufactured by Milestone SRL (Figure 1). (See upper right for Figure 1, click to enlarge.) The only additional materials needed are water and cannabis biomass. The process works on trim or flower, either freshly harvested or fresh frozen or in cured form. The first portion of the work discussed is done using fresh frozen flower material, followed by applying the same process on cured flower material. The strain used in this development work is OGKB 2.0 and the strain choice was because of inventory constraints at the time of development. The intent was to compare the same strain in both fresh frozen form as well as finished shelf-grade material, and OGKB 2.0 was the only strain we had in both forms at the time. Freshly harvested material, without freezing, is not an option because of Nevada state regulations, as it must be frozen within 2 h of harvest and tested by a third party analytical laboratory before being transferred to a production license. The microwave extraction method used was the same for both varieties of material and can be seen in Table I. (See upper right for Table I, click to enlarge.)

Since fresh frozen cannabis material generally has a water content upwards of 70%, no additional water needs to be added in the process. To begin, the frozen cannabis was removed from a -25 °C freezer and allowed to stand at ambient temperature to thaw for 1 h. Once the plant material had warmed, it was dropped evenly into the material vessel. Packing the material with any type of pressure is not recommended because it creates a less efficient distillation. Once the vessel was filled, it was placed into the microwave cavity of the microwave system and the extraction process was started.

Since the plant was tested as frozen material after harvest, there is a reference point for potency and terpene content that can be seen in Table II. (See upper right for Table II, click to enlarge.) This test panel is done on an “as is” basis, so water content has not been factored out. This accounts for the potency and terpene content being at lower-than-average values.

Once the run completed, terpenes were collected from the burette of the microwave system. Since they are not miscible with water, they sit as an oil layer above the water layer. Water contamination is unavoidable during collection. To help mitigate this problem, the water and terpene collection was placed in a griffin beaker and allowed to fully separate at room temperature for 5 min. Once full separation was visually observed, the beaker was placed in a freezer for 1 h. During that time, the water freezes into a solid layer of ice while the terpenes remain in liquid form above the frozen water. The terpene layer can then be decanted into a collection vessel for a pure terpene fraction. This water removal can also be accomplished using a separatory funnel or by running the solution through a bed of sodium sulfate if state regulations allow.

In this batch of fresh frozen OGKB 2.0, the total terpene yield by weight was 0.51%, compared to the analytical result of 0.39% total terpenes seen previously in Table II. These pure terpenes were then sent to a third party analytical laboratory for potency and terpene profiling. The harvested terpenes tested as nondetectable for all cannabinoids. As seen in Table III, the total terpene content was found to be 59.32% total by weight. (See upper right for Table III, click to enlarge.) There are two main reasons that this number does not get near the 100% purity levels expected in the sample. The first is analytical variability found in all testing laboratories. However, that is a small factor when compared to the missing 40% we are looking for. The main issue is that most laboratories only have access to analytical standards containing the 23 major terpenes found in cannabis (7). Since there are hundreds of terpenes found in the plants system, the industry is lacking the analytical standards needed to accurately quantitate the large array of compounds found in cannabis. As seen in the gas chromatography–mass spectrometry (GC–MS) chromatogram in Figure 2, there are numerous peaks circled in red that do not have an associated analytical standard attached to them. (See upper right for Figure 2, click to enlarge.) Without these standards, the compounds cannot be accurately quantified and reported. As the industry matures and develops, more standards will be created and more compounds will be quantifiable. However, for now we are limited to accurately quantifying the standard mix of 23 terpenes, which puts some constraints on us as an industry.

Once the terpene extraction was completed, a random sample of flower was taken from the material vessel and sent out for third party analytical testing for both potency and terpene content. As seen in Table IV, only a small amount of terpenes were found on the plant material. (See upper right for Table IV, click to enlarge.)

The extraction process was then completed on cured OGKB 2.0 for a direct comparison between fresh frozen and cured materials of the same strain. The only change between fresh frozen and cured material is that cured material requires a hydration step to reintroduce water into the plant material before processing. This step is heavily dependent on the moisture content of the plant prior to rehydration; however, the best results have been seen starting with 2 g of water to every 1 g of plant material. If the product becomes over-hydrated, the microwave may have issues reaching optimal distillation temperatures during the run. However, water content can be removed and optimized while the instrument is running by allowing water to periodically drain from the burette. If the material is not hydrated enough, it can burn and release some very unpleasant compounds into the terpenes during distillation, ruining the end product.

The terpene yield by weight seen on cured OGKB 2.0 plant material was 1.59%, again beating the tested analytical value of 1.46% seen on the cured flower. The isolated terpenes were then sent out to a third party analytical laboratory for terpene and potency profiling. The results were similar to the fresh frozen sample sent out, yielding nondetect on cannabinoid levels and a total terpene content of 68.35%. This material produced clear terpenes that can be seen in Figure 3, as most material does; however, varying shades of yellows and oranges have been seen in our laboratory throughout different varieties of cannabis. (See upper right for Figure 3, click to enlarge.)

While valuable, terpenes are only part of the equation in the economics of cannabis. The plant also has a value in the cannabinoids it produces. These molecules are still intact in the plant material after having the terpenes extracted. As seen in Table V, the total potential tetrahydrocannabinol (THC) in the post extraction fresh frozen plant material closely matches the values pre-extraction. (See upper right for Table V, click to enlarge.) Both of the samples tested were chosen at random. One considerable difference is that tetrahydrocannabinolic acid (THCA) is almost fully decarboxylated post-microwave assisted extraction. The plant material is varying shades of brown, a product of the Maillard reaction, and has an extremely high moisture content. We first attempted to simply freeze the material immediately after it was removed from the microwave then extract it using butane, similar to the production of live resin. However, the yields were found to be well below expected values. It was thought that the excess moisture in conjunction with freezing the material was a factor in the yield loss, so a forced air oven was then used to completely dry the microwaved material prior to solvent extraction. This change in process provided the mathematical yields expected after solvent extraction. A sample of the resulting crude oil was sent out for potency testing, further purified into THC distillate, and then sent out for potency testing again. The results can be seen in Table VI. (See upper right for Table VI, click to enlarge.) This process was done to ensure that there were no unexpected side effects in THC production on post-microwave assisted extraction, such as isomerization or potency degradation. No negative side effects were seen throughout processing.

The terpenes isolated can be used in numerous applications. They have a place in aromatherapy and topicals, but our facility uses them almost exclusively in the production of vape carts. We have found that by using cannabis-derived terpenes as the diluting agent for high potency distillate we have been able to produce a vape cartridge completely derived from cannabis that provides an unparalleled flavor profile, an example of which can be seen in Figure 4. (See upper right for Figure 4, click to enlarge.) These terpenes can also be added in higher amounts to other concentrate products (such as shatter or live resin) to produce what is known to the industry as a high terpene full spectrum extract (HTFSE, Figure 5). (See upper right for Figure 5, click to enlarge.) This type of extract sacrifices some cannabinoid potency to achieve an extremely high terpene profile, creating a unique effect and flavor.

Cannabis extraction methods are numerous and ever evolving, with innovations in equipment and technique almost daily. One of those innovations is microwave-assisted extraction. I hope that this article helped shed light on this technique and the possibilities it provides in terpene isolation within the cannabis industry. With new states coming online and opportunities to further research the properties and effects of the cannabis plant continue to open up, the need to efficiently isolate these compounds is only going to increase.

I would like to thank Chris Wren and the team at Medizin Las Vegas for cultivating some of the best cannabis in the world; Levon Shilling and Ryan Boyle from Milestone Srl for their continued support; and DB Labs for their thorough and accurate sample analysis.

Titan MPS Applications Notebook, “Great Results Begin With Good Preparation,” Perkin Elmer, 

www.perkinelmer.com/lab-solutions/resources/docs/APP_Titan-MPS-Applications-Notebook_010773B_01.pdf

www.businessinsider.com/how-do-microwaves-work-2014-6

www.doterra.com/US/en/brochures-magazines-living-winter-2013-2014-distilling-essential-oils

www.restek.com/chromatogram/view/GC_GN1200/terpenes

 is the Vice President of Production for Planet 13 in Las Vegas, Nevada, and is responsible for all concentrate and infused product manufacturing. Markle has over seven years of experience as an analytical chemist in the nutraceutical and cannabis industries. Direct correspondence to: 

A look at the capabilities of microwave-assisted extraction and how the cannabis industry can utilize it

Strain-Specific Isolation of Terpenes Utilizing Microwave-Assisted Extraction

With the introduction of legal cannabis available for sale in the U.S., providing safe, high-quality products defines the industry standard. As part of compliance testing, each state has different requirements for detection of microbial species in cannabis products. An ideal test is one that can be performed quickly with small amounts of cannabis product, is specific for the microbes required, can differentiate between live and dead microbes, and can be automated for high sample throughput. Medicinal Genomics developed a novel quantitative polymerase chain reaction (qPCR)-based test, in a 96-well plate format, that relies on fluorescence to detect amplified deoxyribonucleic acid (DNA). Fluorescence detection indicates the presence of microbial contamination on cannabis. This quantitative PCR method has been adapted for multiple matrices such as flower, leaf, concentrates, and an array of non-flower marijuana infused products (MIP). This review introduces the qPCR assay and compares its performance to culture-based methods.

Recent legislation legalizing the medicinal or recreational use of cannabis or cannabinoid products in the United States and Canada has led to mandated testing of cannabis products for certain microbes. The presence of bacteria and fungi in cannabis poses a potential threat if those microbes include pathogenic species. The current industry standard for detecting harmful microbes on cannabis flower is culture-based testing. However, most culture-based methods were not developed for use in the presence of a complex cannabis matrix. Culture-based yeast and mold tests have shown false positives in cannabis matrices due to off-target bacterial species growth. Most alarming, toxic 

 spp. grows poorly in culture mediums and is severely underreported by current culture-based platforms (1). This review highlights the shortcomings of culture-based methods that have been borrowed from the food industry, and the advantages of using quantitative polymerase chain reaction (qPCR) detection when applied to cannabis matrix.

PCR amplifies a segment of deoxyribonucleic acid (DNA) to create exact copies in an abundance that allows for further analyses. Kerry Mullis invented PCR in 1988, for which he and his colleagues won the Nobel Prize for chemistry in 1993. PCR is extremely sensitive, requiring only a few DNA molecules in a single reaction for amplification across several orders of magnitude of detection. Of importance, qPCR analyses are the design of the primers and probes-the short DNA sequences that determine what part of the target DNA will be amplified. Primers are designed to bind adjacent to the target sequence and are specific to the target DNA such that a single DNA base difference can determine whether binding does or does not occur. This specificity is what makes qPCR a powerful tool for the detection of pathogens in cannabis. This reduces the frequency of false positives in pathogen detection, a frequent problem with current culture-based cannabis testing methods.

qPCR takes advantage of the linearity of DNA amplification to quantify unique sequences in a DNA sample. Using a fluorescent probe reporter, it is possible to measure the amplification of a targeted DNA molecule occurring in real time (Figure 1). (See upper right for Figure 1, click to enlarge.) The real-time increase in fluorescent signal translates into quantification. In brief, if a targeted DNA molecule is present, fluorescence will increase until the signal exceeds a predetermined value. When more DNA is present in the sample, the threshold is exceeded earlier in the application process compared to samples containing less DNA. The quantitative cycle number (Cq) at which the signal curve exceeds the threshold is used to quantify the amount of DNA present in the sample when compared to a known DNA reference standard. This result is then converted to common microbial terms such as colony forming unit (CFU).

Agilent and Medicinal Genomics have partnered to provide sensitive and specific assays for the identification and quantification of microbial species regulated by agencies in the United States and Canada.

Using the Agilent AriaMX Real-Time PCR system, Medicinal Genomics PathoSEEK series of assays detect pathogenic organisms, and has demonstrated robustness, accuracy, and precision for microbial screening in various cannabis flower and cannabinoid product matrices.

To this end, DNA must first be extracted from cannabis plant and microbial cells. To simplify this process, Medicinal Genomics developed SenSATIVAx-a magnetic bead-based DNA extraction kit.

In brief, cannabis flower, leaf, or marijuana-infused product (MIP) is homogenized and, if necessary, allowed to culture in a growth medium, to generate more potential pathogens present on the cannabis product. This culture medium is then subjected to a DNA extraction followed by an optional decontamination step, which rids the sample of any previously amplified DNA.

The extracted and decontaminated DNA sample is then used as a template for the PathoSEEK assay where detection of many of these pathogens is done in multiplex: two to four microbes are targeted in a single PCR reaction. The presence of cannabis DNA and microbial contamination is based on the sample amplification curve achieving an assay-specific fluorescent value within a predetermined number of PCR cycles. Additionally, positive and negative controls show evidence and absence of amplification, respectively. Figure 3 illustrates the assay workflow. (See upper right for Figure 3, click to enlarge.)

An Empirical Comparison of PathoSEEK and Culture-Based Methods

Fifteen medicinal cannabis samples were analyzed using PathoSEEK and two commercially available culture-based methods. To enumerate and differentiate bacteria and fungi present before and after growth on culture-based media, all samples were further subjected to next-generation sequencing (NGS) and metagenomic analyses (MA). 

determines the precise order of nucleotides in a total DNA strand, and 

 analyzes genomes collected in environmental samples. These tools respectfully sequence entire genomes of species’, and profile the diversity of genes in a given sample. Figure 4 illustrates MA data collected directly from plant material before and after culture on 3M petrifilm and culture-based platforms. (See upper right for Figure 4, click to enlarge.)

The results demonstrate substantial shifts in bacterial and fungal growth after culturing on the 3M petrifilm and culture-based platforms. Thus, the final composition of microbes after culturing is markedly different from the starting sample. Most concerning is the frequent identification of bacterial species in systems designed for the exclusive quantification of yeast and mold, as quantified by elevated total aerobic count (TAC) Cq values after culture in the total yeast and mold (TYM) medium. The presence of bacterial colonies on TYM growth plates or cartridges may falsely increase the rejection rate of cannabis samples for fungal contamination. These observations call into question the specificity claims of these platforms.

 spp. grow poorly in culture-based media. One of the common issues is that 

 spp.  clumps over generational growth, so a homogenous spread of the cells in the media is nearly impossible. When 

 is plated, clumped conidia cells form heterogeneous macro-colonies which creates significant issues with quantitation (Figure 5). (See upper right for Figure 5, click to enlarge.) What looks like one large colony is hundreds of colonies that are not countable. Additionally, the clumping nature of 

 spp. spores in media makes it difficult to accurately pipette samples. DNA extraction processes performed before running qPCR assays have the benefit of lysis and DNA purification steps that “de-clump” and break open the cells or spores, providing better sampling techniques and therefore more accurate and reliable results.

Marijuana Infused Products Interference and Impact on Results

Marijuana infused products (MIPs) are a very diverse class of matrices that behave very differently than cannabis flowers. Gummy bears, chocolates, oils, and tinctures all present different challenges to culture-based techniques as the sugars and carbohydrates can radically alter the carbon sources available for growth. To assess the impact of MIPs on colony-forming units per gram of sample (CFU/g) enumeration, we spiked in live 

 cells into various MIPs to measure the qPCR signal. In many of the spiked MIP samples, 

 failed to grow compared to tryptic soy broth (TSB) controls.

This implies the MIPs are interfering with the reporter assay on the films or that the MIPs are antiseptic in nature.

Many MIPs use citric acid as a flavoring ingredient which may interfere with 3M reporter chemistry. In contrast the qPCR signal was constant, implying there is microbial contamination present on the films, but the colony formation or reporting is inhibited.

with and without MIP on 3M coliform films. (See upper right for Figure 6, click to enlarge.) Growth of 

CFU was severely hindered in presence of oil or candy.

To show that the SenSATIVAx DNA extraction method is efficient in different matrices, DNA was spiked into various MIPs as shown in Table I, and at different numbers of DNA copies into chocolate (Table II). (See upper right for Tables I and II, click to enlarge.) The SenSATIVAx DNA extraction kit successfully captures the varying levels of DNA, and our PathoSEEK detection assay can successfully detect that range of DNA. Table III demonstrates that SenSATIVAx DNA extraction can successfully lyse the cells of the microbes that may be present on cannabis for a  variety of organisms spiked onto cannabis flower samples. (See upper right for Table III, click to enlarge.)

More than 60 species in total were used to verify the specificity of the PathoSEEK Microbial Safety Testing Solution, with a minimum of 6 to 10 and a maximum of 45 for each detection assay. The data shows 100% concordance with the current list of species tested. A subset of the data is shown in Table IV. (See upper right for Table IV, click to enlarge.)

Linearity shows the relationship between a group of known dilutions and resulting Cq. To test the linearity of our PathoSEEK assays, we conducted 12, two-fold serial dilutions of a known DNA concentration in triplicate. Figures 7a and 7b show the results of the total aerobic count (TAC) assay and the qPCR efficiency (E) of the assays as inferred by the linear coefficient of determination (R2) which is a measure of how well a dependent variable fits a linear model. (See upper right for Figure 7, click to enlarge.)

Using PathoSEEK to determine the presence or absence of 

 spp. a limit of detection (LOD) was determined by performing triplicate analyses of 12, two-fold serial dilutions starting at two copies through 5000 copies. In each case, a LOD of 10 copies were determined.

Correlation of qPCR results with plating live species will result in an equation that enables conversion of Cq/g to CFU/g as given in equation 1:
	CFU/g = 10 * [(42.185 – Cq Value)/3.691]

Molecular methods often leverage amplification of ribosomal DNA, internal transcribed spacers (ITS) regions (3,6). As a result, these PCR products can detect unculturable organisms and organisms that clump and distort CFU/g enumeration such as 

 species (Figure 9).  (See upper right for Figure 9, click to enlarge.) 

 spp. demonstrate logarithmically slower growth at room temperature than most other yeast. You can see in this comparison to the qPCR PathoSEEK assay results, the CFU plated and the growth on plating media does not appear linear and significantly underestimates 

 spp. growth. However, the plated CFU does compare accurately to the qPCR quantitative cycle (Cq) result for other species.

Accurate methods are required to assess exposure risk across diverse cannabis samples and matrices. Traditional testing methods include culture on nonspecific media, such that many microbial organisms can grow, resulting in false positive results. Further, certain organisms do not grow on media, and their apparent culture and growth are not representative of true organism density, thus causing under-reporting or false negatives. qPCR is being repurposed for its ability to detect small amounts of specific pathogenic microbial organisms potentially present in these myriad sample presentations. To meet this new demand, Medicinal Genomics (MGC) has partnered with Agilent to employ their PCR technology on the Agilent qPCR system. This chemistry includes the SenSATIVAx DNA purification kit and the PathoSEEK qPCR reagents, which include the appropriate positive and negative controls.

C. De Bekker, G.J.  van Veluw, A. Vinck, L.A Wiebenga, and H.A. Wosten, 

(4),1263–7 (2011). PubMed PMID: 21169437. PubMed Central PMCID: 3067247.	

K.J. McKernan, J. Spangler, and L. Zhang et. al., 

https://doi.org/10.12688/f1000research.7507.2

Medicinal Genomics, “PathogINDICAtor qPCR microbial detection Assay on the AriaMX Real-Time PCR System optional decontamination step,” Document EUD-00021 1.4.  (2017). (Medicinal Genomics Corporation).	

K.J. McKernan, J. Spangler, and Y. Helbert et. al.,“Metagenomic analysis of medicinal Cannabis samples; pathogenic bacteria, toxigenic fungi, and beneficial microbes grow in culture-based yeast and mold tests,” 

S.D. Leppanen and H. Ebling, “Optimized Cannabis Microbial Testing: Combined Use of Medicinal Genomics Extraction Methods with the AriaMx qPCR Instrument,” Application Note 5994-0430EN, Agilent Technologies (2018).

 is the Senior Field Applications Scientist and 

 is the Senior Scientist for Agilent Technologies, Inc.  

 is the Senior Applications and Support Manager for Medicinal Genomics. Direct correspondence to:

S.D. Leppanen, H. Ebling, and A. Macherone, 

The print version of this article mistakenly left off Figure 7a and Table IV. Those elements are included in the correct version found here.

This review introduces a quantitative polymerase chain reaction (qPCR) assay and compares its performance to culture-based methods.

Optimized Cannabis Microbial Testing: Combined Use of Extraction Methods and Pathogen Detection Tests Using Quantitative  Polymerase Chain Reaction

The third and final installment of this series reviews how processing parameters affect yields in extraction. Operating conditions, including extraction runtime and processing parameters, have a major influence on the yield obtained from an extraction.

There are many factors influencing yields in extraction. The first article in this three-part series presented a review of starting-material influence on extraction yields: the amount of starting material added to an extraction chamber, in addition to the content of compounds of interest intrinsic to that material, are important (1). Next, part II examined the effects of material preparation: a finer particle size provides better packing efficiency, as well as greater efficiency of solvent-matrix interactions (2). Here, the final installment reviews the influence of operating conditions, elucidating how extraction runtime and processing parameters affect yields in extraction.

An extraction operation should maximize yield while minimizing runtime and operating costs required to achieve the desired result. Consider Figure 1: as an extraction is initiated, the amount of material of interest obtained versus time sharply increases to Point Q, the maximum amount of material of interest extracted per unit time. After this point, the return of material obtained versus time gradually decreases until there is no material remaining (the extraction reaches completion). (See upper right for Figure 1, click to enlarge.)

Three additional points are noted on the graph in Figure 1. Point P, the operating break-even point, defines the moment at which the extraction has recovered enough material to cover the costs incurred to allow operation for that extraction. Point S marks the position of the operating profit line along the extraction curve. The region between Point P and Point S is the profitability region; within it, the return from the product being extracted will more-than pay for all associated costs of the extraction. To the right of Point S, the costs of operation outweigh the return from the material obtained. The undefined Point R is the ideal point to halt the extraction. The precise location of Point R changes depending on factors such as the potency of the feedstock material, whether there is a finite or infinite amount of feedstock material to be processed, and the operation costs unique to each business.

Consider the example scenario outlined in Table I. (See upper right for Table I, click to enlarge.) Because of the way the general extraction curve is shaped, the runtime required to achieve a complete extraction may conflict with the cost of running the equipment or the facility. In this hypothetical production cost-revenue analysis, a day spent performing two extractions to 100% completion would bring $8000 in revenue, whereas carrying out three extractions to 95% completion would turn a profit of $16,500.

To ensure a business is profitable, an operation will want to exceed merely breaking even. There is no magic number for the time an extraction should be allowed to proceed. Rather, runtimes and return of material should be evaluated against facility operating costs as well as those incurred in refinement (post-processing) of the extract. A longer runtime may also provide access to one or more additional components of value or, conversely, may be associated with a higher concentration of undesirable material. It is up to each organization individually to establish the extraction conditions that, for them, make the return of operation worth the effort.

Along with runtime, processing parameters have a significant effect on extraction yields. In performing an extraction, an operator must choose the parameters that result in the highest yield of desirable components and the lowest yield of undesirable components. Furthermore, coextraction of desirable and undesirable components should be minimized to reduce downstream processing. In general, parameters that may affect extraction yield include temperature, pressure, and the solvent-to-feedstock ratio.

Consider Figure 2, which plots hypothetical extraction profiles of three components (components 1, 2, and 3) over three different extraction temperatures (low, medium, and high). (See upper right for Figure 2, click to enlarge.) The low-temperature run (Plot A) returned large amounts of components 1 and 2, which are coextracted; a small amount of component 3 is extracted with separation from other components. The medium-temperature run (Plot B) extracted large amounts of components 1 and 2 and a moderate amount of component 3, all with little to no coextraction. The high-temperature run (Plot C) saw all of components 1, 2, and 3 extracted in large amount; components 2 and 3 were coextracted.

If the operator is only interested in components 1 and 2 but not 3, they should select a low or medium temperature and halt the extraction before component 3 is extracted. If the operator is interested in components 2 and 3 but not 1, they should choose a high-temperature extraction. If the operator is interested solely in component 1, they should select a medium or high-temperature extraction and halt the run before component 2 is extracted.

Divided into three articles, this series has presented in-depth discussions of the factors influencing yields in extraction. To refresh and summarize the full breadth of concepts discussed, it is useful to consider a recipe providing the greatest yield of valuable components.

Beginning with the feedstock, a strain rich in desirable material with a minimum amount of undesirable material is chosen. This is the highest possible yielding option for starting material: the content of components of interest is maximized. Next, reducing particle size ensures that a maximum amount of material can be fit into the extraction chamber and increases efficiency of interaction of the solvent and feedstock matrix: a finer particle size increases packing efficiency and efficiency of solvent-matrix interaction. Extraction parameters will be selected that provide the most material extracted as well as the best separation from the undesirable components: a maximum amount of the component of interest is obtained and its elution is well-separated from unwanted components. Finally, the extraction will be run to completion such that every last bit of valuable material is extracted into the yield: the operation profit line is not a concern, all available material of value will be extracted.

 is a technical and scientific writer with Vitalis Extraction Technology in British Columbia, Canada. 

 is an application science manager with Vitalis Extraction Technology. Direct correspondence to: 

The final installment of this series reviews how processing parameters affect yields in extraction.

Factors Influencing Yields in Extraction, Part III: Return Versus Effort and Associated Processing Parameters

This review article, written from the point of view of an experienced pharmaceutical microbiologist, discusses the risks of microbial contamination for the full range of cannabis-derived products and recommends the most appropriate microbiological quality requirements for each of these products by benchmarking against the microbiological quality recommendations from the American Herbal Products Association (AHPA), the World Health Organization (WHO), European Pharmacopeia (Ph. Eur.), and the U.S. Pharmacopeia (USP) for powdered cannabis, dietary supplements, herbs, botanical products, and nonsterile drug products.

With a rapidly emerging state-regulated industry producing both recreational and medicinal cannabis products, there are many questions surrounding the microbiological attributes of cannabis-derived products. This review article discusses the risk of microbial contamination of these products and the most appropriate microbiological specifications and test methods for the widely different products that range from smoked powdered cannabis buds to chemically defined pharmaceutical drug products. Part of the challenge is that cannabis-derived products based on the consumer (medical and recreational) and mode of use may be viewed as herbal products, recreational drugs, foods, or pharmaceutical drug products. Critical issues to be addressed by state regulators and the cannabis industry alike are the microbiological specifications and test methods for these widely different products.

What are the biggest risks with cannabis-derived products? In medical use, smoking cannabis has been used to control weight loss associated with the human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS), preventing nausea associated with chemotherapy, and alleviating pain associated with a range of illnesses. These patient populations will be more susceptible to microbial infection than recreational users. With respect to mode of administration, the microbiological quality requirement will be widely different for cannabis that is smoked, vaped, topically applied, or eaten. As many medicinal cannabis users have severely compromised immune systems, medical cannabis distributed by licensed producers in the Netherlands and Canada is irradiated for control of the bioburden but not to sterilize the product.

Does the scientific literature report concerns with the microbial contamination of cannabis? This issue was addressed by the author in an earlier publication that may have escaped the cannabis industry’s attention because of its publication in a pharmaceutical trade journal (1). Groups prone to 

 (CPA) associated with medical or recreational use of cannabis (2–12) include immune-compromised individuals receiving chemotherapy and corticosteroids; solid organs and stem cell transplant recipients; and HIV or AIDS patients. In addition to CPA that has a high mortality rate, if not treated early, chronic use of smoked cannabis is associated with 

 (ABPA) (13). An authoritative review on the 

 website (14) stated that the overall risk of 

 appeared low, given the large number of cannabis smokers. An exception noted would be 

 in highly immune-compromised patients. This suggests that medical cannabis that is smoked should have more stringent fungal specifications than other products.

In addition, cannabis may be contaminated with other human pathogens during its cultivation, processing, and distribution. Outbreaks of 

 (17) have been reported. The author expects that this type of contamination may be more likely in field-grown cannabis plants using irrigation water, and poor hygiene practices among harvesters and processors, than greater capitalized greenhouse-grown cannabis operations.

Published surveys of the microbial populations of powdered cannabis in peer-reviewed journals are few and of varying technical rigor. Higher mold numbers using the selective Sabouraud Dextrose Agar (SDA) for cannabis compared to tobacco (200 to 300 versus 10

This study reported that cigarette smoke was negative for mold but cigarettes are tightly rolled and burn at higher temperatures than joints. Other studies have shown that smoke from both burning tobacco and marijuana cigarettes contain fungal spores (19,20). In an evaluation of the quality of medicinal-grade and coffee shop-purchased cannabis in the Netherlands (21), it was reported that irradiated medicinal-grade cannabis contained less than 10 CFU/g enterobacteria and Gram-negative bacteria (bile-tolerant, Gram-negative bacterial count) and less than 100 CFU/g molds and aerobic bacteria (total aerobic microbial count) while 10 samples of coffee shop cannabis ranged from less than 10 to 80,000 CFU/g bile-tolerant, Gram-negative bacterial counts and 120 to 480,000 CFU/g total aerobic microbial counts.

The contaminants from one of the 10 samples were identified as the fecal bacterium 

This section of my review discusses the definition of cannabis-based products as pharmaceuticals per U.S. Pharmacopeia (USP) <

, food classifications, and state cannabis regulations. The hierarchy of risk of microbial infection based solely on the invasiveness of the route of administration would be vaping > smoking > topical application > oral consumption. It is expected that the risk to the cannabis users will be largely determined by their underlying medical status and frequency of usage.


	As far as the author knows, no pharmaceutical drug product is smoked, so how to set a microbiological specification for cannabis buds is more challenging. The literature suggests that microorganisms, especially fungal spores, may survive the burning process, be inhaled into the lungs, and result in infection especially in immunosuppressed users. The microbiological requirements for inhalation products as found in USP 

<1111> Microbiological Examination of Non-Sterile Products: Acceptance Criteria for Pharmaceutical Preparations and Substances for Pharmaceutical Use

 are more stringent and less achievable with a plant-derived material than recommended by standard-setting organizations for botanical products (Table I). (See upper right for Table I, click to enlarge.)

What are the microbial specifications for herbal products? Table II compares the microbiological quality recommendations (total aerobic microbial count, total yeast and mold count, bile-tolerant, Gram-negative bacterial count, and absence of specified microorganisms) from the the American Herbal Products Association (AHPA), the World Health Organization (WHO), European Pharmacopeia (Ph. Eur.), and USP for dietary supplements, herbs, and botanical products. (See upper right for Table II, click to enlarge.)

In 2014, the American Herbal Pharmacopoeia (AHP) issued a revised monograph entitled 

Cannabis Inflorescence-Standards of Identity, Analysis, and Quality Control

 that provided microbial and fungal limits that were claimed to be consistent with applicable state, federal, and international regulations that were based on botanical products. This monograph is highly influential in the cannabis industry (see Table III). (See upper right for Table III, click to enlarge.)

The USP published a stimulus article entitled “The Advisability and Feasibility of Developing USP Standards for Medical Cannabis” that discussed the issues around setting standards for medical cannabis (22).

The monograph cited the U.S. Food and Drug Administration (FDA) 

 (23) as a source of appropriate methods. Another good source of microbial methods would be USP 

<2021> Microbial Enumeration Tests-Nutritional and Dietary Supplements

<2022> Microbiological Procedures For Absence Of Specified Microorganisms­­-Nutritional And Dietary Supplements

. The author believes that the limits recommended in the AHP monograph were qualified in terms that medical cannabis may need more restrictive limits addressing the presence of members of the genus 

, and that the limits recommended will not be suitable for extracted and highly purified cannabinoids where microorganisms would not survive the manufacturing process.

What is missing is a comprehensive survey of the microbial content of cannabis-derived products to serve as a baseline for the development of risk-based microbial specifications, an understanding of the different levels of risk in user populations, and an understanding of the microbiological attributes of products derived by different manufacturing processes. As more U.S. states legalize medical and recreational cannabis products, they are independently setting microbial specifications without this understanding. For example, Table IV contains the microbial limits required by the Colorado Department of Public Health. (See upper right for Table IV, click to enlarge.)

What may be wrong with this approach? These limits emphasize the exclusion of foodborne bacterial pathogens 

 and the numbers of molds in the products. In food microbiology, it is generally viewed as impractical to screen for pathogenic 

 in routine food surveillance programs, and this type of testing would be reserved for outbreak investigations. Setting a limit for the number of 

 per gram of product as indicative of recent fecal contamination would be recommended by the author over specific pathogenic 

 in powdered cannabis may survive despite the low water activity of the material, but would be eliminated during smoking. Handling material before smoking could be a source of enteric infection, whereas 

 spp. may contaminate and survive in olive oil used to extract cannabinoids from cannabis buds. As with 

 in contaminated flour and cake mixes that have caused foodborne infection when eaten raw, baking or frying of the cannabis-laced baked product will destroy this serious bacterial pathogen.

As to the control and monitoring of the fungal content of cannabis-derived products, the question is whether setting a total yeast and mold count (TYMC) limit, that is, not more than (NMT) 10

 CFU/g, is sufficient to promote user safety, or if members of the genus 

 should be specifically excluded because of its role in chronic pulmonary aspergillosis and the potential aflatoxin production. For example, the state of Colorado favors setting a TYMC limit, whereas the state of California favors routine screening for specific 

). If it can be clearly established that 

 is storage contaminant, and not an epiphytic microorganism associated with cannabis plants, then the emphasis should be placed on post-harvest drying and storage as a risk mitigation step, with water content (water activity) added to the cannabis specifications in place of screening for specific 

 species. The reader is directed to a discussion of the distinction between epiphytic fungi, fungal diseases of cannabis plants, and cannabis storage fungi (24). The author recommends the microbiological standards for unprocessed cannabis as shown in Table V. (See upper right for Table V, click to enlarge.)

In contrast to buds, which are smoked, the active ingredients in the cannabis plant may be solvent extracted from the dried, powdered plant material to be smoked, vaporized, eaten, or applied topically.

The extraction method will depend largely on the cannabinoids targeted for purification and the type of concentrate wanted. The three most common extraction procedures are butane, alcohol, or supercritical carbon dioxide extractions (25).

) pressurized to its supercritical state. This converts CO

 into a liquid that passes through a chamber containing powdered cannabis material. The filtrate can be isolated easily by reducing the pressure that evaporates the CO

, leaving a cannabis extract with no solvent.

Sophisticated extractors on the market can also incorporate fractionation to isolate individual cannabinoids, which enables process tuning to isolate desired components, and recover the CO

The resulting material according to the final stages of processing may be called shatter, wax, budder, or crumble.

These systems pressurize butane, propane, or other low molecular weight hydrocarbons to a liquid state. Care must be taken as these solvents are flammable at room temperature. The liquid hydrocarbon passes through a bed of powdered cannabis material and filter, yielding a hydrocarbon extract solution of hydrocarbon and the cannabinoids. Like the CO

 method, a reduction in pressure evaporates the hydrocarbon liquid, yielding a solvent-free cannabinoid. Maintaining the pressurized hydrocarbon in the liquid state requires low temperatures. Recirculating temperature control units that can provide cooling to -60 °C (-76 °F) and below facilitates this process. Heating circulators are also incorporated to increase the liquid butane evaporation to isolate the extract and recycle the butane. The resultant concentrate is frequently described as butane hash oil.

This is the least sophisticated extract method of the three. Food-grade ethanol is used as a solvent to extract cannabis plant material. This can be used with vessels varying from reactors to barrels. A popular process has the ethanol chilled to <-20 °C (-4 °F) either in a cold room or freezer and then pumped into a container of cannabis. After a soak period, the ethanol solution is either filtered or the plant material removed in a “tea bag” fashion. The resultant ethanol mother liquor and extract is then concentrated by removing the ethanol. Typical distillation apparatus used to remove the ethanol include rotary evaporators or a vacuum distillation system. A simple form that allows the alcohol to evaporate is called 

Review of these three extraction and purification procedures strongly indicates that, other than bacterial spores, microorganisms will not survive contact with organic solvents, freezing temperatures without a cryo-protectant, and elevated temperatures associated with distillation. This suggests that cannabis buds and infusions, tinctures, and edibles made from oil-extracted cannabis in terms of microbiological specification should be treated differently than purified cannabinoids in terms of microbial specifications and testing because of the lower risk of microbial contamination (see Table VI). (See upper right for Table VI, click to enlarge.)

An electronic cigarette is a battery-operated vaporizer, which simulates the experience of smoking, without burning tobacco. Their use is commonly termed

. Vaping is the rapidly growing delivery method for nicotine, especially amongst younger people, and will grow amongst cannabis users. The literature reports these electronic vaping devices offer greater efficiency in nicotine delivery than smoking cigarettes, without the inherent dangers of tobacco tars, carbon monoxide, and poly aromatic hydrocarbons. The vehicle of vaping is a heat-generated aerosol of propylene glycol, glycerin, flavoring, and nicotine or cannabinoids. Whereas the combustion temperatures of smoking cannabis ranges from 600–900 °C with around 30% of the cannabinoids lost by pyrolysis, vaping heats the cannabis oils and waxes to 160–230 °C. Cannabinoids vaporize near their boiling points. The boiling points of cannabinoids occur in this range: tetrahydrocannabinol (THC) 157 °C, cannabidiol (CBD) 160–180 °C, and cannabinol (CBN) 185 °C (26). The cannabinoid extraction process, storage in a propylene glycol-glycerin mixture E-solution of low water activity, and the vaporization temperature all make it highly unlikely the heat-generated aerosol will contain viable microorganisms.

Canna-butter, Vegetable Oil, Tea, or Tincture (Edible)

Olive oil may be a common choice for cannabis extracts used for cooking. Studies of the naturally occurring microorganisms in olive oil have been limited and do not give a complete picture for olive oils produced in all of the olive-producing regions of the world (27). For example, Italian researchers (28,29) examined olive oils produced in central Italy, and conducted microbiological analysis for aerobic (standard plate count agar) and lactic acid bacteria (MRS agar), yeasts (SDA), and molds (glucose yeast extract agar with gentamicin and chlorampenicol). They reported that yeasts were consistently present both initially and during storage, molds were occasionally found, and bacteria were never found.

The molds belonged primarily to the genus 

. Using light microscopy, they found that the microorganisms and the solid particles were entrapped in micro drops of vegetation water that were suspended in the olive oil.

Teas may be made by suspending and steeping powdered cannabis in boiling water, which should eliminate vegetative microorganisms. Similarly, tincture made with greater than 50% ethanol would be free of vegetative microorganisms, so routine screening for specified microorganisms could be eliminated.

Route of administration (injection or implantation, gastrointestinal, topical or dermal, mucosal, and inhalation).

Dosage form (aerosols, capsules, creams, emulsions, films, foams, gases, gels, granules, implants, injections, inserts, irrigations, liquids, lotions, lozenges, ointments, pastes, pellets, pills, plasters, powders, soaps and shampoos, solutions, sprays, strips, suppositories, suspensions, tablets, and tapes).

This review article from an experienced pharmaceutical microbiologist discusses the risks of microbial contamination for the full range of cannabis-derived products and recommends the most appropriate microbiological quality requirements for each product.

July/August 2019

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