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The Big FreezeUsing Cryogenic Extraction to Create Golden Perfection

Using Steam to Extract the Botanical Spirit

Distillation of Aromatic Plants

Microwave Terpene

ExtractionStaying True to Your Chemovar and Your Brand

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Cannabis on Collision Course with Science

ISSUE 12 Mar/Apr 2020

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Envisioning Cleaner, Clearer

Extracts through Product Filtration

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Contents

08

06

14

16

20

Envisioning Cleaner, Clearer Extracts through Product Filtration

The Big Freeze – Using Cryogenic Extraction to Create Golden Perfection

22 Meet Our Advisory Board The AC Braddock Chapter


Improving Brand Loyalty with Microwave Terpene Extraction

The Extraction Artist’s Splotch of Oil

Publisher MACE Media Group

CEO Celeste Miranda

Editor-in-Chief Jason S. Lupoi, Ph.D.

Authors Jason S. Lupoi, Ph.D.

Roberto Federico-Perez, Ph.D. Seth Oxhandler

Drew Stahr Rob Brown

AC Braddock

Designer Marko Nedeljkovic

Advertising Julian Azevedo

Bradford Burgess Lisa Dodson

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The organoleptic experiences of different cannabis extraction products are based on their chemistry. The terpenes, of course, provide flavor and aroma in addition to prolific medicinal properties. Take borneol, for example. The isolated terpene (a white powder) is still wonderfully fragrant, although its scent reminds me of the marriage of the fresh woods and a strong, pine-scented cleaning solution. And, unlike the rumors regarding myrcene [1], borneol has been shown to ferry molecules across the blood-brain barrier. [2] Beta-Caryophyllene is peppery and spicy. The terpene isolate (oil) that I have smells like clove. Caryophyllene is resilient throughout the extraction process, and many vape pen products seem to have aromas reminiscent of peppers and cloves.

Medicinally, caryophyllene is very special. It’s been studied in treating addiction [3] and in chronic pain [4], to lightly pepper the discussion with its accolades. These and many other terpenes are taking on more advanced roles within the hemp industry now that cultivators are targeting resin and not fiber production. In the further evolved cannabis product blueprints, terpenes are being added in bulk to mimic a chemovar or in isolated form to achieve a desired physiological outcome (e.g., sleep).

Cannabinoids taste bitter. In a product like a cannabidiol (CBD)-infused water, that slightly bitter taste imparts confidence that the liquid is actually infused with CBD juxtaposed to a glorified version of packaged tap water or equivalent. It’s sad that some commercial CBD products don’t contain CBD. [5] So, that trademark bitterness ought to

at least signal the medicine’s presence.

Other molecules in extracts cause dark colors or vegetal tastes. Depending on the consumer’s palate, one person might favor crude oil products like Rick Simpson Oil (RSO), regardless of its thick mouthfeel and bitter taste. Many people swear by RSO. Another

person, though, might want as much of that bitterness, taste of plant matter, and waxes and chlorophyll, to be stripped out,

resulting in an amber or golden elixir, clean and much more subtle. There’s an

organoleptic solution for everyone.

It’s almost like a splotch of paint, not very beautiful

perhaps, but plentiful with possibilities. The extraction artist doesn’t need to add more color to the painting. Rather, they refine their products to remove unwanted ingredients like pigments or waxes, navigating from blackened to golden hues. What experience are you trying to create? Your adventures as a product designer begin there.

This issue of Extraction Magazine continues our discussion regarding ways of refining cannabis and hemp extracts. From cryogenic ethanol extraction to the use of microwaves for botanical terpene isolation or multi-stage filtration strategies, the extraction artist has many ways to concentrate and beautify their craft.

The Extraction Artist’s Splotch of Oil

6 EXTRACTION MAGAZINE

By Jason S. Lupoi, Ph.D.

References[1] Bresler, T. “Myrcene and the Blood-Brain Barrier: The Universal Claim with the Lack of Scientific Evidence,” Terpenes & Testing Magazine, Uploaded April 1, 2019; Accessed February 24, 2020.

[2] Yu, B. et al. “The Mechanism of the Opening of the Blood–Brain Barrier by Borneol: A Pharmacodynamics and Pharmacokinetics Combination Study”, Journal of Ethnopharmacology, 2013, vol. 150, 2013, p. 1096-1108. [journal impact factor = 3.115; cited by 44]

[3] Xi, Z. et al. “Brain Cannabinoid CB2 Receptors Modulate Cocaine’s Actions in Mice”, Nat Neurosci, vol. 14(9), 2011, p. 1160-1166. [journal impact factor = 21.126; cited by 228]

[4] Klauke, A. et al. “The Cannabinoid CB2 Receptor-Selective Phytocannabinoid beta-Caryophyllene Exerts Analgesic Effects in Mouse Models of Inflammatory and Neuropathic Pain”, European Neuropsychopharmacology, vol. 24(4), 2014, p. 608-620. [journal impact factor = 4.468; cited by 90]

[5] Lupoi, J. “Infinite Chemical Analysis Labs Measures Labeling Discrepancies on 12 CBD Products, Yet Again,” Terpenes & Testing Magazine, Uploaded January 7, 2020; accessed February 24, 2020.

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The psychoactive nature of cannabis is widely understood as an effect of the cannabinoids present in the plant. Cultivation Cultivation focuses not only on enhancing and modulating these effects but also in growing varieties with a better smel and taste to improve their consumer appeal. These sensory properties of cannabis rely on terpene content. The structure and proportion of terpenes result in unique organoleptic profiles that differentiate cannabis plants from one another. The extraction of terpenes from cannabis, therefore, opens the door for the manufacturing of various cannabis derivatives with a distinctive branding.

Principle of Microwave ExtractionMicrowave extraction is an emerging technology that enables obtaining terpene extracts that are true to the original plant profile. The technique works by taking advantage of the polar molecules present in the plant material. Water molecules are the main contributor to microwave absorption. In the presence of microwaves, water molecules exhibit multiple vibrational states that generate heat. The steam released by this process carries over the volatile compounds present in the plant. Since terpenes are immiscible in water, they are easily recovered after condensation in a separate vessel where they form a layer on top of the aqueous phase due to their lower density.

Using microwaves for extraction poses several advantages compared to other techniques. For instance, as opposed to conventional steam distillation that utilizes conduction as a heat transfer method, a more uniform mechanism can be achieved by microwave irradiation, avoiding major temperature gradients since microwaves act on the water molecules in situ. This allows for an improved efficiency and shorter extraction times. [1] In addition, microwave extraction can be used to process fresh or fresh frozen material, which

does not undergo the potential loss of terpenes during the drying or curing process. [2] The use of fresh cannabis with high moisture is restricted in other extraction techniques like CO

2, where water can lead to the formation of carbonic acid.The ETHOS X Microwave Extraction System (Figure 1) integrates the principle of operation described above into a streamlined process. The unit consists of a reactor vessel of variable capacity that is loaded with cannabis and placed into a microwave cavity. The vessel is then attached to an external setup that comprises a stainless-steel condenser connected to a water chiller and a graduated glass collection vessel. Once the microwave program starts, water eventually evaporates and condenses into the graduated glass vessel where the user can keep track of the accumulation of terpenes throughout the run. Water is constantly recirculating back into the reactor to ensure an adequate microwave absorption and a carrier medium for the extracted terpenes.

Extraction ProcedureA typical microwave method includes a series of wattage steps that progressively increase the microwave power supplied to the cannabis material. The system monitors the temperature by using a contactless sensor that reads the bottom of the reactor. The overall microwave program, however, is defined by the wattage supplied at a given time. Method optimization depends on the kind of plant material used, but ultimately consists of determining when condensation occurs in order to reduce the microwave power to sustain the evaporation of water.

An important parameter that dictates the amount of power to be supplied is the amount of material to be processed. Approximate nominal loads for fresh material are around 1, 2.5, or 6 pounds depending on the volume of the vessel (2,

By Roberto Federico-Perez, Ph.D., Milestone Inc

Improving Brand Loyalty with Microwave Terpene Extraction


References[1] Ross, S. et al. “The Volatile Oil Composition of Fresh and Air-Dried Cannabis sativa”, Journal of Natural Products, vol. 59(1), 1996, 9. 49-51. [journal impact factor = 4.257; cited by 68]

[2] Sahraoui, N. et al. “Improved Microwave Steam Distillation Apparatus for Isolation of Essential Oils. Comparison with Conventional Steam Distillation”, Journal of Chromatography A, vol. 1210(2), 2008, p. 229-233. [journal impact factor = 3.858; cited by 81]

[3] Markle, S. “Strain-Specific Isolation of Terpenes Utilizing Microwave-Assisted Extraction,” Cannabis Science and Technology, vol. 2(4), 2019, p. 50-57, 76.

9EXTRACTION MAGAZINE

5, or 12-L, respectively). In a similar way, about 0.5, 1, or 2.5 pounds of cured material can be accommodated in the corresponding vessel. Since water is the microwave-absorbing agent, cured cannabis requires an additional rehydration step. This is normally performed by adding water equivalents to the material in either a 2:1 or 3:1 ratio.

Expected yields for microwave-extracted terpenes are strongly dependent on the quality of the starting material. Anecdotally, these yields range within 0.3-1.0% for fresh or fresh frozen cannabis, and 1.0-1.5% for cured cannabis, as reported by users. These yields are calculated using the original mass as a basis. Although many plants yield colorless terpenes, some cannabis varieties show light yellow to light orange colorations. This has no impact on their smell and flavor profile. Moreover, clarity is a consistent characteristic among all microwave-extracted terpenes, as no particulates or extraneous materials or compounds are carried into the final product.

A relevant feature of microwave extraction is its ability to be selective toward highly volatile compounds (i.e., terpenes), allowing for a pure extract. Heavier compounds with additional functional groups, such as cannabinoids, are retained within the plant material without modification and are not present

in the final extract. Therefore, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) can subsequently be extracted by conventional approaches.

Often these additional methods require dry feedstock to ensure an optimal performance (e.g., CO2 extraction). To achieve this, a common technique that can be coupled to the microwave process is the use of a forced air oven that allows for a fast removal of the remaining moisture in the material. Tables 1 and 2 describe the terpene profile in the original fresh frozen flower and final extract, respectively, and Table 3 shows the cannabinoid content in the flower pre- and post-extraction. [3] Differences in cannabinoid content in the flower pre-and post-extraction can be attributed to variable sample sizes at each stage of the analysis.

Productivity and Microwave-Extracted TerpenesSince many processors are looking for scalable solutions, they utilize the higher throughput option to run as much material as possible in a single run. For a 12-L vessel, a complete run takes about 60 minutes. Additional scalability can be reached by running units in parallel. Table 4 shows a productivity estimation based on use in an average workday. The kind of material used, different ranges of yield, and the number of parallel units are factored into this assessment. As an example,


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running six runs per day with fresh frozen material at an average yield of 0.5% would generate 68 g of terpenes with a single system, or 341 g if using six units.

The figures shown above can be used as general guidelines for calculating the return on investment in this technology. A classic application for cannabis-derived terpenes is vape pen manufacturing. When using distillate rich in THC, a cut of terpenes is added to produce a characteristic taste and flavor to the cartridge, which ultimately leads to a branded product. Although the proportions between distillate and terpene may vary, a conventional 7.5% terpene cut can be used for illustration purposes. Assuming a 1000 g batch of vape cartridge mixture, this proportion translates into 75 g of terpenes for every 925 g of distillate. For a typical cartridge size of 1 g, a thousand cartridges can be produced if there is no loss through packaging. The cost of distillate and the

distribution price of every cartridge fluctuates depending on the market and the regulatory situation of each state, but these two factors ultimately determine the potential profit in this simplified model. Table 5 shows a profit calculation based on different cost and price point levels on these two variables.

With the ongoing interest in cleaner methodologies that are not solvent based due to regulatory and safety concerns, microwave extraction poses an effective alternative to obtain terpenes with a process that is simple and easy to implement. A short processing time and the flexibility of using fresh or cured material results in higher quality terpenes. Since terpenes are one of the main vehicles that generate brand recognition for cannabis derivatives, enhancing extract quality is an excellent approach to generate a loyal customer base for growers and processors seeking to establish their product on the market.

Table 1 Table 2

Table 3Table 4

Table 5

Fresh Frozen MaterialYield 1 Unit 2 Units 3 Units 4 Units 5 Units

0.30% 41g 82g 123g 163g 204g0.50% 68g 136g 204g 272g 341g0.70% 95g 191g 286g 381g 477g

Cured MaterialYield 1 Unit 2 Units 3 Units 4 Units 5 Units

0.30% 41g 82g 123g 163g 204g0.50% 68g 136g 204g 272g 341g0.70% 95g 191g 286g 381g 477g

Flower pre-extractionCompound Mass %

δ-Limonene 0.131β-Myrcene 0.067β-Caryophyllene 0.067Linalool 0.065α-Humulene 0.030β-Pinene 0.024α-Pinene 0.013α-Bisabolol <LOQα-Terpinene <LOQCamphene <LOQCaryophyllene oxide <LOQCis-Nerolidol <LOQCis-Ocimene <LOQδ-3-Carene <LOQEucalyptol <LOQγ-Terpinene <LOQGuaiol <LOQIsopulegol <LOQp-Cymene <LOQTerpinolene <LOQTrans-Nerolidol <LOQTrans-Ocimene <LOQTotal % 0.37

Terpene extractCompound Mass %

δ-Limonene 26.959β-Myrcene 12.829Linalool 6.029α-Humulene 4.927α-Pinene 2.576Trans-Nerolidol 2.258β-Pinene 1.607Cis-Nerolidol 1.182Camphene 0.956α -Bisabolol <LOQα-Terpinene <LOQβ-Caryophyllene <LOQCaryophyllene oxide <LOQCis-Ocimene <LOQδ-3-Carene <LOQEucalyptol <LOQγ-Terpinolene <LOQGuaiol <LOQIsopulegol <LOQp-Cymene <LOQTerpinolene <LOQTrans-Ocimene <LOQTotal % 59.323

CompoundPre-extraction Post-extraction

Mass % Mass %THCa 6.043 0.954

Δ9-THC 0.164 3.905Δ8-THC 0.144 <LOQTHCV <LOQ <LOQCBDa <LOQ <LOQCBD <LOQ <LOQCBDa <LOQ <LOQCBDV <LOQ <LOQCBGa <LOQ <LOQCBG <LOQ <LOQCBN <LOQ <LOQCBC <LOQ <LOQ

Total potential THC 5.464 4.742Total cannabinoids 6.658 5.211

Distillate cost/g Total distillate cost Total 1 g vape carts

Distribution price per 1-g cart Gross revenue Profit

$5.00 $4,625.00 1000 $20.00 $20,000.00 $15,375$10.00 $9,250.00 1000 $40.00 $40,000.00 $30,750$15.00 $13,875.00 1000 $60.00 $60,000.00 $46,125

*Estimations based on 5 lbs of fresh frozen and 2.5 lbs of cured material run every 75 minutes for an 8-hour shift in a 12-L vessel (6 runs per unit)

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At High 5 Edibles in Santa Fe, New Mexico, we developed a protocol that allows us to go from plant material to highly refined cannabis oil from our first pass through our short-path distillation (SPD) head designs in three days without active filtration or winterization. State-certified, 3rd-party lab reports have illustrated results in the 90th percentile for delta-9-tetrahydrocannabinol (THC) concentration.

This process can be done with the simple application of chemistry and for less than every commercial ethanol machine. Furthermore, we use cold in a range where these machines cannot operate. Now, there is a point to focus on. With a look across the spectrum of ethanol extraction machines readily available on the market, I can’t find one that has seals that will hold the cold as low as we go in the near cryogenic range, which we euphemistically call cryogenic extraction.

In the true cryogenic range, -150°C to -272°C, theoretically, molecular motion comes as close as it can to ceasing. There are no machines that operate in that temperature range, and you don’t need one any way. By utilizing thick gauge stainless steel barrels, cold ethanol, dry ice, and liquid nitrogen, we can get there.

To start, I like to pre-freeze the plant material. Freezing tends to make things expand. In the case of cannabis, it forces the oil to extrude through the plant material. So, with that said, let’s keep the frozen material frozen and set aside. We need to prep the solvent.

The solvent (200 proof ethanol, organic, food-grade) does all the work—not a machine. The solvent will dissolve the oil

from the plant material and hold it in suspension. I prefer to have ethanol in a freezer overnight as well. Because we cannot achieve super-cold with a freezer (think of it as just a step down), I like to prep the stainless-steel cans with dry ice so that they are super-cold too.

Place bagged plant material into the cold steel can. Add liquid nitrogen and ice-cold ethanol to the can simultaneously. Keep in mind that extreme cold can have an

By Seth Oxhandler, High 5 Edibles

The Big Freeze – Using Cryogenic Extraction to Create Golden Perfection


extreme reaction. If you are not well versed in the mix ratio, you’re going to want to step back while pouring. If you’re wearing sneakers that are woven, your toes will know you are working with cold.

If you find you’re down in the -135°C to -145°C range, your solvent is cold enough that uncut

plant material will not leach out green. Your “wash,” unless the material comes from an outside grow, should have a golden hue, and not green or any other color. I have had this wash method not work to golden

perfection when I used cannabis grown outdoors because you cannot account for the particles of dirt on the material from the air.

Once you have achieved the lowest temperature possible in your facility, taking into account your geographic location, relative achievable indoor temperature (our first year, we had no air conditioning!), and the elevation, since elevation effects the boiling point, it’s time to prepare your bagged plant material for soaking. I’ve read a lot about the quick ethanol wash method. Personally, I don’t subscribe to it. Because we get our temperatures so low, we allow the plant material to soak until the wash temperature in the vessel rises to approximately -90°C. At that point, we remove the plant material.

This is when we actually walk away. Most facilities actively filter their wash at this point, utilizing everything from vacuum Buchner funnels to larger commercial designs. We utilize passive filtration at this stage for two reasons. First, we’ve found that running liquid through most filters freezes the process. After all, it’s really cold. So, we found we had to wait. Also, active filtration requires active cleaning. Passive filtration requires no additional equipment. We simply wipe out the bottom of the extraction vessels when we are done.

Decarboxylation is achieved while we turn wash into crude through vertical distillation on Day 2 of our protocol. We utilize a vacuumed-sealed design such that, when done with a run, we never break the system down for cleaning. It’s self-cleaning…almost. Because the system is sealed, we simply run a small amount of ethanol through. This both cleans and sterilizes the system for the next run. None of the regimens required for rotary evaporators apply. I admit, sometimes it feels like cheating. But that’s how we make crude for distillate.

Day 3 is probably like anyone else’s short-path refinement day. We utilize our own SPD head designs

on either a 5-L, 12-L, or 22-L boiling flask. Heads range from 4450 mm to 150 mm. The vacuum is powered by an Edwards 45i that ensures that we can pump out enough volume, so we always look forward to refinement days. Looking back at the

progression of our organic ethanol extracts, by going up in THC concentration and down

in temperature, the two points intersect to create the mark of perfection we expect to see in the lab

reports that characterize our extracts.


Visual clarity and the odor profile of hemp and cannabis extracts are the first indicators of product quality. Particulate, cloudiness, and off-odors may indicate poor processing, potential contamination, and spoilage. Because customers have more choices than ever, successful producers must emphasize product safety, quality, and consistency. Extraction processes have evolved as relevant commercial technologies, instrumentation, and sanitary-grade equipment have been adapted to the hemp and cannabis industries. While many advancements have been made regarding commercial hemp and cannabis extraction, commercial-scale extract filtration capabilities are often lagging. Some processors still rely on antiquated, labor intensive techniques, such as over-sized Buchner funnels, residential water filter housing, and home-brew filter columns, which result in inconsistent and unsatisfactory results.

An optimized filtration system requires a comprehensive look at the extraction technique, the planned throughput, and the required finished product characteristics.

An Overview of Extract FiltrationThere is no “better” or “worse” extraction process from the filtration perspective, but the extraction solvent (cold ethanol, supercritical carbon dioxide (CO2), hydrocarbon, (medium chain triglycerides (MCT) oil, etc.) significantly influences the approach to filtration.

As an example, cold ethanol extraction results in a crude extract that contains biomass residue, chlorophyll, and plant lipids. In a batch filtration process, the following filtration arrays are highly effective at refining the crude ethanol extract: (i) Coarse Filtration; (ii) Fine Filtration; (iii) Color Mitigation & Odor Correction; and (iv) Polish Filtration.

This four-stage filtration system assumes that an appropriate biomass containment bag is used during the extraction process to provide a crude extract that is free of bulk biomass and other particulate.

Stage 1 – Coarse Filtration typically uses felt bag or nominally rated cartridge filters to catch loose biomass and bind semi-solid free waxes and lipids that are drawn out during winterization.

Stage 2 – Fine Filtration removes nearly all particulate and plant derived lipids. Pleated synthetic filter cartridges and cellulose/diatomaceous earth (DE)-based media are both highly effective, depending on process scale.

Stage 3 – Color Mitigation & Odor Correction is achieved by exposure to activated carbon, which strips chlorophyll and carburized impurities from the extract to yield a transparent golden amber extract. Activated carbon also removes impurities that are responsible for unpleasant odors or tastes in final products. Note: activated carbon can bind molecules of interest, like cannabidiol or delta-9-tetrahydrocannabinol.

Stage 4 – Polishing Filtration serves as an insurance policy before solvent recovery, ensuring no particles or semi-solids reach the solvent recovery equipment. Generally, this is accomplished with an absolute-rated membrane filter, rated between 1.5- and 0.45-micron.

Note: Nominally rated filters have efficiencies below 95%. Absolute rated filters have efficiencies greater that 99.9%

Compared to ethanol extraction, hydrocarbon and CO2 crude

By Drew Stahr, Filter Products Company

Envisioning Cleaner, Clearer Extracts through Product Filtration


Figure 1 - Schematic overview of a four-stage filtration system

extracts contain less biomass but higher lipid content. The coarse filtration step can often be eliminated, leaving a three-stage filtration process that is similar to the last three stages outlined above.

MCT-derived extracts also benefit from multi-stage filtration, but an added filtration process is required due to differences in extract viscosity and density. Activated carbon and DE is added directly to the extract and homogenized to increase exposure between the extract and the filtration aides in a process known as body-feeding. These body-fed aids are then removed in the multi-stage filtration process.

RecirculationPerhaps the most overlooked area for optimization in extract filtration is recirculation of extracts through the Fine Filtration and Color Mitigation & Odor Correction Filtration stages. Recirculation allows the filtration media to shed loose substrate initially, which is re-captured on subsequent passes through the same filter. This is especially true for activated carbon medias. Recirculation also allows many medias to wet-out, swell slightly, and reach their optimal working state.

In addition to product quality benefits, properly monitored recirculation provides two compelling financial benefits: recirculation through activated carbon ensures that a greater degree of the activation potential of the carbon is utilized before filter change-out; and recirculation through one or more stages before the polish filtration stage significantly reduces the loading on the absolute-rated membrane filters.

Sizing Your Filtration SystemMatching batch and daily extraction throughput are critical when designing and selecting filtration equipment. An undersized system will result in slow throughput, increased maintenance and filter changeout time, and lost employee productivity. Conversely, an oversized system will result in increased extract loss, decreased filtration effectiveness, unnecessary capital and utility expense, and under-utilized filter media.

Fine Tune Your Filtration SystemTo get the most out of your filtration system, it must be fine-tuned at commissioning, but system calibration cannot stop there. Flow rates, recirculation ratios, media types, micron ratings, and differential pressure parameters need to be optimized as your raw materials, extraction parameters, and end-product objectives change. Ignoring these process parameters will translate to inefficiencies and waste in your production process.

About the AuthorDrew Stahr is General Manager and Chief Applications Engineer at Filter Products Company, based in Richmond, Virginia. Drew holds a BSME degree from Mississippi State University and an MBA from the University of Wisconsin. Filter Products Company is a leader in specialty filtration solutions for critical applications, including the food, beverage, and pharmaceutical industries. Filter Products Company serves the hemp and cannabis processing industry with custom filtration solutions, consumable media, and customer-specified biomass containment bags for both OEM and processor clients.

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Steam and/or water distillation is a common scalable method used to extract essential oils from aromatic plants. Essential oils, or the essences of any plant, are complex combinations of volatile aroma chemicals. These oils are used in every consumer product that contains a smell or flavor.

Examples of familiar oils and their end products include peppermint oil, popular for chewing gum and oral care, eucalyptus oil, which is traditionally used in breathing and cough/cold remedies, and tea tree and/or citronella oil, which are often used in anti-fungal and bug repellent products. Each of these powerful essential oils contain dozens of separate molecules but they’re typically made up of one or two key chemical components. For example, peppermint oil is dominated by menthol, tea tree oil by terpinen-4-ol, and eucalyptus oil by 1,8-cineole, aka eucalyptol.

To understand distillation, we must first understand the equipment and how it works. In a typical distillation unit, often referred to as a still, there are five basic but critical components: heat source, pot, condenser, separator, and container.

The heat source is anything that can boil water to generate steam. Whether the process involves steam that’s injected into the pot holding the biomass or water sitting in the pot with the biomass, heat must be generated to turn water into steam to extract the oil from the plant. In the modern United States production of commercial essential oils, the heat source is a boiler fueled by a natural gas, diesel, or gasoline. In developing countries like Morocco, for example, water is boiled using direct fire to a pot of water, producing steam for extraction. The heat source will turn water into steam. Once steam is generated, it’s either injected into a pot, sometimes called a tub or a retort, which is designed to hold the biomass to be distilled into oil.

Pots are typically stationary units where harvest biomass is collected and stored to later be brought to the still. Pots can also be a mobile unit on a trailer in order to harvest biomass

straight from the field. This pot is then taken to the still where it doubles as a distillation pot. The pot is engineered to either be heated at the bottom, in the case of water distillation, or in steam distillation, in which an opening at the bottom connects to a steam line from the boiler to inject steam into the bottom of the pot.

The pot is covered and sealed with a tight lid designed to connect to a vapor line attached to the condenser. As you can imagine, the hot water turns to steam and cooks the aromatic plant, releasing the oil and turning it into a vapor that migrates to the top of the pot, up through the vapor line, and into the condenser.

The condenser is a long winding tube made of copper or stainless steel that is submerged in a container containing cold water. Water and essential oil vapors travel through the vapor line and into the condenser to be cooled and turned back into a liquid. The finished liquid, which is now a mixture of aromatic essential oil and water, flows out of the condenser and into a receiving container.

This container also acts as a separator for the liquid oil and water to, you guessed it, separate! Oil and water are not miscible and do not like each other. Naturally, two layers are created, one oil and one water, and can be easily separated from one another, leaving the producer with finished, steam-distilled essential oil.

By Rob Brown, Extract Consultants




In recent issues, we’ve provided insight into why we’ve chosen specific people to serve on our advisory boards. Some are actively involved in the cannabis industry; others have witnessed their expertise become quite relevant in this nascent, legal industry. Our advisory boards aren’t just for show or to name drop for increased sales. For us, a solid advisory board is like a giant brain that we can tap into when we need to extract nuggets of knowledge. They are co-navigators of our mothership.

You’ll notice in any born teacher the willingness to advise and educate. To them, teaching comes as naturally as the sunrise and sunset. And the really good ones have mastered the knack of taking the complex and reducing it until it can be more readily digested by the layperson. After all, science or technology or medicine aren’t just for an elite fraction of humankind, like some famous Greek philosophers would have had us believe. [1]

In this installment, you’ll meet AC Braddock of Eden Labs, an Extraction Magazine advisory board member whose eloquent way of educating is readily apparent from just taking in a few sentences. And as you’ll soon read, her philosophy is a natural and synergistic fit for EM.

EM: What brought you to the cannabis industry? AC: We live in a world that has forgotten, dismissed, or purposely disparaged the use and protection of the natural world and our integral part in it. Focusing on plants as a source of health and well-being for humans as well as the planet forces us to look at the current science of this relationship medically, environmentally, and socially. We are out of step with our environment and each other as a species.

The pillars of the cannabis industry solve these problems. Cannabis sativa provides medicine, preventative medicine (aka nutraceuticals), clothing, shelter, food, and gives us the ability to repair the damage caused by the prison industrial system, racism, addiction to pharmaceutical drugs, war post-traumatic stress disorder, plastics, etc. We have recently discovered the

endocannabinoid system. What is new is actually ancient. The knowledge of how we are supposed to live is in us and this plant and always has been.

EM: Why do you want to dedicate your education and resources to this industry? AC: This industry is a gift for any creative businessperson, healthcare provider, social activist, the sick, and those seeking more spirituality in their lives. I have given it my all because it has the potential to give back tenfold of what we all put into it.

EM: How have you seen the industry evolve? AC: LOL, that is an entire interview in itself. A lot has happened since 1999. The “industry” began as a medical solution and was fully grounded in health and well-being. Then, around 2014, the focus changed with the lack of access to banking in order to expand. Instead of existing businesses growing organically or seeking funding like any other legal business, they had to seek outside funding sources. Many of these sources gouged the industry with exorbitant interest rates, took large equity positions for small injections of funds, pushed out owners, etc. This influx of green-rush money also debilitated businesses owned by women and people of color who could not compete for those kinds of funding mechanisms. This significantly affected how and why products were being made, which created a market with less informed consumers, low grade products and unstable IPOs [initial public offerings].

Recently, I’ve seen a transition occurring from the highly destructive business practices of early green rushers who ignored why the industry existed to a recent wave of investment from the previously risk averse and crossover from other industries based in health and wellness, farming, environmentalism, and the medical sciences. Integration with another mission-driven industry gives these industries more political power to transform regulations and laws that inhibit the growth of modern, science-driven industries like cannabis.


Meet Our Advisory Board The AC Braddock Chapter

EM: What have been the highlights, both in general, and for you personally? AC: The highlights are seeing so many people finding medical relief from healthy concentrates that came out of our systems and supporting the businesses that made those products. I personally feel grateful to look back on being an integral part of educating the industry on safe extraction technology, the reason for its importance, and the platform to have a voice in the industry’s future development. It was an honor to be nominated for this year’s MJ Biz Industry Impact Award and serve as Board Chair of NCIA and VP of The Cannabis Alliance. Being on the Advisory Board for Extraction Magazine is pretty cool, too. In the coming year, I look forward to working with other industries to bring this industry’s experience and ideals to the table.

EM: Given the industry’s rapid evolution, do you think that the regulations and legislation are going down an appropriate path? AC: No, the current regulations are a travesty. We are STILL in prohibition which is glaringly evidenced by defining a plant genus by a portion of a percent of one of its components (0.3%), by the fact there is a cannabidiol (CBD) market at all, and the Farm Bill which has divided what was once a single industry into two. It is incredible how well the propaganda on this plant was implemented and that it still thrives to this day. It is criminal that it is criminal, and entire countries of people, including our own, don’t understand why.

EM: You’re omnipotent. What would you change about the industry? AC: De-schedule! Extensive anecdotal evidence would be a pathway to medical use without a clinical trial. Every person jailed on a nonviolent charge for possession of cannabis set free and awarded reparations. Women and people of color are provided federal financial aid to start or expand a business.

EM: It’s easy to geek out over cool cannabis science. What have you learned about the cannabis plant, or a product, that really amazed you? AC: Learning in 2015 that cannabis almost completely eliminates bone marrow transplant rejection. Oh! The wonders of this plant! And we are just beginning to understand it and our endocannabinoid system.

EM: Education is obviously vital in this industry. How do you suggest consumers educate themselves? AC: I suggest the industry makes it easier for them. Who is in front of the consumer? Who has direct access? Those sectors of the industry are key in building the industry into what we envision, and not what the consumer thinks it is. Too many consumer-


References[1] Pomeroy, R. “Carl Sagan’s Problems with Plato,” Real Clear Science, posted September 17, 2018; accessed January 20, 2020.

facing companies are following an uneducated market instead of creating an educated one. The general market has no idea what a terpene is or has never heard of tetrahydrocannabivarin (THCV). They don’t understand that a high delta-9-tetrahydrocannabinol (THC) or CBD product is lacking in extended medical use and effect without the other constituents in a chemovar. This is a once-in-a-lifetime opportunity to create an industry. Create a science-based product that can stand the test of time and educate the consumer on why they want it. The consumer needs a trusted source of information. Google only goes so far! If business-to-consumer companies can create or support brands that people can trust by providing an easily digestible, scientific-based education, the entire industry will flourish. (Editor’s Note: Amen.)

EM: If you could tell someone just one thing about cannabis, what would it be? AC: What is new is ancient and if you could only take one plant to a desert island, it should be this one. It will nourish you, cloth you, shelter you, and prevent as well as treat illness.

EM: Do you foresee cannabis extraction evolving much in the future? AC: We already know how to take plants apart and put them back together again. The question is: why are we doing it? What

I hope to see is less manipulation of the plant and more focus on the plant and how it interacts with other living organisms. Then, how do we extract for a heavier concentration of certain components while keeping the chemovar primarily intact? How do we provide a pure, solvent-free product while keeping the chemovar intact? Yes, we can spike a product with single molecules, but to make products with only a few molecules of the chemovar is old science and won’t stand the test of time.

EM: What kinds of technology do you think are needed in any sector of the industry? AC: Recycling or repurposing waste across all sectors and using the plant itself for its packaging, composting, etc.

EM: You do a little bit of crystal gazing. Where do we go from here? What does 2020 bring us? AC: 2020 brings us further global legalization, a continued fight against the stigma of THC and all related THC molecules (tetrahydrocannabinolic acid (THCA), THCV, etc.), and a growing unity with natural products. Most of our future relies on politics and the upcoming elections. Our entire legal industry is based on politics, not on science. If it were based on science, we would not have a CBD market right now or a Farm Bill. We would have a new medical industry based on individualized medicine and preventative health primarily utilizing botanicals.

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Publisher MACE Media Group

CEO Celeste Miranda

Editor-in-Chief Jason S. Lupoi, Ph.D.

Authors: Jason S. Lupoi, Ph.D.

Ben Amirault Eric Farrell

Laura Haupert, Ph.D. Jessa Youngblood

Mike Hogan Kevin O’Brien

Nicco Reggente, Ph.D. Shannon Swantek

Designer Marko Nedeljkovic

Advertising: Julian Azevedo

Bradford Burgess Lisa Dodson

Contents

120806

162024

qPCR for Real-Time Microbial DetectionMicrowave Digestion for Metals Analysis in Cannabis

The Science Behind Plant-Based Solutions in Cannabis Odor Control

Everything is Data

Microorganisms in Cannabis that Can Make You Sick

28 How Does Cannabis-based Medicine Impact Brains with Epilepsy?

30 A Little Enlightenment Will Take You Far

High Speed, Automated DNA Microarray Analysis

The welcoming of spring signals a rejuvenation. Open windows, fresh flowers, fragrant terpenes in the air, beer gardens finally reopened, and, of course, spring cleaning. The old and dusty become refreshed or replaced with the new. The insides of growhouses, extraction facilities, and analytical labs are no different. Sometimes, they need an overhaul, a purge of the antiquated with a surge of novel innovation.

Many exogenous companies and industries now offer their wares to the cannabis industry, acquiescing to the fact that this industry isn’t devilish or stigmatic. Perhaps they were always there or have nascently joined the mushrooming movement. Regardless, there are lots of new options for those looking to energize their businesses with some modern conveniences.

Data is everything – everything is data. That’s not just a mantra for the analytical chemist. The need for data cannot be understated. Big mountains of data. Recent analytical data demonstrated the chemical correlation between hundreds of cannabis cultivars, proving once again that the terpenes differentiated the cultivars, and that consumers were purchasing three chemistries named nearly 400 different things. [1] Data has provided a sobering reality of labeling inaccuracies in the federally legal hemp industry. [2] And that a manufacturer’s diligence and methodical product design preserved a chemovar in a downstream cannabis concentrate formulation. [3]

The ways for obtaining data vary, each with diverse tradeoffs like speed versus accuracy, price gradients, and levels of skill needed. There are also different levels of sophistication and complexity when shopping for analytical tools. Regardless, accuracy should dominate the discussion since precisely measuring the wrong numbers does nothing for you. Just consult the bullseyes.

Defendable data is the creed of any analytical lab looking to stay open for a while. Analytical chemists are always hunting for instrumentation that provides faster, cheaper, and yet, meaningful results. Outside of the lab, on the farm, for example, the use of technology can help maximize crop and cash yields while helping farmers stay true to their roots and mission. Perhaps drones are employed to monitor plant health. Or turnkey chromatography

systems to calibrate the potency of a crop in real-time. Or a gender identification test. [4]

An in-house suite of instrumentation inside an extraction facility can help ensure product batches are compliant and the formulations are accurate prior to being startled by 3rd-party lab testing. This could include gas and liquid chromatography systems for terpene, residual solvent, and cannabinoid quantitation. Or a Karl Fischer titrator to evaluate incoming biomass for water content instead of the standard loss-on-drying ovens that measure mass loss due to baking off volatile chemical constituents like water, but also like terpenes.This issue of Terpenes & Testing Magazine regards the novel and the innovative when considering analytical instrumentation and technology. This issue continues our ongoing sampling of these achievements, including the use of real-time polymerase chain reaction (qPCR) for the evaluation of microbial contamination, a needed practice in the nascent, rather unregulated hemp-derived products industry; the use of microwave digestion for determining metal content in cannabis products; and plant-based options for controlling odor within a growhouse.

Everything is DataBy Jason S. Lupoi, Ph.D.

6 TERPENES & TESTING

Cheap, Fast, and Right – Hunting for the Rare Analytical Trinity

References[1] Reimann-Philipp, U. et al. “Cannabis Chemovar Nomenclature Misrepresents Chemical and Genetic Diversity; Survey of Variations in Chemical Profiles and Genetic Markers in Nevada Medical Cannabis Samples”, Cannabis and Cannabinoid Research, 2019, open access. [journal impact factor = N/A; cited by 2]

[2] Bonn-Miller, M. et al. “Labeling Accuracy of Cannabidiol Extracts Sold Online,” JAMA, vol. 318(17), 2017, p. 1708-1709. [journal impact factor = 51.273; cited by 62]

[3] Reed, R. “You Get the Drift?”, Terpenes & Testing Magazine, Nov-Dec 2019.

[4] DeVito, L. “A Rose by Any Other Name,” Terpenes & Testing Magazine, Uploaded December 11, 2019; Accessed February 24, 2020.

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Microbial contamination of cannabis products represents one of the most significant threats to cannabis consumers, particularly immunocompromised patients who could develop harmful infections. To protect consumers in their jurisdiction, most regulators in the United States and Canada require third-party labs to test cannabis products for certain microbes at certain levels. The targets the regulators set can vary widely.

For example, California requires labs to test for six specific pathogenic species of Aspergillus (A. flavus, A. fumigatus, A. niger, A. terreus), Salmonella, and shiga toxin producing Escherichia coli (STEC). If California labs detect any of those targets in any concentration, the sample fails. On the other hand, Massachusetts regulations set allowable limits for large groups of microorganisms, such as aerobic bacteria, yeast and mold, coliforms and bile-tolerant gram-negative bacteria.

This means that cannabis microbial safety testing methods must be precise enough to detect specific microbial targets and accurately quantify them in order to be used in labs nationwide.

Culture-based Methods Fall ShortThe classic method for detecting microbes on a sample is culture-based testing. However, most culture-based methods were not developed for use in the presence of a complex cannabis matrix, leading to some concerning results. For example, when researchers sequenced the species grown on culture-based yeast and mold tests, they found off-target bacterial species. [1]

Furthermore, since most culture-based techniques rely on viable cells growing in a medium for detection, they do not lyse plant cells open to detect endophytes. Aspergillus is an endophyte that lives inside the plant without causing apparent disease to the plant; but it can cause a deadly infection when inhaled by immunocompromised patients.

It’s clear that culture-based methods borrowed from the food industry are not specific or accurate enough to be used on

cannabis. Deoxyribonucleic acid (DNA)-based methods are much more appropriate because they can actively target DNA sequences that are unique to a certain species or family of microbes. However, not all technologies are equal. Though traditional end-point polymerase chain reaction (PCR) detection methods are an improvement over culture plating, quantitative PCR (qPCR) improves upon PCR in many ways.

What is PCR? Kary Mullis invented PCR in 1988, for which he and his colleagues won the Nobel Prize for Chemistry in 1993. PCR amplifies a segment of DNA to create exact copies in an abundance that allows for further analyses.

Each PCR assay contains short DNA sequences called primers which bind to either end of the target sequence and create a complementary strand. The primers are so specific that they will only bind to DNA that is a perfect match. Even a single DNA base difference will prevent the primers from binding. This specificity reduces the frequency of false positives in pathogen detection, a frequent problem with culture-based cannabis testing methods.

PCR is also extremely sensitive, requiring only a few DNA molecules to produce millions of copies. This amplification process is carried out exponentially over a series of heating and cooling cycles and, as a result, a basic PCR run can be broken up into three phases.

ExponentialDuring this phase, the target DNA doubles with each cycle (assuming 100% reaction efficiency). Exponential amplification occurs because all reagents are fresh and available, and the kinetics of the reaction push the reaction to favor doubling.

LinearAt this point, reagents are becoming scarce and the PCR product is not doubling each cycle.

By Ben Amirault, Medicinal Genomics

qPCR for Real-Time Microbial Detection


Plateau When the line plateaus, the reaction has stopped and no more PCR products are made.

What is qPCR and Why is it Better than Endpoint PCR?qPCR, also called real-time PCR, improves upon endpoint PCR by taking measurements throughout the PCR process. In addition to primers, qPCR assays use probes that emit a fluorescent signal each time the target DNA is copied. The qPCR instrument measures the intensity of that signal after each PCR cycle and plots it on a two-dimensional graph that resembles Figure 1.

qPCR Measures at the Exponential Phase for More Accurate QuantitationThe point at which the fluorescent signal intensity exceeds the PCR threshold is called the cycle quantification (Cq). Samples with a large starting concentration of the target DNA will cross the threshold in fewer cycles than samples with a small amount of target DNA. Therefore, a sample with a Cq value of 15 has more target DNA present than a sample with a Cq of 25.

End-point PCR techniques, such as microarray, analyze the sample in the plateau phase. However, even reactions with the same starting DNA concentration will plateau at a different point due to different reaction kinetics for each sample. Similarly, samples with different starting concentrations could plateau at the same point, thus making end-point PCR a poor

9TERPENES & TESTING

Figure 1: The three phases of a PCR reaction.

PCR

Prod

uct Linear Phase

Exponential Phase

Cycles

Plateau Phase

PCR Threshold


method for quantification compared to qPCR, which measures at the exponential phase (Figure 2).

In Figure 2, you can see that when identical samples are tested with qPCR, the reactions show tight agreement where the plots cross the threshold, making that value much more consistent. Conversely, the point at which the reactions plateau varies widely.

qPCR Results are Delivered in Real-timeBecause lab technicians can monitor the entire qPCR reaction in real time, they can get results before all the PCR cycles have completed. With end-point PCR, lab technicians still have a lot to do after PCR is complete. They must open the PCR plate, remove primers, nucleotides, enzymes, and other impurities from the DNA sample, then run the DNA on a gel or microarray to determine if the reaction was successful. In addition to being labor intensive and time consuming, the act of opening a PCR plate has the potential to contaminate a lab with amplified DNA.

qPCR Provides More Data Points Because qPCR provides measurements following every PCR, lab technicians can ensure the assay performs properly and all three PCR phases occur. If there is a problem with assay design, instrument performance, and/or user error, PCR efficiency will suffer and the DNA product will not double each cycle. As a result, the graph will look more linear than exponential, as in Figure 3. Because end-point PCR only delivers one data point at the end of the PCR reaction, lab technicians are blind to additional data that may indicate a bad test run.

qPCR Probes Add an Additional Level of SpecificityqPCR probes are like primers since they are short DNA sequences that bind to a specific target sequence. This adds an additional layer of specificity to qPCR assays because if probes do not attach, the instrument will not detect the qPCR product. This is useful when designing species-specific assays. In some cases, two qPCR assays may share a primer set, but use different probes to differentiate the species. End-point PCR does not have this additional level of specificity available.

Figure 2: Identical samples produce different quantities of reaction products by the plateau phase of PCR, while Cq values remain consistent.

Cycles

Fluo

rese

nce

(dRn

)

What about DNA from Dead Cells?Primers and probes will amplify any DNA in the sample that matches the target sequence, regardless of viability. This is what we call the “Live-Dead” problem. However, scientists, including the team at Medicinal Genomics, have developed techniques to solve the live-dead problem.

For example, Medicinal Genomics’ Grim Reefer Free DNA Removal Kit is an enzyme-based solution that is added to the sample prior to qPCR to eliminate free DNA from dead cells. This is done by simply adding an enzyme and buffer to the sample and incubating for 10 minutes. Next, lysis buffer is added, which instantaneously inactivates the Grim Reefer enzyme so it does not continue to eliminate DNA when viable cells are lysed.

Another simple solution to determine whether a qPCR failure was due to “dead DNA” is to perform a second qPCR run after a short incubation. If the microbes are living, they should replicate their genomes in 20 minutes to four hours. Therefore, qPCR results post-incubation would show growth.

Better Technology, Better Results PCR testing methods are precise and sensitive enough to detect specific microbial targets at low concentrations; however, only qPCR can accurately quantify them. Furthermore, qPCR is a simpler process than end-point PCR, and it delivers additional data points to provide assurance to lab technicians that results can be trusted.

Figure 3: Linear qPCR graphs indicate poor PCR efficiency which may be due to errors in primer design, instrument performance, or sample prep.

McKernan, K. et. al., “Cannabis microbiome sequencing reveals several mycotoxic fungi native to dispensary grade Cannabis flowers,” F1000 Research, 2016, vol. 4, 2016, p. 1422. [cited by 7]

Reference

11TERPENES & TESTING

PCR Threshold

Cycles

PCR

Prod

uct


With increased state regulations, cannabis growers are required to conduct trace metals testing to ensure safe and high-quality products. This testing encompasses various samples from growers and processors, including soils, fertilizers, plant material, edible products, concentrates, and topicals. Obtaining analytical data required to ensure quality products first starts with the crucial step of sample preparation. Closed-vessel microwave digestion offers an approach for solubilizing samples for analysis by inductively coupled plasma mass spectrometry (ICP-MS). However, there are two very different commercially available designs: rotor-based systems and Single Reaction Chamber (SRC) technology.

Several factors influence the choice for the ideal microwave digestion solution, including the types of acids best suited for elements of interest and the temperatures and pressures required for an optimal digestion. A good understanding of these parameters provides better insight to the pros and cons of each technology.

Principles of Microwave DigestionSample digestion of organic matrices like cannabis products should be carried out using reagents compatible with ICP-OES (optical emission spectrometry) and ICP-MS instrumentation. The reagents used are typically strong oxidizing agents like nitric acid (HNO3) and hydrogen peroxide (H2O2), which are extremely efficient but tend to generate large amounts of carbon dioxide (CO2) and NOx (nitrogen oxides) when the sample decomposes. The microwave system and its components will need to accommodate the high temperature required to digest different organic sample types and handle the subsequent pressures generated. For some samples, the addition of small amounts of hydrochloric acid will help stabilize some elements in solution, particularly mercury (Hg).

Rotor-Based TechnologyWith rotor-based technology, microwaves are directed onto

vessels containing the sample and digestion reagents, which are placed in a rotating carousel. The digestion process is accomplished by raising the temperature and pressure through microwave irradiation as the carousel rotates. This increase in temperature and pressure, together with optimum reagents, increases the speed of the sample’s thermal decomposition and the solubility of metals in solution. Rotor-based systems work extremely well for similar matrices by batching together samples that react similarly. Carrying out the digestion using the same microwave power, temperature, and pressure conditions ensures similar digestion quality across all positions.

To increase throughput, differently sized carousels can be used. However, when dissimilar sample matrices must be digested, productivity could be sacrificed, as each sample type needs to be batched together, which unfortunately precludes different samples being digested together in the same sample run.

Single Reaction Chamber TechnologyTo overcome the limitations of rotor-based systems, we developed Single Reaction Chamber (SRC) technology. Instead of a rotor with individual reaction vessels, the samples are put into vials with loose-fitting caps which sit in a rack that’s lowered into a larger vessel containing a baseload of acidified water. It’s this baseload that absorbs the microwave energy and transfers it to the vials in the chamber. This design allows every vial to react independently within the baseload and ensures that all samples achieve maximum temperature with pressures contained up to 199 bar. No batching of samples is necessary and any combination of sample type and acid chemistry can be run simultaneously in the same chamber. A schematic of the SRC is shown in Figure 1.

Vials with loose-fitting caps are possible because the reaction chamber is pre-pressurized with 40 bar of nitrogen before the start of the microwave program, which acts as

By Eric Farrell, Milestone Inc.

Microwave Digestion for Metals Analysis in Cannabis


a gas cap and keeps all vials independently closed. As the pressure builds, equilibrium is achieved inside and outside the vial. As a result, various vial types including disposable glass, quartz, and polytetrafluoroethylene (PTFE) or any combination of these materials can be used. The use of disposable glass vials essentially eliminates the need to clean vessels, further streamlining the sample preparation process.

Major Differences Between Rotor and SRC TechnologyThe main drawback of rotor-based technology is the required batching of similar matrices and chemistries. Furthermore, the vessels are typically made from PTFE/TFM (modified PTFE), which limits the temperature and pressure. The multiple vessel components also require additional assembly and disassembly, which could impact productivity. Depending on detection limit requirements, the vessel liners may require extra cleaning between runs.

Heavy Metals Testing of Cannabis Plants, Concentrates and Edibles by ICP-MSThe combination of closed-vessel microwave digestion and ICP-MS has become the preferred approach of cannabis testing laboratories to carry out the measurement of heavy metals. The workload, diversity of the samples, and testing protocols used will impact the optimum type of microwave system chosen. To better understand this, we took representative samples of different commercially available cannabis products and digested them by both rotor-based technology (Ethos UP with MAXI-24 High Performance (HP) rotor, Milestone Inc.) and a Single Reaction Chamber system (ultraWAVE, Milestone Inc.) and analyzed them by ICP-MS (Model 7900, Agilent Technologies).

Tables 1 and 2 show the microwave program, acid chemistries, and analytical data for samples digested using rotor-based technology, and Tables 3 and 4 for SRC technology. All samples were fortified with a spike solution containing 20 ppb of all analytes, except for Hg, which was 10 ppb.

Results and DiscussionThe results shown in Tables 2 and 4 demonstrate recoveries of all elements tested between 87% and 106% and relative standard deviations (RSD’s) well below 3%, highlighting the robustness and reproducibility of microwave digestion using rotor-based and SRC technology. A soil reference material (SRM 2711a) was also included as a quality control sample for digestions carried out with the ultraWAVE, demonstrating that a matrix very different from cannabis products could be digested in the same run using this technology.


ConclusionUsing the appropriate microwave digestion technique is critically important for the overall analytical procedure. Modern plasma spectrochemical techniques such as ICP-OES and ICP-MS will generate high-quality, trace element data but rely on an optimal sample preparation technique to achieve it.

The first part of this study demonstrates how a high pressure, rotor-based system results in full recovery and great reproducibility for the most common elements found in cannabis plants and products. Highly reactive samples are completely digested even in large amounts, ensuring reliable analysis and superior recovery of elements, including volatiles such as arsenic and mercury.

SRC technology provides an option for laboratories that might be handling more diverse and complex ranges of samples, avoiding the batching limitations of rotor-based

systems. Therefore, an SRC system offers an optimal solution to digest different sample matrices simultaneously in the same run. In addition to using minimal acid volumes, SRC technology results in 2-3 times higher throughput than rotor-based systems due to a combination of higher sample capacity, use of disposable vials, and faster cool down times, allowing for sample preparation for all cannabis-related products.

A new era of acceptance and legalization has opened new opportunities for cannabis testing laboratories. Standardization of methods for the industry will give regulators the resources and information they need to include common sense regulations to monitor and control the use of cannabis within medicinal and recreational markets. Innovative microwave digestion technologies are a perfect tool for the cannabis industry to increase productivity in performing these methods to address the market’s compliance needs.

Step Time (min) Temp 1 (°C) Temp 2 (°C) Pressure (bar) Power (W)1 20 240 60 110 15002 10 240 60 110 1500

Table 3: SRC microwave program used to digest samples

Table 1: Rotor-based microwave program used to digest samplesStep Time (min) Temp (°C) Power (W)

1 10 160 18002 15 210 18003 10 210 1800

Cannabis Product Analyte As Cd Hg Pb

Cannabis plant material% Recovery

% RSD (n=3)90.31.8

93.40.9

93.82.1

96.71.4

CBD oil% Recovery

% RSD (n=3)91.82.8

88.92.7

101.21.6

94.22.9

Cannabis vape cartridge% Recovery

% RSD (n=3)92.71.5

90.82.1

94.41.1

106.51.4

Cannabis salve% Recovery

% RSD (n=3)94.22.5

94.81.4

103.51.3

90.22.1

Cannabis cookie% Recovery

% RSD (n=3)91.62.8

94.31.0

93.81.3

97.51.4

Cannabis gummy bear% Recovery

% RSD (n=3)98.12.1

97.10.6

97.70.9

96.91.9

Table 2: Recovery of 20 ppb Pb, As, and Cd, and 10 ppb Hg spiked into cannabis products, digested by rotor-based microwave technology, and analyzed by ICP-MS. Reagents used for digestion: 5 mL concentrated nitric acid, 1 mL hydrogen peroxide, ~30%.

Cannabis Product Analyte As Cd Hg Pb

Cannabis plant material% Recovery

% RSD (n=3)91.71.9

93.02.1

98.72.1

88.32.6

CBD oil% Recovery

% RSD (n=3)95.81.8

98.52.3

97.61.1

89.72.2

Cannabis vape cartridge% Recovery

% RSD (n=3)90.81.1

87.32.0

91.81.2

92.01.5

Cannabis salve% Recovery

% RSD (n=3)95.80.3

91.51.1

94.31.4

95.32.2

Cannabis cookie% Recovery

% RSD (n=3)92.82.8

93.80.7

96.11.3

93.31.4

Cannabis gummy bear% Recovery

% RSD (n=3)90.22.1

89.52.0

94.11.0

91.82.2

Soil (SRM 2711a)Leachable Conc. (mg/kg)

% Recovery % RSD (n=3)

8990.4*

2.1

4794.1*

1.9

7.498.7*

1.6

130093.3*

1.1

Table 4: Recovery of 20 ppb Pb, As, and Cd, and 10 ppb Hg spiked into cannabis products, digested by SRC technology, and analyzed by ICP-MS. Reagents used for digestion: 4 mL concentrated nitric acid, 1 mL concentrated hydrochloric acid.* Note: recovery based on leachable certificate values.


As the world celebrated the beginning of a new decade, many residents in Illinois began their first day of 2020, forming long lines waiting to purchase the state’s first legal recreational cannabis.

Illinois is now the eleventh state to legalize recreational sales of cannabis, and cannabis’ first day of legality was a massive success. According to the Chicago Tribune, The Illinois Department of Financial and Professional Regulation reported more than 77,000 transactions netted nearly $3.2 million in cannabis product sales on the inaugural day and nearly $11 million in the initial five days. After the first full week of legal recreational sales, Illinois has experienced widespread supply shortages.

As more states ramp up legal cannabis production, it’s important that their new cultivation businesses learn from the challenges that previous states have had. One primary concern that is often overlooked is odor control.

Growing OdorsStrong odors produced by cannabis cultivation are challenging to manage. These pungent, skunky odors are rarely subtle and can blanket entire residential areas. Some odors from cannabis farms have been detected more than a mile from their source.

Complaints of cannabis odors have increased in some areas by as much as 87% since growing cannabis became legal. In Denver, for example, 30% of odor complaints are cannabis related. [1] Local government officials and clean air agencies are playing catch up, enacting laws and issuing hefty fines to limit odors.

Washington State Senator Judy Warnick recently introduced a bill to create a state task force to evaluate the problem of strong aromas associated with cannabis production and processing. The seven-member task force will consist of representatives from various State agencies, County and City association members, and a representative from the cannabis industry.

However, controlling odors is easier said than done for new cultivation facilities and businesses. Many odor control solutions require sophisticated engineering, expensive permitting, or costly equipment, including multi-million dollar ventilation systems.

Additional Cannabis Odor Control Challenges:

Most municipalities now restrict how commercial cannabis grow operations handle odors.Large-scale ventilation systems that pump untreated air outdoors are

prohibited in some urban areas. Industrial filtration systems can be costly to install, operate, and maintain.Enclosing a greenhouse can cause overheating and harmful rising humidity. Some odor solutions require the use of water to distribute, adding additional costs and equipment.Multiple partners are often needed for equipment, materials setup, and maintenance.

Plant-based Solutions – Formulated for Cannabis Odor ControlWhere more cumbersome methods and masking agents have failed to adequately control cannabis odors, plant-based solutions, like Ecosorb® CNB 100 by OMI Industries, offer natural odor removers designed for controlling cannabis plant odors.

Ecosorb CNB 100 is a blend of purified water, surfactant, and natural plant oils that eliminates odor-causing chemical compounds from cannabis – namely monoterpenes and sesquiterpenes. Ecosorb consists of a proprietary formulation of several essential oils and food-grade surfactants that are biodegradable and safe for people, animals, and plant life. The versatility of essential oils for odor control has made them an acceptable and effective technique.

By Laura Haupert, Ph.D., OMI

The Science Behind Plant-Based Solutions in Cannabis Odor Control

Mode of OperationPlant-based solutions operate by weak electrostatic bonding, gas-phase solubility, and acid-base reactions. The plant-based solutions are applied through various methods to eliminate odors.

The oils are mixed with water and sprayed into the air or onto odorous materials. It is believed the mix in these droplets separate and the oils form a thin film over the water droplet itself and inside the droplet. The exterior “skin” formed by the oils creates an electrostatic charge over its outer surface. This charge attracts the odor molecules onto and into the droplet. Although the water droplet is quite minute, it is still large enough to capture the malodor molecules and affect the neutralization process.

Odor neutralization is accomplished when the odor molecules are fully enveloped by and in the plant-based molecules. This happens through natural plant oil and water reactions.

As an example, α-pinene is a volatile organic compound (VOC) that is a terpene — an odor-causing compound in cannabis. α-pinene is present in other plants, including pine, rosemary, frankincense, cypress, juniper berry, and orange. Some of the essential oils extracted from these plants are effective at attracting and neutralizing odor molecules from cannabis because of their similar chemical makeup.

Using this knowledge, natural odor removers can be specifically designed to eliminate the odorous chemical compounds in cannabis. Since a blend can be engineered for broad-spectrum odor control (it can remove a larger range of odorous compounds), the blend will work better and be more universal than other methods.


Reference[1] Haupert, L. “Smell Science: Addressing Odor Issues in Cannabis Production,” Grow Opportunity, 2018, Retrieved from https://omi-industries.com/wp-content/uploads/2018/10/GrowOp_DL-article.pdf.


Delivery MethodsPlant-based odor neutralizers can be used as constituted or diluted with water based on the delivery method. Distribution systems should be placed where the exhaust exits a growing facility. This eliminates odors before they become concentrated in residential areas.

Note: Ecosorb CNB 100 should not come in direct contact with the cannabis plant, water, or growing soil.

OMI manufacturers dispersion equipment using vapor phase technology (no added water needed). Through vapor phase equipment, dry odor neutralizer is pumped through a perforated pipe distribution system. These systems deliver the neutralizer at rates from 130 cubic feet per minute (CFM) or as high as 2,400 CFM. This makes the dispersion technology adaptable for different locations and applications. Odor neutralizers can also be delivered through misting systems from other manufacturers.

Consider Odor Control from the StartIn most municipalities where cannabis production is legal, there are city or county ordinances that require odor control program

approval before permit approval. When companies apply for permits, they have to draft their game plan, and that plan must include odor mitigation – how it will work, the type of system, and the science behind how it functions.

With odor control becoming standard in many application processes, cultivators must take it seriously or risk citations for noncompliance. Their ability to respond to these citations can easily determine which facility can get off the ground.

Providers of cannabis odor control solutions can work with cultivators and their communities to help alleviate odor issues. For instance, we provide information to growers and local regulators on how our product functions and why it’s safe for employees, work environments, and the surrounding community.

As cannabis production is introduced into more communities, it’s important to consider proactive odor control from a regulatory compliance and community conflict standpoint. As a business owner, ensuring you’re keeping the air stream odor-and chemical-free is being a proper steward of the land your business operates on.


FA C I L I T Y D E S I G N & E N G I N E E R I N G

C O D E C O M P L I A N C E

E Q U I P M E N T S E L E C T I O N , P R O C U R E M E N T, A N D L AY O U T

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Many people are concerned about what kinds of microorganisms might be lurking in their cannabis products. Cultivators and downstream manufacturers are trying their best to understand how these contaminants, or “bugs,” may be getting into their products. Testing labs are working hard to properly screen and identify the worst offenders, even when they are not required to do so by the local jurisdiction. Ultimately, everyone is chasing the same goal: safe cannabis for consumers.

Below are ten types of microorganisms that can be found in cannabis.

10. Mucor spp. This ubiquitous mold is found almost everywhere and rarely presents a problem for most of us. The strains of Mucorales that have the potential to make you sick are most often of the genera Rhizopus, Mucor, and Rhizomuco. A cancer patient diagnosed with this fungal infection died earlier this year and the infection was said to be associated with medicinal cannabis use. [1]

While a mucormycosis or zygomycosis infection is rather rare, the consequences can be very dangerous – especially since those most susceptible to the toxic mold are immunocompromised patients.

9. Penicillium spp. We can thank certain species of this mold genus for saving millions of lives through the advent of the antibiotic penicillin that this organism produces.

However, there are other members of this mold genus that are known as

mycotoxin producers and pathogenic. The most important mycotoxins from Penicillium are ochratoxin A (OTA) and patulin. Many jurisdictions require

screening for the mycotoxins but the parent organism remains largely unchecked. Mycotoxins can co-extract into cannabis concentrates.

The effects of these toxins on human health have not been thoroughly studied; however, OTA has been linked to kidney disease. OTA’s effect on animals is well known and has been proven to be a potent renal carcinogen. [2] Let us not forget, humans are not the only patients and consumers here; the sale of cannabidiol (CBD) to the pet market has grown over 400% since 2017. [3]

8. Klebsiella spp. This is another organism that has been isolated from cannabis. [4] This bacterium is present in soil and sometimes used in agriculture to increase crop yields. Klebsiella bacteria also live in the digestive tract. Patients infected with the bacteria can pass the disease on to others, creating a major concern in medical settings. Infections can be life-threatening for many long-term care patients who may have weakened immune systems or are on long-course antibiotic treatments. Infection can lead to pneumonia, bloodstream infections, wound or surgical site infections, and meningitis.

7. Clostridium botulinum This killer bug is of greatest concern

when talking about edibles such as cannabutter (cannabis-infused coconut oil), peanut butter, or other dense edible material that allows for the

formation of anaerobic, or low oxygen, pockets. This bacterium goes a step

further when the conditions for survival are poor - it releases spores. The spores of C. botulinum have hard coatings which allow them to remain dormant and survive for years. This bacterium is found in soil and should be a

By Jessa Youngblood, Hardy Diagnostics


Microorganisms in Cannabis that Can Make You Sick

major concern for those producing certain cannabis-infused edibles.

6. Pseudomonas aeruginosaThis is a pathogen that thrives in moist soil, plants, and water. A cannabis cultivation facility provides the ideal conditions for proliferation. The problems from this bacterium are wide-ranging and could gravely impact the most vulnerable patients. A case of necrotizing pneumonia was associated with the regular use of cannabis in a water pipe. [5]

The medical issues resulting from a P. aeruginosa include soft tissue infections, bone and joint infections, gastrointestinal infections, respiratory infections, and more. [6] As with most microbial offenders, hospital settings and immune-compromised patients are at the highest risk, especially acquired immunodeficiency syndrome (AIDS) and cancer patients. Some of these patients are looking to cannabis products as part of their treatment plan, highlighting the importance of proving the medicine to be safe. This bug is screened for in a small handful of localities, but it makes sense to broaden the scope of testing.

5. Staphylococcus aureus There is some encouraging early evidence that CBD may help in the battle against drug-resistant bacteria. [7] That said, S. aureus is transmitted from humans onto finished goods.

Methicillin-resistant S. aureus (MRSA) as a foodborne illness has been increasing. [8]

Infections from S. aureus can cause severe food poisoning and gastrointestinal illness. This is of special concern for prepared or ready-to-eat foods. When finished goods are packaged and stored for sale, GMP (good manufacturing practices) environments must be strict and vigilant. While an ingredient might pass initial testing, are critical points in production processes that could introduce contamination understood?

4. Salmonella spp. Outbreaks associated with Salmonella in cannabis have occurred. [9] This pathogen is regularly evaluated in almost every regulated market that requires cannabis testing. Salmonella can

be spread to plants through agricultural processes or through improper handling of other ingredients when preparing edibles. A run-in with Salmonella can result in food poisoning symptoms and severe dehydration. Some serotypes of Salmonella enterica are the cause of the life-threatening illness Typhoid fever. Cannabis labs should be looking for Salmonella in dried cannabis flower and edibles.

3. Escherichia coli E. coli is found in the gut of many animals and easily contaminates soil and water. Natural fertilizers applied to the plant can also be inviting to this bug. Those who become ill from E. coli may experience anything from gastroenteritis to respiratory infections. Certain strains of E. coli produce a toxin that causes severe diarrhea and can, in some cases, cause kidney damage. [10] The presence of E. coli on cannabis products has been confirmed by several independent testing labs, highlighting the importance of testing for E. coli in flower products and edibles.

2. Listeria spp. Listeriosis is an infection caused by the bacteria Listeria monocytogenes. Listeria may infect many systems in the body, such as

the brain, spinal cord membranes, or the bloodstream. It can also lead to

spontaneous abortion in pregnant women. Listeria bacteria can be found in water and soil, both of which are necessary when cultivating cannabis.

Listeria is a common pathogen of concern in food testing and is carefully screened for in many food processing plants. This pathogen also must be screened for in medicines provided to cancer, transplant, and AIDS patients who are at a higher risk for contracting the disease.

1. Aspergillus spp. Aspergillus molds are the most common pathogens of concern in cannabis products. Aspergillus is a potentially pathogenic mold that can cause a lung condition known as Aspergillosis. Aspergillosis is responsible for at least one documented death of an immunocompromised cancer patient who was using cannabis products. [9]


[1] Stone, T. et al. “Pulmonary Mucormycosis Associated with Medical Marijuana Use.” Respiratory Medicine Case Reports, vol. 26, 2019, p.176-179. [journal impact factor = 0.78; cited by N/A]

[2] Bui-Klimke, Travis R, and Felicia Wu. “Ochratoxin A and Human Health Risk: a Review of the Evidence.” Critical Reviews in Food Science and Nutrition, vol. 55(13), 2015, p. 1860-9. [journal impact factor = 6.704; cited by 80]

[3] LaVito, A. “Pets are the hot new cannabis customer as owners use CBD to ease pain and thunderstorm anxiety,” CNBC, posted May 5th, 2019; accessed January 19th, 2020.

[4] Thompson III, G. et al. “A Microbiome Assessment of Medical Marijuana,” Clinical Microbiology and Infection, vol. 23(4), 2017, p. 269-270. [journal impact factor = 6.425; cited by 15]

[5] Kumar, A. et al. “Marijuana “Bong” Pseudomonas Lung Infection: a Detrimental Recreational Experience,” Respirol Case Rep., vol. 6(2), 2018, p. e00293. [journal impact factor = N/A; cited by 2]

[6] Fujitani, S. et al. “Pseudomonas aeruginosa,”

Antimicrobe, open access.

[7] Appendino, G. et al. “Antibacterial Cannabinoids from Cannabis sativa: A Structure-Activity Study,” J. Nat Prod., vol. 71(8), 2008, p. 1427-1430. [journal impact factor = 4.257; cited by 204]

[8] Crago, B. et al. “Prevalence of Staphylococcus aureus and Methicillin-Resistant S. aureus (MRSA) in Food Samples Associated with Foodborne Illness in Alberta, Canada from 2007 to 2010,” Food Microbiol., vol. 32(1), 2012, p. 202-5. [journal impact factor = 3.451; cited by 51]

[9] Taylor, D. et al. “Salmonellosis Associated with Marijuana — A Multistate Outbreak Traced by Plasmid Fingerprinting,” N Engl J Med., vol. 306, 1982, p. 1249-1253. [journal impact factor = 70.670; cited by 139]

[10] Center for Disease Control, “E. coli and Food Safety,” cdc.gov, accessed January 23rd, 2020

[11] Gargani, Y. et al. “Too Many Mouldy Joints – Marijuana and Chronic Pulmonary Aspergillosis,” Mediterr J Hematol Infect Dis., vol. 3(1), 2011, p. e2011005. [journal impact factor = 1.586; cited by 28]

References

Aspergillus has four main species of concern: Aspergillus niger, Aspergillus flavus, Aspergillus terreus, and Aspergillus fumigatus. Many regulated states require testing for Aspergillus but only a few require speciation of the “Big Four.”

These molds are especially troublesome for pediatric and immunocompromised patients. They produce mycotoxins that will persist even if the mold has been killed in the finishing process. Little is known about the virulence of the mycotoxins or how to remediate them, but there are technologies being developed to help us remedy extracts, such as supercritical fluid chromatography.

I consider these the top ten organisms most likely to cause declining health in patients using cannabis products. I’ve had the privilege of working with cannabis labs across every major market including Canada. The labs and growers I have worked with have been open about what they are finding and where. Our industry needs to be sure we are screening the right products for the right organisms in the right way and at the right time. As advocates for cannabis as medicine,

we must be vigilant when it comes to organisms that could compromise the health of patients and consumers.

Jessa Youngblood is the cannabis industry specialist at Hardy Diagnostics. She sits on the AOAC CASP committee for Microbial Contaminants Working Group as well as the NCIA Scientific Advisory Committee. Jessa has a passion for safe access to cannabis medicine and regularly leads webinars and training to support the development of cannabis microbiology testing programs across the US and Canada.

Hardy Diagnostics has been in business since 1980 and is 100% employee owned. The company is ISO 13485 certified and manufactures a complete line of microbiological testing products. With over 9,000 laboratory customers across a broad spectrum of markets, including the clinical, pharmaceutical, and food segments, we understand the environmental monitoring and finished product testing concerns of the cannabis industry. Hardy Diagnostics is uniquely equipped to support and train your quality assurance technicians with all their microbiology testing needs.


12 Numbered Arrays per Slide (9mm spacing)

Glass substratePTFE

barrier

Slide Dimensions

25mm x 75mm x 1mm

Barcode


Enabling Network-Based, National-Scale Microbial Screening Although the primary focus of ongoing commercialization at PathogenDx is to develop and optimize DNA Microarray Tests to detect all bacteria and fungi required by state cannabis and hemp regulation, the long-term goal is to do so in a way that is simple, and cost-effective, is simple, cost-effective, and can be coupled with software that is powerful enough so microbial testing could be used as the basis for national-scale network deployment. In such a network, microbial contamination in Cannabis, Hemp, Food, & Environmental Screening will be monitored every day from air, water, and surfaces in real time at dozens of locations at many sites in the network. Here, we describe the optical and numerical analysis technologies serving as the basis for this vision.

1). DNA Microarray Test to Expedite Simple, Rapid (Fluorescence) Data CollectionA DNA microarray comprises a large panel of different synthetic DNA molecules laid out as a small 2D matrix (i.e., a microarray). In that microarray, each matrix element, called a “probe spot,” is a different DNA, always tagged with a red fluorescent dye designed to be specific for (and will bind to) the DNA in a particular microbe of interest (e.g, Escherichia coli or Aspergillus) (Figure 1).

During the collection and processing of microbes from a sample, the microbial DNA is processed so that it’s abundant enough to be detected and becomes modified with a (green) fluorescent tag. The resulting small volume of fluorescently tagged microbial DNA from each sample is then flooded onto the surface of one of the microarrays, where it can bind to the underlying synthetic DNA probe spots.

For example, if the sample contained E coli, binding occurs at the microarray probe spots designed specifically for E coli. If the sample also contained Salmonella or Aspergillus, binding would also occur on the Salmonella or Aspergillus probe spots (and so on), generating a pattern of (green) fluorescence on the microarray. The resulting 2D pattern is thus a direct measure of the set of microbes which contaminated the sample.

2). Tools for High Speed Fluorescence Data AnalysisBecause the “up-stream” method of microbial sample collection is fast and simple, users can process up to 100 samples per day, generating 100 microarrays worth of fluorescent pattern data, which in aggregate, comprise 100 x 180=18,000 DNA binding tests.

By Mike Hogan and Kevin O’Brien, PathogenDx

High Speed, Automated DNA Microarray AnalysisA Cloud-Enabled, National-Scale Microbial Testing Network

Figure 1: Manufacturing of 12x15 microarrays (12 each) in a standard 1” x 3” glass slide format.

5.25mm

4.125mm

After hybridizationBefore hybridization

To convert fluorescent microarray data analysis into a routine lab process, PathogenDx developed AuguryTM, a software suite which manages the overall process of raw fluorescence image data collection, quantitative image processing, compilation into microbial data and, finally, the reporting, archiving and sharing of the microbial contamination data.

The total time required for imaging all 48 arrays is approximately six minutes, with the resulting-two color data for all 48 arrays stored as a series of 16bit TIFF files which also include the ordinary 1D ink bar code associated with each 1”x 3” slide and well identification on each slide obtained from the image of the well-number embossed at their side in the PTFE (polytetrafluoroethylene) barrier (Figure 2). DNA Binding Data CompilationOnce Augury collects and averages DNA binding data from all 180 spots, the data are used to detect the presence of or to quantify the microbes included in the microarray assay. For “Detection,” the background corrected and averaged

(green, CY3) binding data for any sample being tested is compared to a stored reference threshold value, which are obtained statistically by analyzing thousands of hybridized and unhybridized probes of each type. If the averaged binding data exceeds the threshold, it is recorded as “Detected” and that outcome, along with the raw binding signal data, is stored in a report for the sample under question The Relative Fluorescence Units (RFUs) of each probe are measured and the identity of the probe is determined by consulting a look-up table of all the probes in the array, listed by row and column. This information is then compiled into a report file that is copied into the customer’s Dropbox folder (Figure 3): tools for cloud-based data processing and network-based archiving and delivery. Augury analysis is performed as a cloud-based application, and data are uploaded through a secure network connection. Data from each site is processed through a centralized server with system administration in Tucson (Figure 4). The network of state-regulated PathogenDx labs is growing quickly: Red


Figure 2: Automated spot finding, background correction, and binding signal collection.

250pixels/spot

150-200µm

370µm

The red channel is used to determine the locations and sizes of all the spots. These data are stored as a set of pixels with X and Y values. These specific pixels are then examined on the green channel. The RFU (Relative Fluorescence Units) of any given spot is defined as the median value of all the pixels that make up that spot.


corresponds to 2018 deployment, while green corresponds to 2019 deployment. State regulations for cannabis, hemp, and agricultural testing required by each lab do not allow for data sharing among network sites. Thus, the present test lab network is a simple bi-directional contract between each testing lab and PathogenDx. For 2020, the goal is to modify Augury software to allow regulated data sharing among all sites, thus enabling full network functionality, ideal for the network to be used for Cannabis, Hemp, Food, and Environmental Screening.

SummaryThe environment, especially the microbial load in the air, water, and surfaces around us, has undergone substantial change. Historically, a strong immune system and the availability of antibiotics had mitigated much of the risk of microbial infection from food, water, and our surroundings. But many people now harbor weakened microbial immunity (AIDS, transplantation, chemotherapy, malnutrition) and there is an even greater biological danger due to the new prevalence of multi-drug resistant bacteria and fungi. [1,2] In a recent report, the Centers for Disease Control stated the world has entered the “Post-antibiotic Era”. [3] In that frightening new world, the challenge to those who develop technology is to find ways to monitor large panels of microbial contamination quickly, in detail, and in the field, so that contamination may be characterized and removed.

We believe the tools described here for High Speed, Automated DNA Microarray Analysis will enable the possibility of National-Scale Microbial Testing Networks as a first line of defense to customers and regulators in

the Cannabis, Hemp, Food, and Environmental Screening marketplace, in the face of the clear and present danger imposed by the ”Post-antibiotic Era.”

Figure 3: How microbial data are calculated and reported.

CFU /– Colony Forming Unit/GramBTGN - Bile-tolerant Gram NegativeENT/COL – Enterobacteriaceae/ColiformTAB – Total Aerobic BacteriaTYM – Total Yeast and Mold

References[1] Waclaw, B. “Evolution of Drug Resistance in Bacteria,” Adv Exp Med Biol., 2016, vol. 915, 2016, p. 49-67. [journal impact factor = 2.126; cited by 8]

[2] Montoya M. et al. “Candida auris: The Canary in the Mine of Antifungal Drug Resistance,” ACS Infect Dis., 2019, vol. 5(9), 2019, p. 1487-1492. [journal impact factor = 2.126; cited by 1]

[3] Center for Disease Control, “2019 AR Threats Report, CDC’s Antibiotic Resistance Threats in the United States, 2019 (2019 AR Threats Report)” https://www.cdc.gov/drugresistance/biggest-threats.html

Figure 4: A cloud based AuguryTM lab analysis network


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Epilepsy is a condition that scientists have heavily researched, and multiple lines of evidence indicate that cannabidiol (CBD) is an effective treatment for the brain disorder. [1] What is still unclear, however, is how CBD actually works. While researchers know that CBD can significantly reduce epileptic seizures, scientists are still unsure how the treatment impacts the brain to deliver ideal results.

A 2008 study published in the Journal of Neuroscience could tell us how CBD reduces epileptic seizures. To conduct the research, scientists received access to several brains from deceased people with and without epilepsy. [2] After separating and analyzing the grey matter, the researchers found that epileptic brains had substantially fewer CB1 receptors, an essential component of the endocannabinoid system, in the hippocampus. [2]

Knowing that most seizures manifest in the medial temporal lobe where the hippocampus is located [3], scientists have been reasonable in suspecting that increasing the bioavailability of CBD in the body (and subsequently the hippocampus) might help an epileptic brain nominally function even though it’s missing CB1 receptors that the body should naturally produce. By

increasing the presence of CBD in the hippocampus, the underlying circuitry could theoretically go into overdrive and function the same way it would if it had a normal number of CB1 receptors. It’s like a high-octane fuel in a 4-cylinder engine to help it compete with a V6.

This theory means that epilepsy is potentially linked to malfunctions in the endocannabinoid system that cannabis-based treatments can fix, which is why

CBD can be so effective.But to discover other variables that could play a role in CBD’s ability to decrease epileptic seizures, cannabis

By Nicco Reggente, Ph.D.


How Does Cannabis-based Medicine Impact Brains with Epilepsy?

researchers and physicians must take a few steps.

1. Educate at all levels Studies have shown that physicians have a reluctance toward recommending or prescribing cannabis, owing to their doubts over the evidence that it has significant health benefits and their concerns about adverse side effects. [4] However, to truly understand how CBD reduces epileptic seizures, we need regular seminars and courses that explain how cannabinoids and the endocannabinoid system work. Without more education, the cannabis research community can’t develop more theories that explain why CBD reduces epileptic seizures.

Recently, the University of Maryland developed a graduate program that focuses on teaching students about the medicinal properties of cannabis. Taking monumental steps like this will help provide the foundational knowledge that’s necessary to understand how CBD impacts our bodies and brains and lead to further discoveries. 2. Tailor treatments to individualsAs we move into a new era of personalized medicine, cannabis treatments should follow suit. As the industry strives to learn more about CBD’s impact on epilepsy, physicians need to tailor prescriptions to the individual to see which remedies work based on a person’s genetics and medical conditions.

Doctors describe this step as “Cannabis 2.0”, a movement that focuses on

prescribing specific products based on a person’s particular condition and genetic variations. By taking this step, we can start to thoroughly study how certain remedies like CBD treat brain disorders such as epilepsy when genetics play a role.

More theories will continue to endeavor explaining how CBD reduces epileptic seizures, but we still need additional research and a personalized approach to cannabis to get a complete picture. As scientists continue to conduct studies, one theory we can consider is the idea that epilepsy is associated with defects in the endocannabinoid system, making CBD a potential and ideal solution.


References[1] Press, C. et al. “Parental reporting of response to oral cannabis extracts for treatment of refractory epilepsy,” Science Direct, vol. 30(1), 2015, p. 49-52 [journal impact factor = 2.378; cited by 175]

[2] Ludanyi, A. et al. “Downregulation of the CB1 Cannabinoid Receptor and Related Molecular Elements of the Endocannabinoid System in Epileptic Human Hippocampus,” J. Neurosci. vol 28(12), p. 2976-2990. [journal impact factor = 6.074, cited by 20]

[3] https://www.epilepsy.com/learn/types-epilepsy-syndromes/temporal-lobe-epilepsy-aka-tle

[4] Kondrad, E.; Reid, A. “Colorado Family Physicians’ Attitudes toward Medical Marijuana,” J. Am. Board Fam. Med. vol. 26(1), 2013, p. 52-60. [journal impact factor = 2.511; cited by 135]

Welcome readers to the next edition of our fireside chats with our wonderful advisory board members. This chapter regards Shannon Swantek, Principal Scientist and CEO of Enlightened Quality Analytics, a consulting firm dedicated to helping companies navigate and, indeed, excel through quality management and technical consulting. Shannon has brought over a decade of experience regarding laboratory analysis, compliance, ISO certification, and quality management to the cannabis industry, which is relieving, given the carousel of regulatory additions and changes.

T&T: What brought you to the cannabis industry? What were you doing prior? Why do you want to dedicate your education and resources to this industry?

SS: Prior to working in cannabis, I was a lead assessor for the Oregon Laboratory Accreditation Program (ORELAP). Our program held 200 international labs in compliance with an ISO-based standard that contained more stringent requirements than those contained in ISO/IEC 17025. I had moved to Oregon to be closer to my brother who was an Oregon Medical Marijuana Program cultivator and he always told me before he passed that the industry would need chemists when it was legal for adult-use. Due to his influence, when ORELAP was approached to be the accrediting body for the cannabis labs and I was approached to be on the Rules Advisory Committee (RAC), I jumped at the opportunity to be the Cannabis Laboratory Accreditation Manager.

I spent the next year as the main point of contact for the laboratories. We held training on the ORELAP standard

requirements, and I drafted policies to ensure that the special nature of cannabis could still fit within this national environmental set of standards, the 2009 TNI EL Standards. I partnered with Phenova to create the first “in-matrix” Cannabinoid Proficiency Testing program in the country and worked with the laboratories on blank matrix and alternatives to certain required quality control that needed revision due to the Schedule I limitations on reference materials.

As I worked with several agencies, including the Oregon Department of Agriculture (ODA), the state attorney general’s office, and the governor’s office, to draft the first comprehensive laboratory testing and oversight legislation in the nation, I realized that the cultivation and extraction communities were not aware of the impending implementation of more representative sampling amounts and exhaustive pesticide and solvent lists. I began speaking at processor and other industry group meetings to inform them of upcoming changes and to take their feedback to those making the changes.

Working to support both sides of the newly regulated community was incredibly challenging but experiencing people’s passion for this miraculous plant and medicine on a daily basis embedded in me a commitment to its success and the success of natural medicine being accepted and studied to improve lives on so many fronts that we have yet to explore.

T&T: In your time in the industry, how have you seen it evolve? What have been the highlights, both in general and for you personally?

SS: The industry has changed drastically since Colorado and Washington pioneered adult-use legalization. As each state and region followed suit, I have seen states continue to struggle with regulations involving product safety, product tracking, and other challenges that come with trying to control a federally illegal substance.


A Little Enlightenment Will Take You Far


T&T Advisory Board Interview – The Shannon Swantek Edition

Due to these struggles, the silver lining has been the industry itself starting to become more informed on a national level and more involved in the state regulatory processes. I have seen a market that we called “the wild west” a few short years ago mature exponentially. The industry has gone from trusting word of mouth and reputations formed in the unregulated market, to one of increasing awareness of good practice and accountability, not only to shareholders and investors, but to customers and patients that use the products.

My personal highlight is the recognition of the license-holders of the need for the industry to step up and demonstrate credibility to their communities. People, especially those newly entering the market, are focused on things that weren’t even on the radar several years ago, such as compliant cGMP (current good manufacturing practices) and quality management systems that capture product and organizational risk. I have seen my esteemed scientific colleagues volunteer precious time to collective efforts to standardize laboratory methods to drive more consistent and accurate data. Those efforts are going to make it possible for us to springboard into the next stage of legitimization and federal de-scheduling by giving us the information necessary to make data-based decisions.

T&T: Very well said. Given the industry’s rapid evolution, do you think that the regulations and legislation are going down an appropriate path?

SS: I am looking forward to the day when states no longer struggle to study and regulate this by themselves. I hope that when federal regulations take over, we can go, without extra hampering, to established systems such as cGMP that are already being used in dietary supplements, food, and pharmaceuticals, to capture our product risk throughout the entire process – from farm to product.

T&T: You’re omnipotent. What would you change about the industry? SS: If I was omnipotent, I would go back in time to the days when people in power made decisions that kept this miraculous plant from us for self-serving reasons, like the cotton money driving the decision to outlaw hemp and the Reefer Madness propaganda driving the inequity of arrests and the rise of the prison complex.

I would change the way our industry recognizes itself. People would open their eyes and see their community fused in a

powerful movement instead of the “competing” factions we see now in this industry. We’d show respect and support to our roots and the original pioneers and welcome with open arms our new pioneers in the science and medicine of the

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plant that could not work during the early times. We’d take care of the patients who drove the first medical movements instead of making their medicine more expensive.

Regulating agencies have the resources and time needed to become educated on all aspects of this unique and complex industry in order to keep regulations simple. They would allocate the revenue to address the inequities perpetuated from years of using this plant to arrest minorities and to educate the license-holders and the public on what we are learning.

T&T: It’s easy to nerd out over cool cannabis science. What have you learned about the cannabis plant, or a product, that really amazed you?

SS: The complexity of the matrix blows me away. My favorite thing that I have learned about cannabis, though, regards its uniqueness as a medicine – the ensemble effect, the necessity of a rich chemical profile, and the difficulty of handling a plant in the laboratory when the target analytes are so varied.

The advances I am seeing in laboratories as they strive to maintain sample integrity during sampling, storage, and homogenization have been very creative. This has opened the door for exploring natural medicines with these new techniques. I geek out when I meet people who are not only studying cannabis, but other natural medicines, such as myco-compounds and yeasts, side-by-side, when we did not always have the opportunity or funds to study these medicines before.

T&T: Education is obviously vital in this industry. How do you suggest consumers educate themselves?

SS: This is such a hard question because there is so much misinformation out there. I generally tell them to educate themselves through publications such as this and to ask questions of their product manufacturers, at their dispensaries, and of the laboratories doing testing and to try

to find well-reputed, well-referenced sources whenever possible. When I speak to people trying to source medicine, I usually send them to one of the nurses I know that have been dedicated to this medicine all along. While it’s unfortunate that our medical doctors are bound by risk and sometimes law to steer away from this topic, our nurses have been advocating right alongside our pioneers of the industry and they are often passionate about helping people understand the basics of using this plant as medicine.

T&T: If you could tell someone just one thing about cannabis, what would it be?

SS: You had it right in the last question – I urge them to educate themselves as much as possible.

T&T: As someone who is dedicated to helping business achieve and retain compliance and a strong quality program, which aspects do you find that many new businesses lack?

SS: I think that most people have the right intentions and commitment to

quality and compliance but that they are often caught up in the speed at which the industry moves and the pressure to get licensed and producing right away. I find new businesses lack the time and leaders early in their timeline to implement quality systems with the quality officer position filled, procedures written, and employees trained on standardized practices. I urge everyone to consider these essential core items as early as possible to ensure a consistent and easy to maintain compliant quality management system.

T&T: You do a little bit of crystal gazing. Where do we go from here? What does 2020 bring us?

SS: I think we are going to see a change in the federal landscape. I believe de-scheduling of cannabis will play an important role in the political landscape and that the federal agencies are poised and prepared to take over when this change happens. I believe that 2020 brings the need to stay flexible, active in communities on a state and federal level, and informed of upcoming changes.


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