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An Evaluation of Sample Preparation Techniques for Cannabis Potency Analysis

By Kelsey Cagle, Frank L. Dorman, Jessica Westland
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Sample preparation is an essential part of method development and is critical to successful analytical determinations. With cannabis and cannabis products, the analyst is faced with a very challenging matrix and targets that may range from trace level through percent level thus placing considerable demands on the sample preparation techniques.1 The optimal sample preparation, or “extraction”, method for potency analysis of cannabis flower was determined using a methanol extraction coupled with filtration using regenerated cellulose filters. 

In the United States (US), Canada, and other countries where medicinal and/or adult recreational cannabis has been legalized, regulatory entities require a panel of chemical tests to ensure quality and safety of the products prior to retail sales2. Cannabis testing can be divided into two different categories: Quality and Safety. Quality testing, which includes potency analysis (also known as cannabinoid testing or cannabinoid content), is performed to analyze the product in accordance with the producer/grower expectations and government regulations. Safety testing is conducted under regulatory guidelines to ensure that consumers are not exposed to toxicants such as pesticides, mycotoxins, heavy metals, residual solvents and microbial contaminates.

Potency testing evaluates the total amount of cannabinoid content, specifically focusing on tetrahydrocannabinol (THC) and cannabidiol (CBD). In the US, the biggest push for accurate total THC is to differentiate between hemp (legally grown for industrial or medicinal use), which is defined as cannabis sativa with a THC limit ≤ 0.3 %, and cannabis (Cannabis spp.), which is any cannabis plant with THC measured above 0.3 %3. Potency testing is typically performed by liquid chromatography (LC) with UV detection to determine the quantity of major cannabinoids.

In addition to reporting THC and CBD, their respective precursors are also important for reporting total potency. Tetrahydrocannabinolic acid (THCA) is the inactive precursor to THC while cannabidiolic acid (CBDA) is the precursor to CBD.4,5

Methods and Materials

Sample Preparation

All samples were homogenized using an immersion blender with a dry material grinder. The nominal sample amounts were 200 mg of flower, 500 mg of edibles, and 250 mg of candy samples.

Potency Extraction Method (1)

Twenty milliliters (mL) of methanol (MeOH) was added to each sample. The samples were mechanically shaken for 10 minutes and centrifuged for 5 minutes.

Potency Extraction Method (2)

Ten mL of water was added to each sample. The samples were mechanically shaken for 10 minutes. 20 mL of acetonitrile (ACN) was then added to each sample and vortexed. An EN QuEChERS extraction salt packet was added to the sample. The samples were placed on a mechanical shaker for 2 minutes and then centrifuged for 5 minutes.

Each extract was split and evaluated with two filtration/cleanup steps: (1) a regenerated cellulose (RC) syringe filter (Agilent Technologies, 4 mm, 0.45 µm); (2) a PFTE syringe filter (Agilent Technologies, 4 mm, 0.45 µm). The final filtered extracts were injected into the ultra-performance liquid chromatograph coupled with a photodiode array detector (UPLC-PDA) for analysis.

Figure 1: Calibration curve for THC potency

Calibration

Standards were obtained for the following cannabinoids at a concentration of 1 mg/mL: cannabidivarin (CBDV), tetrahydrocannabivarin (THCV), cannabidiol (CBD), cannabigerol (CBG), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabinol (CBN), tetrahydrocannabinol (9-THC), cannabichromene (CBC), tetrahydrocannabinol acid (THCA). Equal volumes of each standard were mixed with MeOH to make a standard stock solution of 10 ug/mL. Serial dilutions were made from the stock to make concentrations of 5, 1, and 0.5 ug/mL for the calibration curve (Figure 1).

Instrumental Method

All instrument parameters were followed from Agilent Application Note 5991-9285EN.8 A UPLC with a PDA (Waters Corp, Milford, MA) detector was employed for potency analysis. An InfinityLab Poroshell 120 EC-C18, 3.0 x 50 mm, 2.7 um column (Agilent Technologies, Wilmington, DE) was utilized for compound separation. The organic mobile phase composition was 0.05 % (v/v) formic acid in HPLC grade MeOH and the aqueous mobile phase composition was 0.1 % (v/v) formic acid in HPLC grade water. The mobile phase gradient is shown in Table 1. The flow rate was 1 mL/min (9.5 minute total program), injection volume was 5 uL, and column temperature was 50 °C.

Table 1: LC mobile phase gradient for potency samples6

Discussion and Results

Table 2 summarizes the relative standard deviations (% RSD) were found for the THC calibrator (at 1 ug/mL) and one extract of a homogeneous sample (utilizing 7 replicates).

Table 2- %RSD values for the instrument response precision for THC in both the calibrations and the homogeneous extract.

The cannabinoid potency of various cannabis plant and cannabis product samples were determined for the various extraction techniques In the chromatograms THC was observed ~8.08 minutes and CBD was observed ~4.61 minutes (Figure 2).

Figure 2: Chromatogram of the 10ug/mL calibrator for potency/cannabinoid analysis

Total potency for THC & CBD were calculated for each sample using the equations below. Equation 1 was used because it accounts for the presence of THCA as well as the specific weight difference between THC and THCA (since THCA will eventually convert to THC, this needs to be accounted for in the calculations).

Table 3 shows the % THC and the total THC potency values calculated for the same flower samples that went through all four various potency sample preparation techniques as described earlier. Figure 3 also provides LC chromatograms for flower sample 03281913A-2 and edible sample 03281912-1.

Table 3-THC and Total THC potency values for the same cannabis flower sample processed through the combination of extractions and cleanups.
Figure 3: Potency/Cannabinoid analysis chromatogram for flower sample 03281913A-2 (red trace) and edible sample 03281912-1 (green trace).

The results indicated that with the “Potency Extraction Method 2” (ACN/QuEChERS extraction) coupled with the RC filter provided a bias of 7.29 % greater for total THC % over the other extraction techniques. Since the other 3 techniques provided total THC values within 2% of each other, the total THC of the sample is more likely ~14%.

Since the sample dilution for the above data set reduced the CBD content, an undiluted sample was run and analyzed. This data is reported in Table 4.

Table 4- CBD and Total CBD potency values for the same cannabis flower sample processed through different sample preparation techniques.

The CBD results indicated that with the “Potency Extraction Method 1” (methanol extraction) coupled with RC filter, allowed for a greater CBD recovery. This may indicate the loss of CBD with an ACN/QuEChERS extraction.

With an average ~14% total THC and 0.06% total CBD for a homogenous cannabis flower sample, the optimal sample preparation extraction was determined to be a methanol extraction coupled with filtration using a regenerated cellulose filter. Since potency continues to remain at the forefront of cannabis regulatory testing it is important to utilize the right sample prep for your cannabis samples.

References

  1. Wang M, Wang YH, Avula B, Radwan MM, Wanas AS, Mehmedic Z, et al. Quantitative Determination of Cannabinoids in Cannabis and Cannabis Products Using Ultra-High-Performance Supercritical Fluid Chromatography and Diode Array/Mass Spectrometric Detection. Journal of Forensic Sciences 2016;62(3):602-11.
  2. Matthew Curtis, Eric Fausett, Wendi A. Hale, Ron Honnold, Jessica Westland, Peter J. Stone, Jeffery S. Hollis, Anthony Macherone. Cannabis Science and Technology, September/October 2019, Volume 2, Issue 5.
  3. Sian Ferguson. https://www.healthline.com/health/hemp-vs-marijuana. August 27, 2020.
  4. Taschwer M, Schmid MG. Determination of the relative percentage distribution of THCA and 9-THC in herbal cannabis seized in Austria- Impact of different storage temperatures on stability. Forensic Science International 2015; 254:167-71.
  5. Beadle A. CBDA Vs CBD: What are the differences? [Internet]. Analytical Cannabis. 2019 [cited 2020 Apr 22]; https://www.analyticalcannabis.com/articles/cbda-vs-cbd-what-are-the-differences-312019.
  6. Storm C, Zumwalt M, Macherone A. Dedicated Cannabinoid Potency Testing Using the Agilent 1220 Infinity II LC System. Agilent Technologies, Inc. Application Note 5991-9285EN

Hardware Platforms in Cannabis: A Q&A with Mike McDonald, President and CEO of Ammonite

By Aaron Green
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More and more we are seeing the development of proprietary hardware platforms in cannabis. With proprietary technology in hand, manufacturers often lean on MSOs, LPs and other brand partners to grow their business through existing sales channels.

We spoke with Mike McDonald, President and CEO at Ammonite, to learn more about the history of the Dablicator™ platform and Ammonite’s North American brand partner strategy. Mike formed Ammonite as a spin-off company from Jetty Extracts after getting to know the founders in a real estate transaction. Prior to Ammonite, Mike was an operator in the manufacturing and product development space, having helped to launch the Giant bicycle brand as well as growing and eventually selling the Atlas Snowshoe Company to K2 Sports.

Aaron: How did you get involved in cannabis?

Mike: Well, like a lot of folks in the industry, my background is pretty eclectic. I come primarily from an operator’s perspective – I’ve been in manufacturing, product development and company growth for my whole career. I lived in Taiwan for several years and helped to launch the Giant bicycle brand worldwide. I was also involved with a ski business that was started at Stanford as a thesis project called Atlas Snowshoe Company. Fast-forward, we built it into the largest snowshoe brand and activity in the US and later sold it to K2 Sports. So, I’ve always been involved in the growth of product-related businesses.

Mike McDonald, President and CEO at Ammonite

I’ve also done some real estate development as well; I actually sold our building to the Jetty guys, which is how we met. In that process, I got involved with their company, helped Jetty reorganize its business model, raise some money, and then just got addicted to the whole industry and really found it fascinating. I liked the team at Jetty and couldn’t resist jumping in, and now I’ve been full-time in the business for over three years.

Aaron: How did you get involved in Ammonite?

Mike: Ammonite is actually a spin out company from Jetty Extracts, which is one of the largest brands in California. Our main Ammonite product is called the Dablicator™ Oil Applicator, which was originally invented at Jetty as a medical device for cancer patients. We saw a big demand for it as a private label partnership product, so we decided to spin out a separate hardware company and really focus on developing unique IP and CBD and cannabis related hardware.

Aaron: What trends are you following in the industry?

Mike: Certainly the MSOs of the world are really expanding and the top three to five are making a mark with growth and more sophistication in the market. I think the social equity movement is really a big component that we’re all excited about in the industry. You’re seeing the larger players really put their money where their mouth is around that. We’ve always been a big part of that in California.

Specifically, regarding trends in the cannabis space, Colorado and California are probably the two most mature markets. We generally say what’s happening in California and Colorado eventually make their way out to the rest of the world. Vaping was invented in California and Colorado, and now it’s a huge part of the business where before, four or five years ago, the market was mostly flower-centric.

There’s a trend away from inhalables, with more awareness around lung-related illnesses and of course COVID, so we’re seeing a big growth in edibles, drinks and so forth. Interestingly enough, although it’s an inhalable, infused pre-rolls are a big growth sector as well. Jetty is actually launching an infused pre-roll program in February.

Folks are looking for ways to get their medicine without smoking – and this has definitely led to a growth in the oil application business. Oil application has traditionally been delivered via a syringe. Dablicator™ oil applicator is essentially an improved, more convenient syringe. On the medical side, patients have been taking oil sublingually, putting it in food and drink and so forth for years because a lot of them can’t smoke. As that trend transfers over to the adult use market, oil application is becoming really big. You can take it sublingually; you can put it in your food or beverage. On the recreational side, you can add it to your loose flower or joints, or of course, dab it directly onto your rig via the heat resistant tip.

Further, you’re probably familiar with a lot of these portable dab rigs that are taking off, like the G Pen Roam and the Puffco Peak and a variety of others. So now you can dab on the go with your standard wax and shatter in a jar. It’s just not the most convenient way if you’re up on a hike or on a mountain bike ride. So now, with a portable dab rig and something like the Dablicator™ oil applicator, you can have a really convenient mess-free way to enjoy cannabis. The big growth in concentrates and areas that aren’t necessarily inhalables is where our product hardware really fits in.

Aaron: How did you come up with the idea for the Dablicator?

Mike: The Jetty team had a friend that had brain cancer. He was doing a lot of chemotherapy and was having trouble eating and keeping weight on and he couldn’t smoke. So, the guys at Jetty began to bring him cannabis oil, which he was able to use ingesting it from a spoon initially and it really helped him with his pain, his anxiety and his appetite. In that process, we realized that there wasn’t really a great way to deliver oil. Syringes were there, but they were kind of sketchy and they weren’t convenient.

So, the Jetty team developed a better mousetrap. Several iterations later, this Dablicator™ product was ready for patients. In fact, it became a big part of the Jetty Shelter Project, a non-profit where the team delivers cannabis to cancer patients, and it was a very much sought-after product delivery device in that world. So, it was developed inside of a need on the medical side and it’s really sort of grown inside the expansion on the adult-use side.

Aaron: Can you explain how the Dablicator™ oil applicator works from a perspective of form and function?

Mike: Pre-Dablicator™ you would use a syringe type product – for direct oil application, sublingual application, or as an add on to your flower. The difference between Dablicator™ oil applicator and a traditional syringe is that Dablicator™ is a twist and plunge product. Imagine a pen filled with oil, but instead of inhaling it, you’re able to dispense it through a tip that is heat resistant, which means you can apply directly to your dab rig nail. You’re able to put it in your pocket without fear of cannabis oil leakage. It’s discreet, precise, compact and portable.

Aaron: How does the user dose using Dablicator™ oil applicator?

Mike: Basically, there’s measurements on the plunger of 55 milligrams apiece – one click is 55 milligrams, and you can dispense as many clicks as you like. What’s cool about the product itself is if you’ve clicked too many times accidentally, you can back it off and the excess oil won’t dispense. You can go to dablicator.com and see demo videos as well.

Aaron: Dablicator™ oil applicator started as a Jetty Extracts spin-off. I see you are now white labeling for other oil brands. How do you go about selecting your partners?

Mike: We call it our brand partner program. It’s not too dissimilar to what other hardware manufacturers, like PAX and GPen, are doing. We’ve got a patented and innovative device where our brand partners, MSOs and leading brands throughout the US and Canada, can take their existing vape and tincture oils and offer them in Dablicator™ oil applicator hardware.

Our focus is signing up major, well respected brands and MSOs on to the “platform,” meaning they are able to immediately offer between six and ten new SKUs to their consumers. They take their existing oils, put them into a custom branded Dablicator™ hardware unit and add their custom branded packaging. It’s a full turnkey solution. For example, one of our partners, 710 Labs, is developing their RSO and were shopping for a delivery method specifically geared towards medical patients. Within eight weeks, we had a custom program for them and delivered hardware, and we assisted on the packaging front as well.

Our partners have to be reputable folks that are interested in developing or delivering oil in a unique and innovative way. Frankly, our early partners are those that see where the growth is. 710 Labs is on the platform, as well as Surterra in Florida, Ancient Roots in Ohio, and we’ve got multiple conversations going to some of the other MOSs and the LPs in Canada.

Aaron: Are the brand partners loading the oil applicator themselves?

Mike: We customize the product for them and then ship them unassembled and empty. In their lab, they use the same machinery and equipment they use to fill their vape cartridges. They then fill their Dablicator™, assemble it, package it and ship it out just like any other product that they’re processing and manufacturing.

Aaron: What kind of oils are suitable for Dablicator™?

Mike: Pretty much any oil that’s going into a vape cart is suitable and then some. Some of our customers, including Jetty, started out with a THC distillate. Live resin is becoming a big product category in California as well as solventless oils. Dablicator™ oil applicator can accommodate everything from distillate to live resin to solventless to RSO and even full spectrum CBD. If it can flow, if it doesn’t crystallize up like shatter and sugars and diamonds, you can put it into Dablicator™, even the thickest of oils. It’s designed to contain any kind of liquids that are flammable.

Aaron: What geographies are you currently in?

Mike: We’re in multiple states throughout the US and actually just signed up with an LP in Canada. We only launched the program in August of 2020, and today we’ve got partners California, Colorado, Ohio, Arizona, Missouri, Florida, soon to be Michigan, Illinois, and throughout Canada.

Aaron: Any plans for international expansion beyond North America?

Mike: We’re getting inquiries on a regular basis from all over the place, including internationally. We’re in conversations with some folks down in Brazil. Spain is actually a big cannabis market and we’re having some conversations with some folks there. The inquiries are coming in faster than we can process the relationships, but right now our major focus is on North America.

Aaron: What are your goals with Ammonite?

Mike: We are developing a category, right? So today, oil dispensing isn’t top of mind. Today, if you want oil, you go into a dispensary and say, “Hey, give me those syringes.” My goal is that a year from now, you can walk into Harborside in Oakland and you see a wall of different branded Dablicator™ oil applicators. The goal is to really turn the oil dispensing business into a category, and then position Dablicator™ oil applicator as the best and leading product in that category.

Aaron: What are you personally interested in learning more about?

Mike: Well, I’ve got two teenagers – two daughters, as a matter of fact, a freshman and a senior – and they’re being homeschooled right now. So that’s been quite an interesting development!

I think on the cannabis side, it’s just fascinating what it is as a business model. It’s the most recent multi-billion-dollar opportunity in consumer products. You only get a chance to participate in something like that maybe once in a lifetime. I’m really looking forward to seeing it become more adopted into the mainstream and it’s already becoming that way from a consumer perspective. I am watching the cannabis market become legal from a federal perspective, hoping that the social equity component of the industry really stays with it.

I’ve been in a lot of businesses over the years; I feel like one of the gray hairs in this business that is actually an operator versus someone who came over from the financial side. I am continuing to learn, grow and work with great people and this has been a really amazing experience for me.

Aaron: Okay, great. Mike, that’s the end of the interview. Thank you for your time today!

Choosing Filling Machinery for CBD and THC Products

By Michelle Pudlo
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As the legalization of cannabis continues to spread across the U.S., both THC and CBD products are rapidly growing in popularity, and we can expect that popularity to increase in the coming years. The cannabis industry alone is expected to account for nearly $16.9 billion in revenue this year.

Subsequently, there is a rising need among infused product manufacturers for sufficient filling machinery for CBD and THC products. These products, including CBD oil cartridges, require filling equipment that can provide quick turnaround, detailed parts and simple changeover and cleanup, among other factors. Let’s go over the different types of filling machinery used for these products.

Vial Filling Machines

For small vial packages made of glass, metal or plastic, vial filling machines are available. Often used for a variety of pharmaceutical products, they’re now suitable for filling liquid THC and CBD oil. Vial fillers are also often suitable for filling liquid products of varying viscosity levels, with either the installation of peristaltic pumps or volumetric filling stations.

Rotary Fillers

Rotary fillers can also fill containers at high speeds and with quick turnaround, and are ideal for filling various types and sizes of containers made of materials such as plastic, metal or glass. A good rotary filler will be able to meet the demands of high-speed environments consistently and with accuracy.

Fixed or Variable Volume Cartridge Filling ToolsAs the industry develops more demand for high-quality filling and other types of equipment, more machines are likely to be manufactured or configured specifically with these types of products in mind.

Fixed and variable volume cartridge filling tools often feature a single-handed operation and are used to rapidly fill cartridges for THC and CBD oils used for vaping. With fixed volume fillers, you’ll be able to designate a specific and consistent volume, while variable volume models allow for different fill volumes for applications requiring versatility.

Automatic Fillers

Automatic filling machines will be able to fill a large number of products at varying speed settings, without the need for manual operation. These machines can fill many different types of products with consistency that helps maintain optimal productivity. As with other fillers, automatic fillers are often customizable in a wide variety of configurations.

Filling Syringes

For concentrates, filling syringes are ideal in many cases. Many patients are in need of a specific dosage of oil, and a syringe can allow for accuracy through the inclusion of measurement indicators. Many dispensaries sell syringe units, so this type of packaging method is likely to continue to rise in popularity.

Other Types of Equipment for Liquid Cannabis Packaging

In addition to reliable filling systems, manufacturers should make sure every other aspect of their packaging lines is covered with high-quality equipment. Facilities will require a variety of conveyors to transport products from one end of the line to the other, cleaners to ensure that bottles or other containers are clean prior to filling, and labelers to apply custom labels to packaging, among other machinery.

With one or more of these types of liquid fillers in a facility, companies can maintain accuracy and efficiency throughout their operations when filling CBD or THC products. As the industry develops more demand for high-quality filling and other types of equipment, more machines are likely to be manufactured or configured specifically with these types of products in mind.