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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

Recent Developments in Supercritical CO₂ Winterization

By Aaron Green
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Supercritical carbon dioxide (CO2) extraction is a processing technique whereby CO2 is pressurized under carefully controlled temperatures to enable extraction of terpenes, cannabinoids and other plant molecules. Once the extract is obtained the crude is often subjected to an ethanol winterization process to remove chlorophyll, fats and waxes.

Green Mill Supercritical is a Pittsburgh-based manufacturing and engineering company focused on cannabis and hemp extraction. The company offers a range of CO2 extraction equipment where users can tune and control their extraction methods. They recently announced  a technology advance enabling winterization in-process, which has the potential to remove the need for ethanol winterization.

We spoke with Jeff Diehl, director of marketing at Green Mill Supercritical, to learn more about the new process. Jeff was working in the tech industry in San Francisco in 2017 when he was invited to join Green Mill by his cousin, Jeremy Diehl, who is the founder and CTO.

Aaron Green: Before we get to your new technology, can you explain what industry trends you are watching?

Jeff Diehl: A big thing that I watch is the premium extract space. More and more consumers are demanding higher premium extracts. They want differentiated products. They want products that are safe and that have some kind of meaningful connection to the specific plant from which they came. Right now, CO2 plays a small role in the market for those products. Most premium products are generated through hydrocarbon extraction. So, I am watching how people are using CO2 to create the next generation of safe, premium products.

Aaron: What is the normal process for a CO2 extraction today?

Jeff Diehl, director of marketing at Green Mill Supercritical

Jeff: The current CO2 extraction process generally consists of two major phases to producing your final extract. In the first phase, you have extraction where you get your crude product. The second phase is post-extraction where you do cleanup to get your refined oil. Within that post-extraction phase, most operations include an ethanol-based winterization process.

Aaron: What does the winterization step do, exactly?

Jeff: Winterization is about removing waxes. Your main extraction is considered crude because it’s got a lot of materials from the plant that you don’t want. The large majority of unwanted material is waxes. Winterization is the process of using a solvent, traditionally ethanol, to separate the waxes from the cannabinoids. There are multiple challenges inherent in ethanol-based winterization that introduce cost, time and product loss. It’s terribly inefficient. Plus, there will always be residual ethanol left in your final product, and that’s not something consumers appreciate.

Aaron: You’ve recently announced a new process at Green Mill that moves the winterization step into the supercritical CO2 equipment. Can you explain how that works?

Jeff: With our process, which we call Real-Time Winterization, there is no ethanol involved in winterization anymore. It is all done with CO₂ during the primary extraction. That’s the major advance of our process and although it has been attempted before, no one has succeeded at doing it in a viable way. You take a process which is normally four days – one day for CO2 extraction and three days for ethanol winterization – and you do it all in less than a day. We have automated software, sensors and pumps that makes this all possible.

Aaron: How does the quality of the resulting product compare with the new process?

Jeff: You can see the difference right away, if you’re at all familiar with extraction. It just looks clean and bright. Lab analysis has been very positive thus far, but we continue to run tests. Our R&D team has done multiple tests, mostly on hemp and CBD. That’s because we don’t have a license for THC. We’re currently engaging with a licensed partner so that we can collect more data on THC-containing products, so we can give exact numbers. But with CBD, we’ve done multiple tests to validate the method and the technology, and are seeing consistently excellent results in regards to both purity of the product and efficiency of the process.

Aaron: How do yields compare between the processes?

Hemp CBD extract straight out of a Green Mill SFE Pro running Real-Time Winterization.

Jeff: From the data that we’ve seen in the industry, it looks like when you winterize with ethanol, you leave anywhere from 5 to 10% of your cannabinoids behind in the waxes. That’s just lost. With Real-Time Winterization using CO2 we have seen recovery rates as high as 99%. We are continuing to investigate that result with testing to make sure it was not an outlier, but in any case, recovery rates look promising.

Aaron: One of the other issues with ethanol is taxes and the ability to find food grade supply. Do you have any perspective you can share on that?

Jeff: There are a number of advantages to moving away from ethanol. The sheer quantity of ethanol is a factor. There are a lot of regulations and fire requirements around managing large quantities of ethanol. The ethanol winterization process itself is not just one process. There are multiple stages, from mixing, to freezing, to filtering, to removing the solvent. These are all opportunities for things to go wrong, so you’re always managing those risks. Multiple large pieces of equipment, including fume hoods, filter skids, cryo freezers and rotary evaporators, are expensive and require heavy management.

I think Elon Musk said the best process is no process. Anytime in an industrial process when you can remove steps in the process, that’s the direction you want to go in. And, that’s what we’ve done. With this recent work, we have effectively removed post processing for certain categories of end product.

Aaron: Do you have any patents on the new process?

Jeff: We have a patent pending on both the method and the equipment, which is allowing us to talk about this as much as we are.

Aaron: So, how does this work if somebody already owns an existing piece of Green Mill equipment? Is this something that can be retrofitted? Is it a software upgrade?

Jeff: There are two components. One is an equipment upgrade, which can be done retroactively for existing customers, and one is a methodology upgrade, which we assist our customers with. The automation software inherently can handle the settings that you need to run the methodology. In fact, it’s that software and the rest of our existing tech stack, the proprietary pump, the triple inline fractionation, the precision and stability of the overall system, that is what made this winterization advance possible.

Aaron: Where are you rolling this out first? And do you plan to go international?

Jeff: International is definitely in the plan, since we’ve already sold systems abroad. We are currently getting ready to announce the opening of our beta program with the new technology. So, we’re not ready to sell this widely at this time, but we are taking submissions from companies that want to get in early and join us at the forefront of CO₂ extraction innovation.

Aaron: Okay, great. Thanks Jeff, that’s the end of the interview.

Essential Cannabis Businesses Must Protect Employees and Customers During COVID-19 With Sanitation and Social Distancing Practices

By David Laks
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Unlike their retail neighbors who have been forced to move inventory online to survive, many cannabis businesses are considered essential and remain open during the current pandemic. With that, though, comes a tremendous responsibility to maintain optimal protocol for safe operations and customer shopping.

Whether you run a retail or production operation, allow only essential vendors (i.e. delivery, service companies) into the facility and have non-essential staff telecommute, when possible. Some businesses may want to consider splitting shifts for the management team as well.

Each state and local municipality will have their own rules when it comes to protocols for open retail establishments. Where those are more stringent than the following recommendations, adhere to the more stringent rule.

Cannabis Production Facility Best Practices

While not being face-to-face with cannabis customers on a daily basis, production facilities are the first and possibly only ones to handle the raw product the customer will eventually consume. For this reason, it’s important to conduct a refresh training session on sanitation procedures and new COVID-19 protocol for all production employees. Consider the following critical procedures for cannabis production facilities:

  • Review current production sanitation procedures and adjust accordingly, focusing on high touch points and potentially contaminated surfaces. Include office items such as keyboard, phones, and kitchen areas.
  • Review the business’ call-in sick policy and make sure employees know they can – and should – do so if they’re under the weather.
  • Sanitize high touch points every 30 minutes or less.
  • Instruct employees to wash hands with soap and water for 20 seconds after blowing their nose, coughing, sneezing, going to the bathroom, before eating and when touching any communal surface, including door handles and surfaces. Wear personal protective equipment (PPE) at all times while working with raw product, including gloves and masks.

    control the room environment
    PPE can reduce the risks of spreading disease
  • If an employee coughs or sneezes in a production area, instruct them to do so into the elbow of their outer garment, and immediately change following proper donning techniques. Instruct them to avoid touching their face.

Cannabis Retail Facility Best Practices

Retail cannabis establishments must realize first and foremost that those with compromised immune systems may be frequenting their store to purchase medical cannabis. Consider, evaluate and appropriately publicize protocol relative to employee interactions with customers, including:

  • Enable mobile or order-ahead features along with curbside pickup and contact-less delivery, when possible. Where this isn’t an option, limit the number of customers in the store at a time.
  • Consider moving to appointment-only operations, or restricted hours for those over 65.
  • Reduce store visits by recommending patients order their prescription for the maximum allowable 60 days.
  • Designate an employee to champion personal sanitation and social distancing. Create an entry sanitation station and require all customers to use it upon entry. Maintain social distance of 6-ft. minimum between customers. Place markings on the floor to designate this.
  • Limit sales to only sealed products.
  • Sanitize high touch points twice an hour, including ID check booths, display cases, phones, keyboards, etc. and provide adequate PPE for all, including gloves, masks, etc.
  • Install separation barriers, like thick plastic or plexiglass at each cashier station.

The requirements of keeping an essential business open will vary by location and will likely change as the COVID-19 pandemic evolves. Regularly check for changes to the rules of your local jurisdiction and adapt accordingly.

IR Spectrum of 2,4-Dichlorophenol in different physical states
From The Lab

Gas Chromatography/Infrared Spectroscopy: A Tool For the Analysis of Organic Compounds in Cannabis

By John F. Schneider
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IR Spectrum of 2,4-Dichlorophenol in different physical states

Editor’s Note: The author will be teaching a 1/2 day short course on this topic at PITTCON in Philadelphia in March 2019.


The combination of gas chromatography and infrared spectroscopy (GC/IR) is a powerful tool for the characterization of compounds in complex mixtures. (1-5) Gas chromatography with mass spectroscopy detection (GC/MS) is a similar technique, but GC/MS is a destructive technique that tears apart the sample molecules during the ionization process and then these fragments are used to characterize the molecule. In GC/IR the molecules are not destroyed but the IR light produced by molecular vibrations are used to characterize the molecule. IR spectrum yields information about the whole molecule which allows the characterization of specific isomers and functional groups. GC/IR is complementary to GC/MS and the combination results in a powerful tool for the analytical chemist.

A good example of the utility of GC/IR vs GC/MS is the characterization of stereo isomers. Stereo isomers are mirror images such as a left hand and a right hand. In nature, stereo isomers are very important as one isomers will be more active then its mirror image. Stereo isomers are critical to medicinal application of cannabis and also a factor in the flavor components of cannabis.

GC/MS is good at identifying basic structure, where GC/IR can identify subtle differences in structure. GC/MS could identify a hand, GC/IR could tell you if it is a left hand or right hand. GC/MS can identify a general class of compounds, GC/IR can identify the specific isomer present.

Why GC/IR?

Gas chromatography interfaced with infrared detection (GC/IR), combines the separation ability of GC and the structural information from IR spectroscopy. GC/IR gives the analyst the ability to obtain information complementary to GC/MS. GC/IR gives the analyst the power to perform functional group detection and differentiate between similar molecular isomers that is difficult with GC/MS. Isomer specificity can be very important in flavor and medical applications.

 IR Spectrum of 2,4-Dichlorophenol in different physical states

IR Spectrum of 2,4-Dichlorophenol in different physical states

Gas chromatography with mass spectrometry detection (GC/MS) is the state-of-the-art method for the identification of unknown compounds. GC/MS, however, is not infallible and many compounds are difficult to identify with 100 % certainty. The problem with GC/MS is that it is a destructive method that tears apart a molecule. In infrared spectrometry (IR), molecular identification is based upon the IR absorptions of the whole molecule. This technique allows differentiation among isomers and yields information about functional groups and the position of such groups in a molecule. GC/IR complements the information obtained by GC/MS.

Interfaces

Initial attempts to couple GC with IR were made using high capacity GC columns and stopped flow techniques. As GC columns and IR technology advanced, the GC/IR method became more applicable. The advent of fused silica capillary GC columns and the availability of Fourier transform infrared spectrometry made GC/IR available commercially in several forms. GC/IR using a flow cell to capture the IR spectrum in real time is known as the “Light Pipe”. This is the most common form of GC/IR and the easiest to use. GC/IR can also be done by capturing or “trapping” the analytes of interest eluting from a GC and then measuring the IR spectrum. This can be done by cryogenically trapping the analyte in the solid phase. A third possibility is to trap the analyte in a matrix of inert material causing “Matrix Isolation” of the analyte followed by measuring the IR spectrum.

Infrared Spectroscopy

The physical state of the sample has a large effect upon the IR spectrum produced. Molecular interactions (especially hydrogen bonding) broadens absorption peaks. Solid and liquid samples produce IR spectra with broadened peaks that loses much of the potential information obtained in the spectra. Surrounding the sample molecule with gas molecules or in an inert matrix greatly sharpens the peaks in the spectrum, revealing more of the information and producing a “cleaner” spectrum. These spectra lend themselves better to computer searches of spectral libraries similar to the computer searching done in mass spectroscopy. IR spectral computer searching requires the standard spectra in the library be of the same physical state as the sample. So, a spectrum taken in a gaseous state should be searched against a library of spectra of standards in the gaseous state.

IR of various phases:

  • Liquid Phase – Molecular interactions broaden absorption peaks.
  • Solid Phase – Molecular interactions broaden absorption peaks.
  • Gas Phase – Lack of molecular interactions sharpen absorption peaks.
  • Matrix Isolation – Lack of molecular interactions sharpen absorption peaks.

IR Chromatograms

GC/IR yields chromatograms of infrared absorbance over time. These can be total infrared absorbance which is similar to the total ion chromatogram (TIC) in GC/MS or the infrared absorbance over a narrow band or bands analogous to selected ion chromatogram. This is a very powerful ability, because it gives the user the ability to focus on selected functional groups in a mixture of compounds.

Conclusion

Gas chromatography with infrared detection is a powerful tool for the elucidation of the structure of organic compounds in a mixture. It is complementary to GC/MS and is used to identify specific isomers and congeners of organic compounds. This method is greatly needed in the Cannabis industry to monitor the compounds that determine the flavor and the medicinal value of its products.


References

  1. GC–MS and GC–IR Analyses of the Methoxy-1-n-pentyl-3-(1-naphthoyl)-Indoles: Regioisomeric Designer Cannabinoids, Amber Thaxton-Weissenfluh, Tarek S. Belal, Jack DeRuiter, Forrest Smith, Younis Abiedalla, Logan Neel, Karim M. Abdel-Hay, and C. Randall Clark, Journal of Chromatographic Science, 56: 779-788, 2018
  2. Simultaneous Orthogonal Drug Detection Using Fully Integrated Gas Chromatography with Fourier Transform Infrared Detection and Mass Spectrometric Detection , Adam Lanzarotta, Travis Falconer, Heather McCauley, Lisa Lorenz, Douglas Albright, John Crowe, and JaCinta Batson, Applied Spectroscopy Vol. 71, 5, pp. 1050-1059, 2017
  3. High Resolution Gas Chromatography/Matrix Isolation Infrared Spectrometry, Gerald T. Reedy, Deon G. Ettinger, John F. Schneider, and Sid Bourne, Analytical Chemistry, 57: 1602-1609, 1985
  4. GC/Matrix Isolation/FTIR Applications: Analysis of PCBs, John F. Schneider, Gerald T. Reedy, and Deon G. Ettinger, Journal of Chromatographic Science, 23: 49-53, 1985
  5. A Comparison of GC/IR Interfaces: The Light Pipe Vs. Matrix Isolation, John F. Schneider, Jack C. Demirgian, and Joseph C. Stickler, Journal of Chromatographic Science, 24: 330- 335, 1986
  6. Gas Chromatography/Infrared Spectroscopy, Jean ‐ Luc Le Qu é r é , Encyclopedia of Analytical Chemistry, John Wiley & Sons, 2006