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Chris English
The Practical Chemist

Accurate Detection of Residual Solvents in Cannabis Concentrates

By Chris English
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Chris English

Edibles and vape pens are rapidly becoming a sizable portion of the cannabis industry as various methods of consumption popularize beyond just smoking dried flower. These products are produced using cannabis concentrates, which come in the form of oils, waxes or shatter (figure 1). Once the cannabinoids and terpenes are removed from the plant material using solvents, the solvent is evaporated leaving behind the product. Extraction solvents are difficult to remove in the low percent range so the final product is tested to ensure leftover solvents are at safe levels. While carbon dioxide and butane are most commonly used, consumer concern over other more toxic residual solvents has led to regulation of acceptable limits. For instance, in Colorado the Department of Public Health and Environment (CDPHE) updated the state’s acceptable limits of residual solvents on January 1st, 2017.

Headspace Analysis

Figure 1: Shatter can be melted and dissolved in a high molecular weight solvent for headspace analysis (HS). Photo Courtesy of Cal-Green Solutions.

Since the most suitable solvents are volatile, these compounds are not amenable to HPLC methods and are best suited to gas chromatography (GC) using a thick stationary phase capable of adequate retention and resolution of butanes from other target compounds. Headspace (HS) is the most common analytical technique for efficiently removing the residual solvents from the complex cannabis extract matrix. Concentrates are weighed out into a headspace vial and are dissolved in a high molecular weight solvent such as dimethylformamide (DMF) or 1,3-dimethyl-3-imidazolidinone (DMI). The sealed headspace vial is heated until a stable equilibrium between the gas phase and the liquid phase occurs inside the vial. One milliliter of gas is transferred from the vial to the gas chromatograph for analysis. Another approach is full evaporation technique (FET), which involves a small amount of sample sealed in a headspace vial creating a single-phase gas system. More work is required to validate this technique as a quantitative method.

Gas Chromatographic Detectors

The flame ionization detector (FID) is selective because it only responds to materials that ionize in an air/hydrogen flame, however, this condition covers a broad range of compounds. When an organic compound enters the flame; the large increase in ions produced is measured as a positive signal. Since the response is proportional to the number of carbon atoms introduced into the flame, an FID is considered a quantitative counter of carbon atoms burned. There are a variety of advantages to using this detector such as, ease of use, stability, and the largest linear dynamic range of the commonly available GC detectors. The FID covers a calibration of nearly 5 orders of magnitude. FIDs are inexpensive to purchase and to operate. Maintenance is generally no more complex than changing jets and ensuring proper gas flows to the detector. Because of the stability of this detector internal standards are not required and sensitivity is adequate for meeting the acceptable reporting limits. However, FID is unable to confirm compounds and identification is only based on retention time. Early eluting analytes have a higher probability of interferences from matrix (Figure 2).

Figure 2: Resolution of early eluting compounds by headspace – flame ionization detection (HS-FID). Chromatogram Courtesy of Trace Analytics.

Mass Spectrometry (MS) provides unique spectral information for accurately identifying components eluting from the capillary column. As a compound exits the column it collides with high-energy electrons destabilizing the valence shell electrons of the analyte and it is broken into structurally significant charged fragments. These fragments are separated by their mass-to-charge ratios in the analyzer to produce a spectral pattern unique to the compound. To confirm the identity of the compound the spectral fingerprint is matched to a library of known spectra. Using the spectral patterns the appropriate masses for quantification can be chosen. Compounds with higher molecular weight fragments are easier to detect and identify for instance benzene (m/z 78), toluene (m/z 91) and the xylenes (m/z 106), whereas low mass fragments such as propane (m/z 29), methanol (m/z 31) and butane (m/z 43) are more difficult and may elute with matrix that matches these ions. Several disadvantages of mass spectrometers are the cost of equipment, cost to operate and complexity. In addition, these detectors are less stable and require an internal standard and have a limited dynamic range, which can lead to compound saturation.

Regardless of your method of detection, optimized HS and GC conditions are essential to properly resolve your target analytes and achieve the required detection limits. While MS may differentiate overlapping peaks the chances of interference of low molecular weight fragments necessitates resolution of target analytes chromatographically. FID requires excellent resolution for accurate identification and quantification.

Applications for Tissue Culture in Cannabis Growing: Part 1

By Aaron G. Biros
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Dr. Hope Jones, chief scientific officer of C4 Laboratories, believes there are a number of opportunities for cannabis growers to scale their cultivation up with micropropagation. In her presentation at the CannaGrow conference recently, Dr. Jones discussed the applications and advantages of tissue culture techniques in cannabis growing.

Dr. Hope Jones, chief scientific officer at C4 Labs

Dr. Jones’ work in large-scale plant production led her to the University of Arizona Controlled Environment Agriculture Center (CEAC) where she worked to propagate a particularly difficult plant to grow- a native orchid species- using tissue culture techniques. With that experience in tissue culture, hydroponics and controlled environments, she took a position at the Kennedy Space Center working for NASA where she developed technologies and protocols to grow crops for space missions. “I started with strawberry TC [tissue culture], because of the shelf life & weight compared with potted plants, plus you can’t really ‘water’ plants in space- at least not in the traditional way,” says Dr. Jones. “Strawberries pack a lot of antioxidants. Foods high in antioxidants, I argued, could boost internal protection of astronauts from high levels of cosmic radiation that they are exposed to in space.” That research led to a focus on cancer biology and a Ph.D. in molecular & cellular biology and plant sciences, culminating in her introduction to the cannabis industry and now with C4 Labs in Arizona.

Working with tissue culture since 2003, Dr. Jones is familiar with this technology that is fairly new to cannabis, but has been around for decades now and is widely used in the horticulture industry today. For example, Phytelligence is an agricultural biotechnology company using genetic analysis and tissue culture to help food crop growers increase speed to harvest, screen for diseases, store genetic material and secure intellectual property. “Big horticulture does this very well,” says Dr. Jones. “There are many companies generating millions of clones per year.” The Department of Plant Sciences Pomology Program at the Davis campus of the University of California uses tissue culture with the Foundation Plant Services (FPS) to eliminate viruses and pathogens, while breeding unique cultivars of strawberries.

A large tissue culture facility run in the Sacramento area that produces millions of nut and fruit trees clones a year.

First, let’s define some terms. Tissue culture is a propagation tool where the cultivator would grow tissue or cells outside of the plant itself, commonly referred to as micropropagation. “Micropropagation produces new plants via the cloning of plant tissue samples on a very small scale, and I mean very small,” says Dr. Jones. “While the tissue used in micropropagation is small, the scale of production can be huge.” Micropropagation allows a cultivator to grow a clone from just a leaf, bud, root segment or even just a few cells collected from a mother plant, according to Dr. Jones.

The science behind growing plants from just a few cells relies on a characteristic of plant cells called totipotency. “Totipotency refers to a cell’s ability to divide and differentiate, eventually regenerating a whole new organism,” says Dr. Jones. “Plant cells are unique in that fully differentiated, specialized cells can be induced to dedifferentiate, reverting back to a ‘stem cell’-like state, capable of developing into any cell type.”

Cannabis growers already utilize the properties of totipotency in cloning, according to Dr. Jones. “When cloning from a mother plant, stem cuttings are taken from the mother, dipped into rooting hormone and two to five days later healthy roots show up,” says Dr. Jones. “That stem tissue dedifferentiates and specializes into new root cells. In this case, we humans helped the process of totipotency and dedifferentiation along using a rooting hormone to ‘steer’ the type of growth needed.” Dr. Jones is helping cannabis growers use tissue culture as a new way to generate clones, instead of or in addition to using mother plants.

With cannabis micropropagation, the same principles still apply, just on a much smaller scale and with greater precision. “In this case, very small tissue samples (called explants) are sterilized and placed into specialized media vessels containing food, nutrients, and hormones,” says Dr. Jones. “Just like with cuttings, the hormones in the TC media induce specific types of growth over time, helping to steer explant growth to form all the organs necessary to regenerate a whole new plant.”

Having existed for decades, but still so new to cannabis, tissue culture is an effective propagation tool for advanced breeders or growers looking to scale up. In the next part of this series, we will discuss some of issues with mother plants and advantages of tissue culture to consider. In Part 2 we will delve into topics like sterility, genetic reboot, viral infection and pathogen protection.

emerald test retail

Emerald Scientific Proficiency Test Approved for Lab Accreditation & Regulatory Compliance

By Aaron G. Biros
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Emerald Scientific’s Inter-Laboratory Comparison and Proficiency Test (ILC/PT) was recently approved in Washington as an official cannabis lab PT program, according to a press release. The Emerald Test program measures the accuracy of individual labs as well as comparing their results to other labs for indicators of variability and performance improvement.

Washington requires certified cannabis labs to participate in proficiency testing and Emerald Scientific’s tests is the only approved program in 4 out of 5 of the categories: potency, pesticide, heavy metals and residual solvent analysis. The most recent round of The Emerald Test showed broad improvements in many of the testing categories.

Perry Johnson, a third-party lab accreditation service for ISO/IEC 17025 also decided that The Emerald Test “meets the audit criteria for the proficiency test participation requirement for the accreditation,’ according to the press release. The proficiency test is a key component of quality assurance, which is a major requirement for labs seeking ISO 17025 accreditation. “The Emerald Scientific PT ensures that the cannabis testing labs are performing their function to the best of their ability,” says Reggie Gaudino Ph.D., vice president of Science, Genetics and Intellectual Property at Steep Hill Labs. “Any lab that isn’t participating and exceeding the minimal passing requirements should be viewed as suspect. It’s that important.”

According to the press release, Emerald Scientific’s spring 2017 program has expanded from 5 to 6 tests. The residual solvents and pesticide analysis portions offer more comprehensive testing that previously. “The other tests include 2 microbial panels and a Potency Test, which measures 5 cannabinoids including THC, THCA, CBD, CBDA, and CBN,” says the press release. “New this spring is the Heavy Metals Test, which is offered in 2 parts, one solution for cannabis heavy metals and the other in a hemp matrix.”

More than 60 labs are expected to participate. Results will be released at the National Cannabis Industry Association’s Cannabis Business Summit and Expo on June 13, 2017. For more information please visit www.emeraldtest.com or email sales@emeraldscientific.com.

Going Beyond POS: Innovations in Dispensary Software

By Aaron G. Biros
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In a highly competitive market, dispensaries use wide product selections, competitive prices, rewards and loyalty programs to stay relevant and attract new customers. Many of those tools used to make the retail space more efficient require analytics to stay on top of their performance metrics.

At their SE 7th Ave location in Portland, Oregon, Cannabliss & Co. uses Baker software to better connect with their customers and track sales. According to Kevin Mahoney, manager of that dispensary, they use Baker’s software for things like their online menu, online ordering, text alerts and a rewards program.

Cannabliss & Co. SE 7th Ave location
Cannabliss & Co. SE 7th Ave location

Located in an historic firehouse built in 1913, Cannabliss & Co. was Oregon’s very first medical cannabis dispensary. Now that they offer both recreational and medical cannabis, their product inventory has expanded, their sales have grown and they have a wider customer base.

IMG_7545After using Baker’s software platform for almost a year now, Mahoney says he has seen great ROI on text alerts and the analytics. The online ordering and menu features have not only highlighted sales trends, but have made budtender-customer interactions easier. “We don’t want our budtender using the menu as a focal point of the conversation, but this allows for us to highlight particular specials or strains on our menu that gets eye attention right when the customer gets in,” says Mahoney. “Moving past the point of sale, it allows another conversation to happen organically, which keeps the customer engaged.”

On average, Baker sees conversion rates close to a 5% range per campaign. “That check in option is phenomenal; we get to see how many people actually came into the store from any given text alert,” says Mahoney. “In my mind, text alerts are preferable to email alerts; they can’t be marked as spam, it is easy to delete or opt out and takes much less time.”

Kevin Mahoney at his SE 7th Ave location
Kevin Mahoney at his SE 7th Ave location

Mahoney says the online ordering feature that Baker offers is a big selling point too. “Having an ordering service is absolutely terrific,” says Mahoney. “They can come in and out in less than five minutes with their full order by using the online ordering portal.” Mahoney says they see a real draw in this feature because it lets customers treat their dispensary like a takeout window at a restaurant.

Baker just launched a software platform designed for delivery service that a dispensary in Bend, Oregon has been using for two months now. With Portland legalizing cannabis delivery services recently, Mahoney is eyeing Baker’s software for his online ordering and delivery. “When the time comes, that is something we are very interested in pursuing.”

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Analytics allow users to track the success of campaigns

In August of 2016, Baker secured $1.6 million in seed funding, led by Former Salesforce Executive Michael Lazerow, according to a press release. “Baker has created a solution that is clean and easy to use and can help dispensary owners engage their shoppers like never before – online, mobile, social and in-store,” says Lazerow. “I witnessed first-hand how Salesforce supercharges its customers’ businesses and I’m inspired to see Baker driving the entire cannabis industry forward with this same intelligent approach.” In 18 months of business, Baker has worked with hundreds of dispensaries, helping them build better connections with over 100,000 customers. At Baker, we believe the cannabis shopping experience should be as comfortable and personalized as it has become in every other retail environment,” says Joel Milton, chief executive officer at Baker. “With expertise in cannabis, data and technology we have created an industry-specific tool that allows dispensaries and brands engage with customers and build brand loyalty through a personalized shopping experience.”

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Text alerts are customizable and easy to send out

According to Eli Sklarin, director of marketing at Baker, the number one reason why patients and customers choose a dispensary is because of products on the shelf. “We originally started the platform in 2014 so people could order ahead and wouldn’t have to wait in lines at the dispensary,” says Sklarin. “In 2015, we saw more dispensaries than fast food establishments in many cities. Once inventory started to settle down, we saw a need for the dispensary to better connect with their customers.” The three core products that Baker offers are online ordering, connect SMS & email and the check in & loyalty program.

Their entire suite of software options is specific to the cannabis retail space. “Our customizable program is designed to help dispensaries catch customers and keep them coming back,” says Sklarin. “The software can give a snapshot of who their customers are, insights into the overall health of their dispensary, sales per day of the week, monthly promotions and other basic analytics that help them understand their customers.” Things like strain alerts can help retain customers, allowing dispensaries to notify certain groups of customers when products are back in stock. Whether it’s a customer who prefers a particular brand of edibles or concentrates, these software tools can help dispensaries get the right message to the right customer.

Annual AOCS Meeting Spotlights Cannabinoid Analytics

By Aaron G. Biros
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The AOCS Annual Meeting is an international science and business forum on fats, oils, surfactants, lipids and related materials. The American Oil Chemist’s Society (AOCS) is holding their meeting in Orlando, Florida from April 30 to May 3, 2017. Last year’s meeting included discussions on best practices and the pros and cons of different extraction techniques, sample preparation, proficiency testing and method development, among other topic areas.

Posters on display for the duration of the Annual Meeting will discuss innovative solutions to test, preparing samples, discovering new compounds and provide novel information about the compounds found in cannabis. David Egerton, vice president of technical operations at CW Analytical (a cannabis testing laboratory in Oakland, CA), is preparing a poster titled Endogenous Solvents in Cannabis Extracts. His abstract discusses testing regulations focusing on the detection of the presence of solvents, despite the fact that endogenous solvents, like acetone and lower alcohols, can be found in all plant material. His study will demonstrate the prevalence of those compounds in both the plant material and the concentrated oil without those compounds being used in production.rsz_am17-editorialpic-cij

The conference features more than 650 oral and poster presentations within 12 interest areas. This year’s technical program includes two sessions specifically designed to address cannabinoid analytics:

Lab Proficiency Programs and Reference Samples

How do you run a lab proficiency program when you cannot send your samples across state lines? What constituents do you test for when state requirements are all different? Are all pesticides illegal to use on cannabis? What pesticides should be tested for when they are mostly illegal to use? How do you analyze proficiency results when there are no standard methods? Learn about these and other challenges facing the cannabis industry. This session encourages open and active discussion, as the cannabis experts want to hear from you and learn about your experiences.

Method Development

The need for high-quality and safe products has spurred a new interest in cannabinoid analytics, including sample preparation, pesticide, and other constituent testing. In this session, a diverse group of scientists will discuss developing analytical methods to investigate cannabis. Learn the latest in finding and identifying terpenes, cannabinoids, matrix effects, and even the best practice for dissolving a gummy bear.

Cynthia Ludwig speaking at last year's meeting
Cynthia Ludwig speaking at last year’s meeting

Cynthia Ludwig, director of technical services at AOCS, says they are making great progress in assembling analytical methods for the production of the book AOCS Collection of Cannabis Analytical Methods. “We are the leading organization supporting the development of analytical methods in the cannabis industry,” says Ludwig. “Many of the contributors in that collection will be presenting at the AOCS Annual Meeting, highlighting some of the latest advances in analyzing cannabis.” The organization hopes to foster more collaboration among those in the cannabis testing industry.

In addition to oral and poster sessions, the 2017 Annual Meeting will feature daily networking activities, more than 70 international exhibitors, two special sessions, and a Hot Topics Symposia which will address how current, critical issues impact the future of the fats and oils industry.

Colorado Cannabis Lab Methods Updated for Microbial Testing

By Aaron G. Biros
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The Colorado Department of Public Health and Environment’s (CDPHE) Marijuana Laboratory Inspection Program issued a bulletin on January 30th regarding updates required for licensed cannabis testing labs. The updated method for microbial contaminant testing includes a longer incubation period in yeast and mold testing.BannerForEnf

“After careful consideration of emerging data regarding the use and effectiveness of 3M Total Yeast and Mold Rapid Petrifilms in marijuana, CDPHE has concluded that 48 hours is not a sufficient incubation period to obtain accurate results,” the letter states. “Based upon the review of this information, marijuana/marijuana products require 60-72 hours of incubation as per the manufacturer’s product instructions for human food products, animal feed and environmental products.” The letter says they determined it was necessary to increase the incubation period based on data submitted from several labs, along with a paper found in the Journal of Food Protection.

An incubator (Right) at TEQ Analytical Labs
An incubator (Right) at TEQ Analytical Labs

According to Alexandra Tudor, manager of the microbiology department at TEQ Analytical Labs (a cannabis testing lab in Aurora, CO), the update is absolutely necessary. “The incubation time extension requirement from CDPHE offers more reliable and robust data to clients by ruling out the possibility of a false yeast and mold result during analysis,” says Tudor.

Alexandra Tudor, microbiology department manager at TEQ Analytical Labs
Alexandra Tudor, microbiology department manager at TEQ Analytical Labs

“3M, the maker of Petrifilm, recommends an incubation time of 48-72 hours, but during TEQ’s method validation procedure, we learned that 48-hour incubation was not sufficient time to ensure accurate results. Although some laboratories in industry had been incubating for the minimum amount of time recommended by the manufacturer, the 48-hour incubation time does not provide a long enough window to ensure accurate detection of microbiological contaminants present in the sample.” Tudor says the update will help labs provide more confident results to clients, promoting public health sand safety.IMG_6386-2

As a result of the update in testing methodology, cultivators and infused product manufacturers in Colorado need to submit a batch test for yeast and mold. The point of requiring this batch test is to determine if the producer’s process validation is still effective, given the new yeast and mold testing method.

emerald test retail

Analyzing The Emerald Test Results: Cannabis Labs Making Progress

By Aaron G. Biros
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emerald test retail

The Emerald Test advisory panel recently convened to review the results from the Fall 2016 round of the semi-annual Inter-Laboratory Comparison and Proficiency Test (ILC/PT), ahead of the third annual Emerald Conference just a few weeks away. After reviewing and analyzing the results, the panel noticed a significant improvement across the board over their Spring 2016 round of proficiency testing.rsz_emerald-scientific_letterhead-1

Emerald Scientific’s ILC/PT program is a tool laboratories use to check how accurate their testing capabilities are compared to other labs. A lab receiving The Emerald Test badge indicates their testing meets the criteria established by the panel to demonstrate competency. This means that they were within two standard deviations of the consensus mean for all analytes tested, according to Wes Burk, vice president of Emerald Scientific. He says the labs performed better than expected on both the microbial and pesticide tests.

Wes Burk, vice president of Emerald Scientific.
Wes Burk, vice president of Emerald Scientific.

emerald test retailEach lab has access to raw, anonymized data including a consensus mean, z-scores and kernel density plots. This round measured how well 35 cannabis labs perform in testing for potency, pesticides, residual solvents and microbial contaminants such as E. coli, Salmonella, Coliform, yeast and mold.

The advisory panel includes: Robert Martin, Ph.D., founder of CW Analytical, Cynthia Ludwig, director of technical services at AOCS, Rodger Voelker, Ph.D., lab director, OG Analytical, Tammie Mussitsch, QA manager at RJ Lee Group, Shawn Kassner, senior scientist at Neptune & Company, Inc., Jim Roe, scientific director at Steep Hill Labs, Chris Hudalla, Ph.D., founder and chief scientific officer at ProVerde Labs, Sytze Elzinga, The Werc Shop and Amanda Rigdon, Chief Technical Officer at Emerald Scientific.

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Amanda Rigdon, chief technical officer at Emerald Scientific

According to Amanda Rigdon, chief technical officer at Emerald Scientific, the labs performed very well in potency, residual solvents and microbial testing PTs. This is the first year the proficiency testing includes pesticides. “All of the labs did a great job identifying every pesticide in our hemp-based PT, but some more work will most likely have to be done to bring quantitative results in line,” says Rigdon. “Since this was the first pesticide PT we had offered, we were pretty conservative when choosing analytes and their levels. For the most part, analytes and levels were taken from the Oregon pesticide list, which is widely recognized to be the most reasonable and applicable pesticide list out there to date.” They covered pesticides of high concern, like abamectin and Myclobutanil, but also included a wide range of other pesticides that labs are expected to encounter.

Shawn Kassner, senior scientist at Neptune
Shawn Kassner, senior scientist at Neptune & Company, Inc.

Shawn Kassner, senior scientist at Neptune & Company, Inc., believes microbial contamination proficiency testing should be a priority for improving public health and safety going forward. Although five participating labs did not receive badges for the microbial contamination PTs, panel members say the overall performance was really quite good. “Microbiology testing are essential analyses for all cannabis products and it’s just slower in regulatory implementation than potency testing,” says Kassner. “The risk of Salmonella and E. coli to an individual using a medical cannabis product could be very life threatening. Microbiology contamination is a huge concern for any public health agency, which is why we have seen that microbiology testing is usually the first analytical test required after potency.” Kassner notes that there were few outliers and with each Emerald PT program, he is seeing an improvement in overall laboratory performance.

For The Emerald Test’s next round, the panel hopes to make some improvements in the test’s robustness and consistency, like obtaining assigned values for all samples and comparing to a consensus mean. “We want to develop permanent badge criteria, streamline the appeals process and possibly implement a qualitative performance review in the pesticide PT,” says Burk. For the next round of pesticide PTs, they want to build a better list of pesticides to cover more states, allowing labs to pick a set based on their state’s regulations. Burk says they also want to collect data on whether or not matrix-matched curves were used for pesticides.

Rodger Voelker, Cynthia Ludwig and Shawn Kassner, all members of the advisory panel, will be speaking at the Emerald Conference, discussing some of their findings from this round of proficiency testing. The Emerald Conference will take place February 2nd and 3rd in San Diego, CA.

The Practical Chemist

Potency Analysis of Cannabis and Derivative Products: Part 2

By Rebecca Stevens
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As mentioned in Part 1, the physiological effects of cannabis are mediated by a group of structurally related organic compounds known as cannabinoids. The cannabinoids are biosynthetically produced by a growing cannabis plant and Figure 1 details the biosynthetic pathways leading to some of the most important cannabinoids in plant material.

Potency figure 1
Figure 1: The biosynthetic pathway of phytocannabinoid production in cannabis has been deeply studied through isotopic labeling experiments

The analytical measurement of cannabinoids is important to ensure the safety and quality of cannabis as well as its extracts and edible formulations. Total cannabinoid levels can vary significantly between different cultivars and batches, from about 5% up to 20% or more by dry weight. Information on cannabinoid profiles can be used to tailor cultivars for specific effects and allows end users to select an appropriate dose.

Routine Analysis vs. Cannabinomics 

Several structurally analogous groups of cannabinoids exist. In total, structures have been assigned for more than 70 unique phytocannabinoids as of 2005 and the burgeoning field of cannabinomics seeks to comprehensively measure these compounds.¹

Considering practical potency analysis, the vast majority of cannabinoid content is accounted for by 10-12 compounds. These include Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabigerol (CBG), Δ9-tetrahydrocannabivarian (THCV), cannabidivarin (CBDV) and their respective carboxylic acid forms. The cannabinoids occur primarily as carboxylic acids in plant material. Decarboxylation occurs when heat is applied through smoking, vaporization or cooking thereby producing neutral cannabinoids which are more physiologically active.

Potency Analysis by HPLC and GC

Currently, HPLC and GC are the two most commonly used techniques for potency analysis. In the case of GC, the heat used to vaporize the injected sample causes decarboxylation of the native cannabinoid acids. Derivatization of the acids may help reduce decarboxylation but overall this adds another layer of complexity to the analysis² ³. HPLC is the method of choice for direct analysis of cannabinoid profiles and this technique will be discussed further.

A sample preparation method consisting of grinding/homogenization and alcohol extraction is commonly used for cannabis flower and extracts. It has been shown to provide good recovery and precision² ³. An aliquot of the resulting extract can then be diluted with an HPLC compatible solvent such as 25% water / 75% acetonitrile with 0.1% formic acid. The cannabinoids are not particularly water soluble and can precipitate if the aqueous percentage is too high.

To avoid peak distortion and shifting retention times the diluent and initial mobile phase composition should be reasonably well matched. Another approach is to make a smaller injection (1-2 µL) of a more dissimilar solvent. The addition of formic acid or ammonium formate buffer acidifies the mobile phase and keeps the cannabinoid acids protonated.

The protonated acids are neutral and thus well retained on a C18 type column, even at higher (~50% or greater) concentrations of organic solvent² ³.

Detection is most often done using UV absorbance. Two main types of UV detectors are available for HPLC, single wavelength and diode array. A diode array detector (DAD) measures absorbance across a range of wavelengths producing a spectrum at each point in a chromatogram while single wavelength detectors only monitor absorbance at a single user selected wavelength. The DAD is more expensive, but very useful for detecting coelutions and interferences.

References

  1. Chemical Constituents of Marijuana: The Complex Mixture of Natural Cannabinoids. Life Sciences, 78, (2005), pp. 539
  2. Development and Validation of a Reliable and Robust Method for the Analysis of Cannabinoids and Terpenes in Cannabis. Journal of AOAC International, 98, (2015), pp. 1503
  3. Innovative Development and Validation of an HPLC/DAD Method for the Qualitative and Quantitative Determination of Major Cannabinoids in Cannabis Plant Material. Journal of Chromatography B, 877, (2009), pp. 4115

Rebecca is an Applications Scientist at Restek Corporation and is eager to field any questions or comments on cannabis analysis, she can be reached by e-mail, rebecca.stevens@restek.com or by phone at 814-353-1300 (ext. 2154)

amandarigdon
The Nerd Perspective

Pesticide Detection in Cannabis: Lab Challenges and Why Less Isn’t Always More

By Amanda Rigdon
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amandarigdon

Almost as soon as cannabis became recreationally legal, the public started to ask questions about the safety of products being offered by dispensaries – especially in terms of pesticide contamination. As we can see from the multiple recalls of product there is a big problem with pesticides in cannabis that could pose a danger to consumers. While The Nerd Perspective is grounded firmly in science and fact, the purpose of this column is to share my insights into the cannabis industry based on my years of experience with multiple regulated industries with the goal of helping the cannabis industry mature using lessons learned from other established markets. In this article, we’ll take a look at some unique challenges facing cannabis testing labs, what they’re doing to respond to the challenges, and how that can affect the cannabis industry as a whole.

Photo: Michelle Tribe, Flickr
Photo: Michelle Tribe, Flickr

The Big Challenge

Over the past several years, laboratories have quickly ‘grown up’ in terms of technology and expertise, improving their methods for pesticide detection to improve data quality and lower detection limits, which ultimately ensures a safer product by improving identification of contaminated product. But even though cannabis laboratories are maturing, they’re maturing in an environment far different than labs from regulated industry, like food laboratories. Food safety testing laboratories have been governmentally regulated and funded from almost the very beginning, allowing them some financial breathing room to set up their operation, and ensuring they won’t be penalized for failing samples. In contrast, testing fees for cannabis labs are paid for by growers and producers – many of whom are just starting their own business and short of cash. This creates fierce competition between cannabis laboratories in terms of testing cost and turnaround time. One similarity that the cannabis industry shares with the food industry is consumer and regulatory demand for safe product. This demand requires laboratories to invest in instrumentation and personnel to ensure generation of quality data. In short, the two major demands placed on cannabis laboratories are low cost and scientific excellence. As a chemist with years of experience, scientific excellence isn’t cheap, thus cannabis laboratories are stuck between a rock and a hard place and are feeling the squeeze.

Responding to the Challenge

One way for high-quality laboratories to win business is to tout their investment in technology and the sophistication of their methods; they’re selling their science, a practice I stand behind completely. However, due to the fierce competition between labs, some laboratories have oversold their science by using terms like ‘lethal’ or ‘toxic’ juxtaposed with vague statements regarding the discovery of pesticides in cannabis using the highly technical methods that they offer. This juxtaposition can then be reinforced by overstating the importance of ultra-low detection levels outside of any regulatory context. For example, a claim stating that detecting pesticides at the parts per trillion level (ppt) will better ensure consumer safety than methods run by other labs that only detect pesticides at concentrations at parts per billion (ppb) concentrations is a potentially dangerous claim in that it could cause future problems for the cannabis industry as a whole. In short, while accurately identifying contaminated samples versus clean samples is indeed a good thing, sometimes less isn’t more, bringing us to the second half of the title of this article.

Less isn’t always more…

Spiral Galaxy Milky Way
The Milky Way

In my last article, I illustrated the concept of the trace concentrations laboratories detect, finishing up with putting the concept of ppb into perspective. I wasn’t even going to try to illustrate parts per trillion. Parts per trillion is one thousand times less concentrated than parts per billion. To put ppt into perspective, we can’t work with water like I did in my previous article; we have to channel Neil deGrasse Tyson.

The Milky Way galaxy contains about 100 billion stars, and our sun is one of them. Our lonely sun, in the vastness of our galaxy, where light itself takes 100,000 years to traverse, represents a concentration of 10 ppt. On the surface, detecting galactically-low levels of contaminants sounds wonderful. Pesticides are indeed lethal chemicals, and their byproducts are often lethal or carcinogenic as well. From the consumer perspective, we want everything we put in our bodies free of harmful chemicals. Looking at consumer products from The Nerd Perspective, however, the previous sentence changes quite a bit. To be clear, nobody – nerds included – wants food or medicine that will poison them. But let’s explore the gap between ‘poison’ and ‘reality’, and why that gap matters.

FDAIn reality, according to a study conducted by the FDA in 2011, roughly 37.5% of the food we consume every day – including meat, fish, and grains – is contaminated with pesticides. Is that a good thing? No, of course it isn’t. It’s not ideal to put anything into our bodies that has been contaminated with the byproducts of human habitation. However, the FDA, EPA, and other governmental agencies have worked for decades on toxicological, ecological, and environmental studies devoted to determining what levels of these toxic chemicals actually have the potential to cause harm to humans. Rather than discuss whether or not any level is acceptable, let’s take it on principle that we won’t drop over dead from a lethal dose of pesticides after eating a salad and instead take a look at the levels the FDA deem ‘acceptable’ for food products. In their 2011 study, the FDA states that “Tolerance levels generally range from 0.1 to 50 parts per million (ppm). Residues present at 0.01 ppm and above are usually measurable; however, for individual pesticides, this limit may range from 0.005 to 1 ppm.” Putting those terms into parts per trillion means that most tolerable levels range from 100,000 to 50,000,000 ppt and the lower limit of ‘usually measurable’ is 10,000 ppt. For the food we eat and feed to our children, levels in parts per trillion are not even discussed because they’re not relevant.

green apple with slice isolated on the white background.

A specific example of this is arsenic. Everyone knows arsenic is very toxic. However, trace levels of arsenic naturally occur in the environment, and until 2004, arsenic was widely used to protect pressure-treated wood from termite damage. Because of the use of arsenic on wood and other arsenic containing pesticides, much of our soil and water now contains some arsenic, which ends up in apples and other produce. These apples get turned into juice, which is freely given to toddlers everywhere. Why, then, has there not an infant mortality catastrophe? Because even though the arsenic was there (and still is), it wasn’t present at levels that were harmful. In 2013, the FDA published draft guidance stating that the permissible level of arsenic in apple juice was 10 parts per billion (ppb) – 10,000 parts per trillion. None of us would think twice about offering apple juice to our child, and we don’t have to…because the dose makes the poison.

How Does This Relate to the Cannabis Industry?

The concept of permissible exposure levels (a.k.a. maximum residue limits) is an important concept that’s understood by laboratories, but is not always considered by the public and the regulators tasked with ensuring cannabis consumer safety. As scientists, it is our job not to misrepresent the impact of our methods or the danger of cannabis contaminants. We cannot understate the danger of these toxins, nor should we overstate their danger. In overstating the danger of these toxins, we indirectly pressure regulators to establish ridiculously low limits for contaminants. Lower limits always require the use of newer testing technologies, higher levels of technical expertise, and more complicated methods. All of this translates to increased testing costs – costs that are then passed on to growers, producers, and consumers. I don’t envy the regulators in the cannabis industry. Like the labs in the cannabis industry, they’re also stuck between a rock and a hard place: stuck between consumers demanding a safe product and producers demanding low-cost testing. As scientists, let’s help them out by focusing our discussion on the real consumer safety issues that are present in this market.

*average of domestic food (39.5% contaminated) and imported food (35.5% contaminated)

The Practical Chemist

Building the Foundation of Medical Cannabis Testing – Understanding the Use of Standards and Reference Materials – Part 2

By Joe Konschnik
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In the last article I referred to the analogy of the analytical reference material being a keystone of the laboratory foundation, the stone upon which all data relies. I then described the types of reference materials and their use in analytical testing in general terms. This article will describe the steps required to properly manufacture and deliver a certified reference material (CRM) along with the necessary documentation.

A CRM is an exclusive reference material that meets strict criteria defined by ISO Guide 34 and ISO/IEC 17025.  ISO is the International Organization for Standardization and IEC is the International Electrotechnical Commission. These organizations work together to set globally recognized standards. In order for a reference material to be labeled as a CRM it must 1) be made with raw or starting materials which are characterized using qualified methods and instruments, 2) be produced in an ISO-accredited lab under documented procedures, and 3) fall under the manufacturer’s scopes of accreditation. Verifying a CRM supplier has these credentials is easily done by viewing their certificates which should include their scopes of accreditation. Restek_accredit

There are many steps required to produce a CRM that meets the above three criteria.  The first step requires a review of the customer’s, or end-user’s requirements to carefully define what is to be tested, at what levels and which analytical workflow will be used.  Such information enables the producer to identify the proper compounds and solvents required to properly formulate the requested CRM.

The next step requires sourcing and acquiring the raw, or starting materials, then verifying their compatibility and stability using stability and shipping studies in accordance with ISO requirements. Next the chemical identify and purity of the raw materials must be characterized using one or more analytical techniques such as: GC-FID, HPLC, GC-ECD, GC-MS, LC-MS, refractive index and melting point. In some cases, the percent purity is changed by the producer when their testing verifies it’s different from the supplier label. All steps are of course documented.

restek_CRMThe producer’s analytical balances must be verified using NIST traceable weights and calibrated annually by an accredited third party provider to guarantee accurate measurement. CRMs must be prepared using Class A volumetric glassware, and all ampules and vials used in preparation and final packaging must be chemically treated to prevent compound degradation during storage. Next, CRMs are packaged in an appropriate container, labeled then properly stored to maintain the quality and stability until it’s ready to be shipped. All labels must include critical storage, safety and shelf life information to meet federal requirements. The label information must be properly linked to documentation commonly referred to as a certificate of analysis (COA) which describes all of the above steps and verifies the traceability and uncertainty of all measurements for each compound contained in the CRM. Restek_CRM2

My company, RESTEK, offers a variety of documentation choices to accompany each CRM. Depending on the intended use and data quality objectives specified by the end-user, which were defined way back at the first step, three options are typically offered:  They include gravimetric only, qualitative which includes gravimetric, and fully quantitative which includes all three levels of documentation. The graphic to the right summarizes the three options and what they include. 

It’s important to understand which level you’re purchasing especially when ordering a custom CRM from a supplier. Most stock CRMs include all three levels of documentation, but it’s important to be sure.

Understanding what must be done to produce and deliver a CRM sets it apart from other reference material types, however it’s important to understand there are some instances where CRMs are either not available, nor required and in those situations other types of reference materials are perfectly acceptable.

If you have any questions or would like more details about reference materials please contact me, Joe Konschnik at (800) 356-1688 ext. 2002 by phone, or email me at joe.konschnik@restek.com.