Tag Archives: methodology

Swetha Kaul, PhD

Colorado vs. California: Two Different Approaches to Mold Testing in Cannabis

By Swetha Kaul, PhD
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Swetha Kaul, PhD

Across the country, there is a patchwork of regulatory requirements that vary from state to state. Regulations focus on limiting microbial impurities (such as mold) present in cannabis in order for consumers to receive a safe product. When cultivators in Colorado and Nevada submit their cannabis product to laboratories for testing, they are striving to meet total yeast and mold count (TYMC) requirements.In a nascent industry, it is prudent for state regulators to reference specific testing methodologies so that an industry standard can be established.

TYMC refers to the number of colony forming units present per gram (CFU/g) of cannabis material tested. CFU is a method of quantifying and reporting the amount of live yeast or mold present in the cannabis material being tested. This number is determined by plating the sample, which involves spreading the sample evenly in a container like a petri dish, followed by an incubation period, which provides the ideal conditions for yeast and mold to grow and multiply. If the yeast and mold cells are efficiently distributed on a plate, it is assumed that each live cell will give rise to a single colony. Each colony produces a visible spot on the plate and this represents a single CFU. Counting the numbers of CFU gives an accurate estimate on the number of viable cells in the sample.

The plate count methodology for TYMC is standardized and widely accepted in a variety of industries including the food, cosmetic and pharmaceutical industries. The FDA has published guidelines that specify limits on total yeast and mold counts ranging from 10 to 100,000 CFU/g. In cannabis testing, a TYMC count of 10,000 is commonly used. TYMC is also approved by the AOAC for testing a variety of products, such as food and cosmetics, for yeast and mold. It is a fairly easy technique to perform requiring minimal training, and the overall cost tends to be relatively low. It can be utilized to differentiate between dead and live cells, since only viable living cells produce colonies.

Petri dish containing the fungus Aspergillus flavus
Petri dish containing the fungus Aspergillus flavus.
Photo courtesy of USDA ARS & Peggy Greb.

There is a 24 to 48-hour incubation period associated with TYMC and this impedes speed of testing. Depending on the microbial levels in a sample, additional dilution of a cannabis sample being tested may be required in order to count the cells accurately. TYMC is not species-specific, allowing this method to cover a broad range of yeast and molds, including those that are not considered harmful. Studies conducted on cannabis products have identified several harmful species of yeast and mold, including Cryptococcus, Mucor, Aspergillus, Penicillium and Botrytis Cinerea. Non-pathogenic molds have also been shown to be a source of allergic hypersensitivity reactions. The ability of TYMC to detect only viable living cells from such a broad range of yeast and mold species may be considered an advantage in the newly emerging cannabis industry.

After California voted to legalize recreational marijuana, state regulatory agencies began exploring different cannabis testing methods to implement in order to ensure clean cannabis for the large influx of consumers.

Unlike Colorado, California is considering a different route and the recently released emergency regulations require testing for specific species of Aspergillus mold (A. fumigatus, A. flavus, A. niger and A. terreus). While Aspergillus can also be cultured and plated, it is difficult to differentiate morphological characteristics of each species on a plate and the risk of misidentification is high. Therefore, positive identification would require the use of DNA-based methods such as polymerase chain reaction testing, also known as PCR. PCR is a molecular biology technique that can detect species-specific strains of mold that are considered harmful through the amplification and analysis of DNA sequences present in cannabis. The standard PCR testing method can be divided into four steps:

  1. The double stranded DNA in the cannabis sample is denatured by heat. This refers to splitting the double strand into single strands.
  2. Primers, which are short single-stranded DNA sequences, are added to align with the corresponding section of the DNA. These primers can be directly or indirectly labeled with fluorescence.
  3. DNA polymerase is introduced to extend the sequence, which results in two copies of the original double stranded DNA. DNA polymerases are enzymes that create DNA molecules by assembling nucleotides, the building blocks of DNA.
  4. Once the double stranded DNA is created, the intensity of the resulting fluorescence signal can uncover the presence of specific species of harmful Aspergillus mold, such as fumigatus.

These steps can be repeated several times to amplify a very small amount of DNA in a sample. The primers will only bind to the corresponding sequence of DNA that matches that primer and this allows PCR to be very specific.

PCR testing is used in a wide variety of applications
PCR testing is used in a wide variety of applications
Photo courtesy of USDA ARS & Peggy Greb.

PCR is a very sensitive and selective method with many applications. However, the instrumentation utilized can be very expensive, which would increase the overall cost of a compliance test. The high sensitivity of the method for the target DNA means that there are possibilities for a false positive. This has implications in the cannabis industry where samples that test positive for yeast and mold may need to go through a remediation process to kill the microbial impurities. These remediated samples may still fail a PCR-based microbial test due to the presence of the DNA. Another issue with the high selectivity of this method is that other species of potentially harmful yeast and mold would not even be detected. PCR is a technique that requires skill and training to perform and this, in turn, adds to the high overall cost of the test.

Both TYMC and PCR have associated advantages and disadvantages and it is important to take into account the cost, speed, selectivity, and sensitivity of each method. The differences between the two methodologies would lead to a large disparity in testing standards amongst labs in different states. In a nascent industry, it is prudent for state regulators to reference specific testing methodologies so that an industry standard can be established.

Designing Your Continuing Cannabis Education Program

By RJ Starr
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As many states’ medical cannabis programs are already in full swing and several are launching or nearing their one-year or biennial maturation periods, medical cannabis dispensaries and cannabis cultivation and processing facilities should be fine-tuning their Continuing Cannabis Education Program, or CCEP, and be ready for inspection by state agencies.

While states with medical cannabis programs administer them through various agencies such as Department of Medicine/Health, Department of Pharmacy, Department of Commerce, Alcoholic Beverage Control, each has their own minimum requirements for continuing education in the medical cannabis space, and each structures their program in the direction within which that particular regulatory agency leans. Each state’s personality also brings an influential component as well; for example, a state with a highly visible opioid crisis may place greater emphasis on substance abuse training.

Suffice it to say that while there is certainly insight to be gained from knowing your particular state, there are certain elements of an ongoing professional development program that should be considered in each CCEP. This article will explore a few of the elements integral to any successful human capital and professional development plan from a vantage of compliance, and will offer some insight into the exceptional training methodology designed by Midwest Compassion Center and Bloom Medicinals.

There are a number of key considerations in developing a Continuing Cannabis Education Program, and a thoughtful CCEP should be developed specifically to meet the needs of both the organization and its employees. This can be done by a needs assessment consisting of three levels: organizational, occupational, and individual assessments.

  1. Needs assessment and learning objectives. This part of the framework development asks you to consider what kind of training is needed in your organization. Once you have determined the training needed, you can set learning objectives to measure at the end of the training.
    1. Organizational assessment. In this type of needs assessment, we can determine the skills, knowledge and abilities our cannabis dispensaries need in order to meet their strategic objectives. This type of assessment considers things such as changing laws, demographics and technology trends. Overall, this type of assessment looks at how the organization as a whole can handle its weaknesses while promoting strengths.
    2. Occupational (task) assessment. This type of assessment looks at the specific tasks, skills, knowledge and abilities required of our employees to do the jobs necessary within our dispensaries.
    3. Individual assessment. An individual assessment looks at the performance of an individual employee and determines what training should be accomplished for that individual.
  2. Consideration of learning styles. Making sure to teach to a variety of learning styles is important to development of training programs.
  3. Delivery mode. What is the best way to get your message across? Is classroom or web-based training more appropriate, or should one-on-one mentoring be used? Successful training programs should incorporate a variety of delivery methods.
  4. How much money do you have to spend on this training? This does not only include the cost of materials, but the cost of time. Consideration should also be given to the costs associated with not investing in training: CFO asks CEO, “What happens if we invest in developing our people and then they leave us?” CEO: “What happens if we don’t, and they stay?”
  5. Delivery style. Will the training be self-paced or instructor led? What kinds of discussions and interactions can be developed in conjunction with this training? The delivery style must take into account people’s individual learning styles. A balance of lectures, discussions, role-playing, and activities that solidify concepts are considered part of delivery style.
  6. Audience. Who will be part of this training? Do you have a mix of roles, such as accounting people and marketing people? What are the job responsibilities of these individuals, and how can you make the training relevant to their individual jobs? The audience for the training is an important aspect when developing your CCEP. This can allow the training to be better developed to meet the needs and the skills of a particular group of people.
  7. Content. What needs to be taught? How will you sequence the information? The content obviously is an important consideration. Learning objectives and goals for the training should be established and articulated before content is developed.
  8. Timelines. How long will it take to develop the training? Is there a deadline for training to be completed, and if so, what risk analysis can be used to determine the consequences of not meeting that deadline? After content is developed, understanding time constraints is an important aspect. Will the training take one hour or a day to deliver? What is the timeline consideration in terms of when people should take the training?
  9. Communication. How will employees know the training is available to them? Letting people know when and where the training will take place is part of communication.
  10. Measuring effectiveness. How will you know if your training worked? What ways will you use to measure this? The final aspect of developing a training framework is to consider how it will be measured. At the end, how will you know if the trainees learned what they needed to learn?

A thorough review of your state’s rules and regulations should take place quarterly, with one or more specific employees designated to stay abreast of changes. If your regulatory authority has implemented requirements that trainings must be approved in advance, know that as well, and keep your Continuous Cannabis Education Program up-to-date and ready for inspection.

The Practical Chemist

Instrumentation for Heavy Metals Analysis in Cannabis

By Chris English
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Determination of Toxic Metals in Cannabis

Heavy metals are common environmental contaminants often resulting from mining operations, industrial waste, automotive emissions, coal fired power plants, amount other sources. Several remediation strategies exist that are common for the reduction/elimination of metals in the environment. Phytoremediation is one method for removing metals from soil, utilizing plants to uptake metals which then bioaccumulate in the plant matter. In one study, cesium concentrations were found to be 8,000 times greater in the plant roots compared to the surrounding water in the soil. In 1998, cannabis was specifically tested at the Chernobyl nuclear disaster site for its ability to remediate the contaminated soil. These examples demonstrate that cannabis must be carefully cultivated to avoid the uptake of toxic metals. Possible sources would not only include the growing environment, but also materials such as fertilizers. Many states publish metal content in fertilizer products allowing growers to select the cleanest product for their plants. For cannabis plant material and concentrates several states have specific limits for cadmium (Cd), Lead (Pb), Arsenic (As) and Mercury (Hg), based on absolute limits in product or daily dosage by body weight.

Analytical Approaches to Metals Determination

Inductively Coupled Plasma, Ionized Argon gas stream. Photo Courtesy: Sigma via Wikimedia Commons

Flame Atomic Absorption Spectroscopy (Flame AA) and Graphite Furnace Atomic Absorption Spectroscopy (GFAA) are both techniques that determine both the identity and quantity of specific elements. For both of these techniques, the absorption in intensity of a specific light source is measured following the atomization of the sample digestate using either a flame or an electrically heated graphite tube. Reference standards are analyzed prior to the samples in order to develop a calibration that relates the concentration of each element relative to its absorbance. For these two techniques, each element is often determined individually, and the light source, most commonly a hollow cathode lamp (HLC) or electrodeless discharge lamp (EDL) are specific for each element. The two most common types of Atomic Emission Spectroscopy (AES) are; Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) and ICP-Mass Spectrometry (ICP-MS). Both of these techniques use an argon plasma for atomization of the sample digestates. This argon plasma is maintained using a radio frequency generator that is capable of atomization and excitation of the majority of the elements on the periodic table. Due to the considerably higher energy of the plasma-based instruments, they are more capable than the flame or furnace based systems for measurement of a wide range of elements. Additionally, they are based on optical emission, or mass spectrometric detection, and are capable of analysis of all elements at essentially the same time.

Technique Selection

Flame AA is easy to use, inexpensive and can provide reasonable throughput for a limited number of elements. However, changes to light sources and optical method parameters are necessary when determining different metals. GFAA is also limited by similar needs to change the light sources, though it is capable of greater sensitivity for most elements as compared to flame AA. Runtimes are on the order of three minutes per element for each sample, which can result in lower laboratory throughput and greater sample digestate consumption. While the sensitivity of the absorption techniques is reasonable, the dynamic range can be more limited requiring re-analyses and dilutions to get the sample within the calibration range. ICP-OES allows the simultaneous analysis of over 70 elements in approximately a minute per sample with a much greater linear dynamic range. ICP-OES instruments cost about 2-5 times more than AA instruments. ICP-MS generally has the greatest sensitivity (sub-parts-per-trillion, for some elements) with the ability to determine over 70 elements per minute. Operator complexity, instrument expense and MS stability, as well as cost are some of the disadvantages. The US FDA has a single laboratory validated method for ICP-MS for elements in food using microwave assisted digestion, and New York State recently released a method for the analysis of metals in medical cannabis products by ICP-MS (NYS DOH LINC-250).

The use of fertilizers, and other materials, with low metal content is one step necessary to providing a safe product and maintaining customer confidence. The state-by-state cannabis regulations will continue to evolve which will require instrumentation that is flexible enough to quickly accommodate added metals to the regulatory lists, lower detection limits while adding a high level of confidence in the data.

Shimadzu Launches Cannabis Analyzer for Potency

By Aaron G. Biros
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On Monday, March 6th, Shimadzu Scientific Instruments, a leading laboratory analytical instrumentation manufacturer, announced the launch of a new product focused on cannabis, according to a press release. Their Cannabis Analyzer for Potency is essentially a high-performance liquid chromatograph (HPLC) packaged with integrated hardware, software, workflows and all the supplies. The supplies include an analytical column, guard columns, mobile phase and a CRM standard mixture.canAnalyzerImg1

The instrument is designed to test for 11 cannabinoids in less time and with greater ease than traditional HPLC instruments. In the press release, they claim “operators are now able to produce accurate results with ease, regardless of cannabis testing knowledge or chromatography experience.” One very unique aspect of the instrument is the lack of experience required to run it, according to Bob Clifford, general manager of marketing at Shimadzu. “We have our typical chromatography software [LabSolutions] with an overlay that allows the user to analyze a sample in three simple steps,” says Clifford. Those in the cannabis industry that have a background in plant science, but not analytical chemistry, could run potency analyses on the instrument with minimal training. “This overlay allows ease of use for those not familiar with chromatography software,” says Clifford.

An overlay of a flower sample with the standards supplied in the High-Sensitivity Method package.
An overlay of a flower sample with the standards supplied in the High-Sensitivity Method package.

The instrument can determine cannabinoid percentages per dry weight in flower concentrates and edibles. “Once you open the software, it will get the flow rate started, heat the column up and automatically begin to prep for analysis,” says Clifford. Before the analysis begins, information like the sample ID number, sample name, sample weight, extraction volume and dilution volume are entered. After the analysis is complete all the test results are reported for each sample.

Because laboratories wouldn’t have to develop quantitative testing methodology, they argue this instrument would save a lot of time in the lab. “After one day of installation and testing, users are equipped with everything they need to obtain cannabis potency results,” states the press release. According to Clifford, method development for potency analysis in-house can take some labs up to three months. “We can bring this instrument to the lab and have it ready for testing almost immediately,” says Clifford. “The methods for this instrument were developed by a team of twenty scientists working on different platforms at our Innovation Center and was tested for ruggedness, repeatability and quantitative accuracy.”

Screenshots from the software on the instrument
Screenshots from the software on the instrument

The instrument’s workflow is designed to meet three methods of analysis depending on testing needs. The High Throughput method package can determine quantities of ten cannabinoids with less than eight minutes per sample. The method was developed in collaboration with commercial testing laboratories. The High Sensitivity method package adds THCV to that target analyte list with ten minutes per analysis. The method provides the sharpest chromatographic peaks and best sensitivity. The High Resolution method package offers full baseline resolution for those 11 cannabinoids in less than 30 minutes per analysis and the ability to add cannabinoids to that target list if regulations change.

The press release states the interface should allow users to reduce the number of steps needed in the analysis and simplify the workflow. The instrument comes with a three-year warranty, preventative maintenance plan and lifetime technical support.

From The Lab

QuEChERS 101

By Danielle Mackowsky
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Sample preparation experts and analytical chemists are quick to suggest QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) to cannabis laboratories that are analyzing both flower and edible material for pesticides, mycotoxins and cannabinoid content. Besides having a quirky name, just what makes QuEChERS a good extraction technique for the complicated matrices of cannabis products? By understanding the chemistry behind the extraction and the methodology’s history, cannabis laboratories can better implement the technology and educate their workforce.

QuEChERS salt blends can be packed into mylar pouches for use with any type of centrifuge tubes
QuEChERS salt blends can be packed into mylar pouches for use with any type of centrifuge tubes

In 2003, a time when only eight states had legalized the use of medical cannabis, a group of four researchers published an article in the Journal of AOAC International that made quite the impact in the residue monitoring industry. Titled Fast and Easy Multiresidue Method Employing Acetonitrile Extraction/Partitioning and “Dispersive Solid-Phase Extraction” for the Determination of Pesticide Residues in Produce, Drs. Michael Anastassiades, Steven Lehotay, Darinka Štajnbaher and Frank Schenck demonstrate how hundreds of pesticides could be extracted from a variety of produce samples through the use of two sequential steps: an initial phase partitioning followed by an additional matrix clean up. In the paper’s conclusion, the term QuEChERS was officially coined. In the fourteen years that have followed, this article has been cited over 2800 times. Subsequent research publications have demonstrated its use in matrices beyond food products such as biological fluids, soil and dietary supplements for a plethora of analytes including phthalates, pharmaceutical compounds and most recently cannabis.

QuEChERS salts can come prepacked into centrifuge tubes
QuEChERS salts can come prepacked into centrifuge tubes

The original QuEChERS extraction method utilized a salt blend of 4 g of magnesium sulfate and 1 g of sodium chloride. A starting sample volume of 10 g and 10 mL of acetonitrile (ACN) were combined with the above-mentioned salt blend in a centrifuge tube. The second step, dispersive solid phase extraction (dSPE) cleanup, included 150 mg of magnesium sulfate and 25 mg of primary secondary amine (PSA). Subsequent extraction techniques, now known as AOAC and European QuEChERS, suggested the use of buffered salts in order to protect any base sensitive analytes that may be critical to one’s analysis. Though the pH of the extraction solvent may differ, all three methods agree that ACN should be used as the starting organic phase. ACN is capable of extracting the broadest range of analytes and is compatible with both LC-MS/MS and GC-MS systems. While ethyl acetate has also been suggested as a starting solvent, it is incompatible with LC-MS/MS and extracts a larger amount of undesirable matrix components in the final aliquot.

All laboratories, including cannabis and food safety settings, are constantly looking for ways to decrease their overhead costs, batch out the most samples possible per day, and keep their employees trained and safe. It is not a stretch to say that QuEChERS revolutionized the analytical industry and made the above goals tangible achievements. In the original publication, Anastassiades et al. established that recoveries of over 85% for pesticides residues were possible at a cost as low as $1 per ten grams of sample. Within forty minutes, up to twelve samples were fully extracted and ready to be analyzed by GC-MS, without the purchase of any specialized equipment. Most importantly, no halogenated solvents were necessary, making this an environmentally conscious concept. Due to the nature of the cannabis industry, laboratories in this field are able to decrease overall solvent usage by a greater amount than what was demonstrated in 2003. The recommended starting sample for cannabis laboratories is only one gram of flower, or a tenth of the starting volume that is commonly utilized in the food safety industry. This reduction in sample volume then leads to a reduction in acetonitrile usage and thus QuEChERS is a very green extraction methodology.

The complexity of the cannabis matrix can cause great extraction difficulties if proper techniques are not used
The complexity of the cannabis matrix can cause great extraction difficulties if proper techniques are not used

As with any analytical method, QuEChERS is not perfect or ideal for every laboratory setting. Challenges remain in the cannabis industry where the polarity of individual pesticides monitored in some states precludes them from being amenable to the QuEChERS approach. For cannabis laboratories looking to improve their pesticide recoveries, decrease their solvent usage and not invest their resources into additional bench top equipment, QuEChERS is an excellent technique to adopt. The commercialization of salt blends specific for cannabis flowers and edibles takes the guesswork out of which products to use. The growth of cannabis technical groups within established analytical organizations has allowed for better communication among scientists when it comes to best practices for this complicated matrix. Overall, it is definitely worth implementing the QuEChERS technique in one’s cannabis laboratory in order to streamline productivity without sacrificing your results.

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.