Tag Archives: Testing

Terpene_KAS2
From The Lab

The Other Side of Cannabis: Terpenes

By Dr. Zacariah Hildenbrand, Allegra Leghissa, Dr. Kevin A. Schug
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Terpene_KAS2

Have you ever wondered why all beers have that strong, characteristic smell? Or why you could tell the smell of cannabis apart from any other plant? The answer is simple – terpenes.

These 55,000 different molecules are responsible for a majority of the odors and fragrances around us, from a pine forest, to the air diffuser in your house 1–3. They all share the same precursor, isoprene, and because of that, they are all related and have similar molecular structures. Unfortunately, it is this uncanny similarity that makes their analysis so challenging; we still lack a complete list of which terpenes expected to be found in each given plant species 1,2.

Many different methods have been developed in an effort to provide a time-optimized and straightforward analysis. Gas chromatography (GC) is usually center stage due to the volatility of the terpenes. Therefore, there is significant concern with the type of GC detector used 2.

The flame ionization detector (FID) is a good quantitative detector for GC, but qualitatively it does not provide any information, except for retention time; the differentiation between terpene species is achieved solely by use of retention indices (RI), which are based on elution times from a particular GC stationary phase. The best part of the FID is its low cost, reliability, and relatively easy interface, which make it an effective tool for quality control (QC) but less so with respect to research and discovery 2.

The primary choice for a research setting is the mass spectrometer (MS) detector. It is more expensive and complicated than FID, but importantly, it provides both good quantitative capabilities, and it provides mass spectra for each species that elutes from the chromatograph. However, for terpene analysis, it may still not be the best detector choice. Since terpene class molecules share many structural and functional similarities, even their fragmentation and sub-sequential identification by MS may lead to inconsistent results, which need to be confirmed by use of RI. Still, MS is a better qualitative analysis tool than the FID, especially for distinguishing non-isobaric terpenes 2.

Recently, new technology based on vacuum ultraviolet spectroscopy (VUV) has been developed as a new GC detector. The VUV detector enables analysis of virtually all molecules; virtually all chemical compounds absorb light in the range in the 125-240 nm wavelength range probed by the detector, making it an essentially universal detector 4–11. Previously, spectroscopic absorption detectors for GC have lacked sufficient energy to measure absorption of most GC-amenable species. The VUV detector fills a niche, which is complementary to MS detection in terms of the qualitative information it provides.

Terpene_KAS2
Figure 1: A, Section of the chromatographic separation of a terpenes standard mix; B, highlight of the co-eluting terpenes, camphor and (-)-isopulegol; C, differences in the absorbance spectra of camphor and (-)-isopulegol.

With the VUV detector, each compound exhibits its own unique absorbance spectrum. Even isomers and isobars, which are prevalent in terpene mixtures and can be difficult to distinguish different species by their electron ionization mass spectra, can be well differentiated based on their VUV spectra 6,9,10.  Nevertheless, because analytes exhibit different spectra, it is not required to achieve a perfect chromatographic separation of the mixture components. Co-eluting peaks can be separated post-run through the use of library spectra and software inherent to the instrument 4,10. This ability is called “deconvolution”, and it is based on the fact that two co-eluting terpenes will give a peak with an absorbance spectrum equal to the sum of the two single absorbance spectra 4. Figure 1 shows the deconvolution process for two co-eluting terpenes, camphor and (-)-isopulegol. Due to their different absorbance spectra (Figure 1C), it is possible to fully separate the two peaks in post-run, obtaining sharp peaks for both analytes 6.

The deconvolution process has been shown to yield precise and accurate results. Thus, chromatographic resolution can be sacrificed in favor of spectroscopic resolution; this enables the development of methods with faster run times. With the ability to deconvolve unresolved peaks, a long temperature ramp to chromatographically separate all isomeric terpenes is not required 6. Additionally, the presence of coeluting components, which might normally go undetected with some GC detectors, can be easily judged based on comparison of the measured spectra with pure reference spectra contained in the VUV spectral library.

The other issue in terpenes analysis is the extraction process. Terpenes can be extracted with the use of solvents (e.g., methanol, ethanol, hexane, and cyclohexane, among others), but the process is usually time-consuming, costly and not so environmentally-friendly 2. The plant needs to be manually crushed and then aliquots of solvent are used to extract components from the plant, ideally at least 3 times and combined to achieve acceptable results. The problem is that some terpenes may respond better to a certain solvent, making their extraction easier and more optimized than for others 2. The choice of solvent can cause discrimination against the extraction some terpenes, which limits the comprehensiveness of analysis.

Headspace is another technique that can be used for the sample preparation of terpenes. Headspace sampling is based on heating the solid or liquid sample inside a sealed vial, and then analyzing the air above it after sufficient equilibration. In this way, only volatile analytes are extracted from the solid/liquid sample into the gas phase; this allows relatively interference-free sampling 12–14.

How do we know whether our extraction analysis methods are correct and comprehensive for a certain plant sample? Unfortunately, there is not a complete list of available molecules for each plant species, and even if two specimens may smell really similar to our nose, their terpenes profiles may be notably different. When working with a new plant material, it is difficult to predict the extraction efficiency for the vast array of terpenes that may be present. We can only perform it with different extraction and detection methods, and compare the results.

The route for a comprehensive and fast analysis of terpenes is therefore still long; however, their intoxicating aromas and inherent medicinal value has provided a growing impetus for researchers around the world. Considering the evolving importance of Cannabis and the growing body of evidence on the synergistic effects between terpenes and cannabinoids, it is likely that newly improved extraction and analysis methods will be developed, paving the way for a more complete list of terpene species that can be found in different cultivars. The use of new analytical technologies, such as the VUV detector for GC, should aid considerably in this endeavor.


References:

[1]          Breitmaier E., Terpenes: Flavors, Fragrances, Pharmaca, Pheromones. John Wiley & Sons 2006.

[2]          Leghissa A., Hildenbrand Z. L., Schug K. A., A Review of Methods for the Chemical Characterization of Cannabis Natural Products. J. Sep. Sci.2018, 41, 398–415 .

[3]          Benvenuto E., Misra B. B., Stehle F., Andre C. M., Hausman J.-F., Guerriero G., Cannabis sativa: The Plant of the Thousand and One Molecules. Front. Plant Sci2016, 719, DOI: 10.3389/fpls.2016.00019.

[4]          Schug K. A., Sawicki I., Carlton D. D., Fan H.,Mcnair H. M.,Nimmo J. P., Kroll P.,Smuts J.,Walsh P., Harrison D., Vacuum Ultraviolet Detector for Gas Chromatography. Anal. Chem.2014, 86, 8329–8335 .

[5]          Fan H.,Smuts J., Walsh P.,Harrison D., Schug K. A., Gas chromatography-vacuum ultraviolet spectroscopy for multiclass pesticide identification. J. Chromatogr. A2015, DOI: 10.1016/j.chroma.2015.02.035.

[6]          Qiu C.,Smuts J., Schug K. A., Analysis of terpenes and turpentines using gas chromatography with vacuum ultraviolet detection. J. Sep. Sci.2017, 40, 869–877 .

[7]          Leghissa A., Smuts J., Qiu C., Hildenbrand Z. L., Schug K. A., Detection of cannabinoids and cannabinoid metabolites using gas chromatography-vacuum ultraviolet spectroscopy. Sep. Sci. Plus2018, 1.

[8]          Bai L.,Smuts J., Walsh P., Fan H., Hildenbrand Z., Wong D., Wetz D., Schug K. A., Permanent gas analysis using gas chromatography with vacuum ultraviolet detection. J. Chromatogr. A2015,1388, 244–250 .

[9]          Skultety L., Frycak P., Qiu C.,Smuts J., Shear-Laude L., Lemr K., Mao J. X., Kroll P., Schug K. A., Szewczak A., Vaught C., Lurie I., Havlicek V., Resolution of isomeric new designer stimulants using gas chromatography – Vacuum ultraviolet spectroscopy and theoretical computations. Anal. Chim. Acta2017, 971, 55–67 .

[10]       Bai L., Smuts J., Walsh P., Qiu C., McNair H. M., Schug K. ., Pseudo-absolute quantitative analysis using gas chromatography–vacuum ultraviolet spectroscopy–a tutorial. Anal. Chim. Acta2017, 953, 10–22 .

[11]       Schenk J., Nagy G., Pohl N. L. B., Leghissa A., Smuts J., Schug K. A., Identification and deconvolution of carbohydrates with gas chromatography-vacuum ultraviolet spectroscopy. J. Chromatogr. A2017, 1513, 210–221 .

[12]       Van Opstaele F., De Causmaecker B., Aerts G., De Cooman L., Characterization of novel varietal floral hop aromas by headspace solid phase microextraction and gas chromatography-mass spectrometry/olfactometry. J. Agric. Food Chem.2012, 60, 12270−12281 .

[13]       Hamm S., Bleton J., Connan J., Tchapla A., A chemical investigation by headspace SPME and GC-MS of volatile and semi-volatile terpenes in various olibanum samples. Phytochemistry2005,66, 1499–1514 .

[14]       Aberl A., Coelhan M., Determination of volatile compounds in different hop Varieties by headspace-trap GC/MS-in comparison with conventional hop essential oil analysis. J. Agric. Food Chem.2012, 60, 2785−2792 .

HACCP

Hazard Analysis and Critical Control Points (HACCP) for the Cannabis Industry: Part 2

By Kathy Knutson, Ph.D.
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HACCP

HACCP is a food safety program developed in the 1960s for the food manufacturing industry, mandated for meat, seafood and juice and adopted by foodservice for the safe serving of meals at restaurants. With state requirements for the safe production of cannabis-infused products, namely edibles, facilities may be inspected against HACCP principles. The cannabis industry and state inspectors recognize the need for safe edible manufacture. Lessons can be learned from the food industry, which has advanced beyond HACCP plans to food safety plans, starting with procurement and including the shipment of finished product to customers.

In my work with the food industry, I write HACCP and food safety plans and deliver training on food safety. In Part 1 of this series, I wrote about the identification of hazards, which is the first step in HACCP plan development. Before we continue with the next HACCP step, I will discuss Good Manufacturing Practices (GMPs). GMPs are the foundation on which HACCP is built. In other words, without GMPs in place, the facility will not have a successful HACCP program. GMPs are required in the food, dietary supplement and pharmaceutical industries, all under the enforcement of the federal Food and Drug Administration (FDA). Without federal regulation for cannabis edible manufacture, there may not be state-mandated requirements for GMPs. Let me warn you that any food safety program will not succeed without proper control of GMPs.HACCP

GMPs cover all of your programs and procedures to support food safety without having a direct, instant control. For example, when brownies are baked as edibles, food safety is controlled by the time and temperature of baking. A written recipe and baking procedure are followed for the edible. The time and temperature can be recorded to provide documentation of proper baking. In the food industry, this is called a process preventative control, which is critical to food safety and is part of a HACCP plan. Failure of proper time and temperature of baking not only leads to an unacceptable product in terms of quality, but results in an unsafe product that should not be sold.

Back to GMPs. Now think of everything that was done up to the steps of mixing and baking. Let’s start with personnel. Facilities for edibles have hiring practices. Once an employee is hired, the employee is trained, and training will include food safety procedures. When working at the job after training, the employee measuring ingredients will demonstrate proper grooming and hand washing. Clean aprons, hairnets, beard nets and gloves will be provided by the facility and worn by the employee. The same goes for the employee that bakes and the employee that packages the edible. One category of GMPs is Personnel.

Edibles facilities are not foodservice; they are manufacturing. A second GMP category is cleaning and sanitizing. Food safety is controlled through proper cleaning and sanitizing of food contact surfaces (FCS). The edible facility will have in place the frequency and methods for cleaning all parts of the facility- outside, offices, restrooms, break room and others. GMPs cover the general cleaning procedures and procedures for cleaning receiving, storage; what we would consider processing to include weighing, process steps and packaging; finished product storage and shipping. Management of the facility decides the methods and frequency of cleaning and sanitizing with greater care given to processing. Without proper cleaning and sanitizing, a facility cannot achieve food safety.

I could go on and on about GMPs. Other GMPs include water safety, integrity of the buildings, pest control program, procurement, sewage disposal and waste disposal. Let’s transition back to HACCP. In Part 1 of this series, I explained identification of hazards. Hazards are one of three types: biological, chemical and physical.

At this point, I am not surprised if you are overwhelmed. After reading Part 1 of this series, did you form a food safety team? At each edibles facility, there should be at least one employee who is trained externally in food safety to the standard that foodservice meets. Classes are offered locally and frequently. When the facility is ready, the next step of training is a HACCP workshop for the food industry, not foodservice. Edibles facilities are not foodservice; they are manufacturing. Many colleges and associations provide HACCP training. Finally, at the least, one employee should attend a workshop for Preventive Controls Qualified Individual.

To institute proper GMPs, go to ConnectFood.com for a GMP checklist. Did you draw up a flow diagram after reading Part 1? With a flow diagram that starts at Receiving and ends at Shipping, the software at ConnectFood.com takes you through the writing steps of a HACCP or food safety plan. There are many resources out there for GMPs, so it can get overwhelming. ConnectFood.com is my favorite resource.

The next step in HACCP development after identification of hazards is to identify the exact step where the hazard will be controlled. Strictly speaking, HACCP only covers process preventive controls, which typically start with a weigh step and end with a packaging step. A facility may also have a step where temperature must be controlled for food safety, e.g. cooling. In HACCP, there are commonly two process preventive controls:

  • Biological hazard of Salmonella and Escherichia coli: the heat step
  • Physical hazard of metal: metal detector

Strictly speaking, HACCP does not include cleaning, sanitizing and supplier approval for procurement of ingredients and packaging. I hope you see that HACCP is not enough. There have been hundreds of recalls and outbreaks due to problems in non-processing steps. The FDA requires food manufactures to go beyond HACCP and follow a written food safety plan, which includes hazards controlled at these steps:

  • Biological hazard of Listeria monocytogenes: cleaning and sanitizing of the processing environment and equipment
  • Physical hazards coming in with ingredients: supplier approval
  • Physical hazard of glass and hard plastic: Here I am thinking of glass breaking or plastic pieces flying off buckets. This is an internal hazard and is controlled by following written procedures. The written document is a Standard Operating Procedure (SOP).
  • Chemical hazard of pesticides: supplier approval
  • Chemical hazard of mycotoxins: supplier approval
  • Chemical hazard of allergens: supplier approval, label check at Receiving and product labeling step

Does a cannabis edible facility honestly not care or not control for pesticides in ingredients because this is not part of HACCP? No. There are two ways for procurement of ingredients in which pesticides are controlled. Either the cannabis cultivation is controlled as part of the samebusiness or the facility works with a supplier to confirm the ingredient meets pesticide tolerances. Strictly speaking, this control is not part of HACCP. For this and many other reasons, HACCP is a good place to start the control of food safety when built on a solid foundation of GMPs. In the same way the food industry is required to go beyond HACCP with a food safety plan, the cannabis industry must go beyond HACCP.

My thoughts will be shared in a webinar on May 2nd hosted by CIJ and NEHA. I encourage you to listen in to continue this discussion.Please comment on this blog post below. I love feedback!

Is There a Looming Supply Bottleneck in California?

By Aaron G. Biros
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California’s regulated adult use cannabis market has been up and running for around four months now and rumors of a potential supply bottleneck on the horizon are beginning to circulate. There are a number of factors that could have an impact on the cannabis supply in the market, most of which stem from changes in the distribution channels now that the state is implementing new regulations.

Those include a slow rollout in licensing cannabis businesses, new testing requirements, the supply carryover period prior to January 1stas well as new labeling and packaging regulations. In this piece, we are going to examine some of those rumors, see if there might be some truth to them and provide some guidance for what businesses can do to prepare for this.

A Slow Start to Licensing

This one is perhaps the most obvious factor to impact the supply chain in California. Much of the delays in licensing cannabis businesses came from the issue of local control, where businesses needed to get approval from their municipality before getting a state license. In the first month of the new market, it took Los Angeles weeks longer than other counties to begin licensing dispensaries. Whereas San Diego retailers saw a massive influx of customers right away, forcing them to buy up product to meet the high demand. Smaller producers also had trouble getting licenses as quickly as some of the larger ones.

Basically it all boils down to a slow start for the new market, according to Diane Czarkowski, co-founder of Canna Advisors. “The state is requiring businesses to get their local licenses before they can get their state license and that will create a delay in operators being able to bring products to market,” says Czarkowski. She says this is pretty typical of new markets, or when a market experiences dramatic changes quickly. “It could be a brand-new market, like in Hawaii, where the operators were ready with product, but there were no labs to test the products, which caused delays.” In addition to the licensing roll out being slow to start, the temporary licenses initially awarded to businesses are set to expire soon, by the end of April.

Stricter Rules to Come

The same logic goes for the testing regulations. New testing and labeling requirements, according to the Bureau of Cannabis Control regulating the market, will be phased in throughout 2018.

CA cannabis testing chart
California’s plan for phasing in testing requirements.

The state has already phased in cannabinoids, moisture content, residual solvent, pesticide, microbial impurities and homogeneity testing to some extent. On July 1st, the state will add additional residual solvent and pesticide testing as well as foreign material testing. At the end of 2018, they plan on requiring terpenoids, mycotoxins, heavy metals and water activity testing. All of those tests cost money and all of those tests could impact suppliers’ ability to bring product to market. “Oftentimes regulations require different types of testing to be done to products without recognizing that adequately completing those tests requires different methods, equipment, and standards,” says Czarkowski. “Most labs do not have all of the necessary components, and they are very costly. Producers could wait weeks to get test results back before they know if they can sell their products.”

Back when we spoke with Josh Drayton, deputy director of the California Cannabis Industry Association, about the upcoming changes to the California market, he voiced his concerns with the coming testing rules. “A lot of testing labs are concerned they are unable to test at the state’s threshold for some of these contaminants and pesticides; the detection limits seem very low,” says Drayton. “The testing portion will take years to work out, I am sure we will remove and add different pesticides and contaminants to the list.” California’s testing industry is, however, capable of adapting to changing rules, as they’ve done in the past on more than one occasion. It should be noted that many labs in the state are on the cutting edge of testing cannabis, working with The Bureau to implement the new rules.

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Roy Bingham, CEO of BDS Analytics

Cannabis products made prior to December 31st, 2017, did not need to comply with the stricter testing rules that are coming in the next few months. This carryover period allowed dispensaries to have products on the shelves when the new market launched in the beginning of 2018. Retailers knew this rule meant they needed to stockpile product in the event of a supply bottleneck, and it appears much of that product is now sold and running out, according to Roy Bingham, founder and chief executive officer of BDS Analytics. “The true impact of licenses is starting to be felt since the carryover from December buying prior to the licensed market has been sold,” says Bingham. “Some of the major brands have consciously not applied for licenses. Some of that has to do with the flexibility the government has given them to wait.”

A fourth reason for a potential bottleneck could also come from packaging and labeling rules. “There will have to be many modifications to products to ensure they follow the new potency regulations, and many formulations will have to be modified in order to meet new regulations,” says Czarkowski. Distributor licenses, according to The Bureau, have a number of compliance documentation requirements, such as arranging for all product testing, quality assurance and packaging and label accuracy. Everything has to be packaged before it gets to a dispensary, which is a new rule California businesses need to comply with.

Pricing is the Indicator

There are a handful of reasons why prices could increase; some of them are more defined than others, the biggest factor being the tax burden passed on to consumers, where reports showed up to a 40% increase from last year. A price increase in the future could also come from The Bureau implementing testing regulations throughout 2018, as mentioned previously.

If prices were to surge enormously and very quickly, that might be an indicator that a shortage is fast approaching. A dramatic increase in price over this year could squeeze margins for smaller producers, forcing retailers to pass that burden on to consumers as well.“So yes, the rumors are true.”

According to Roy Bingham, there has been a significant increase in pricing in all categories at the retail level. “In January and February, we are seeing about 10% increases per month in average retail prices,” says Bingham. “If we look at concentrates in California during 2017, they averaged about $34 by the end of the year, whereas it was about $31 at the start of 2017. So in January, prices have increased up to $38, which is a bit above trend, but in fact we were seeing a trend upwards before January 1st as well.” Comparing that with edibles pricing, Bingham says we see a clear jump at the start of 2018. “It was basically flat in 2017, averaging $14 roughly almost straight-line across, dipped in December, then in January it jumped to $17 and then to $18 in February, a big increase and significantly more than concentrates,” says Bingham. He also says flower was hovering around $9 per gram in December 2017, but surged above $10 in February 2018.

According to Cannabis Benchmarks, the California wholesale averages surged in the summer of 2017 up to $1,631 by September, then reached their lowest point in December, with their spot index at $1,368. The Cannabis Benchmarks report underlines some important reasons for the changes in pricing, but they also attribute it to the new licensing system.

“Increasing operating expenses for businesses preparing to enter California’s licensed system in 2018 were key to propping up supply side rates in the first six months of 2017. New compliance requirements were being instituted to varying degrees by local governments, while market participants warily eyed draft regulations from state officials for guidance as to how to prepare their sites and facilities to meet under-construction regulatory mandates.”

Their report highlights some very important aspects of the supply chain. “Again, it is likely that the increased costs faced by operators up and down the supply chain exert some upward pressure on wholesale rates, preventing them from steep year-over-year declines that were observed in some of the other major Western markets,” reads the Cannabis Benchmarks report.

So How Can Businesses Prepare?

Well to start, producers should make sure their operations and product are clean and safe. Making sure your product will pass a pesticide test should be top of mind. Dispensaries should also be wise in selecting their suppliers, performing supplier quality audits or some form of verification that they meet your standards is key in a consistent supply chain.

Dr. Jon Vaught, chief executive officer of Front Range Biosciences, believes tissue culture could be a viable solution for some California producers. Using tissue culture, as a form of propagation instead of mothers and clones can be cleaner, cheaper and more efficient, thus allowing growers to keep up with demand and prevent a shortage.

Dr. Jon Vaught headshot
Dr. Jon Vaught, CEO of Front Range Biosciences

Dr. Vaught says growers could look to tissue culture as a means to “mitigate risk to their supply chain and mitigate the risk of potential loss and improve their ability to efficiently grow their plant.” Maintaining a disease-free, sterile environment is a huge advantage in the cannabis market. “The real use of tissue culture is to provide disease free, clean, certified material, that has gone through a QA program,” says Dr. Vaught. “In greenhouses, the ability to control your environment is also critical because your margin of error is high. Variations in sunlight, weather, humidity all of these things have an impact in your plants. Technology can help monitor this.”

We’ve covered the basics of tissue culture previously on CIJ, with Dr. Hope Jones chief science officer of C4 Laboratories. She echoes many of Dr. Vaught’s points, firmly believing that, having existed for decades, tissue culture is an effective propagation tool for advanced breeders or growers looking to scale up.It is a complex supply chain that requires systems thinking.

It is important to note they don’t think growers should try this at home. Work with professionals, get the necessary funding, the training and facilities required if this is a project that interest you. “There’s a pretty big barrier to entry there,” Dr. Vaught urges. “The ability to manage thousands or millions of plants in a greenhouse increases risk, whereas in the lab, you’ve got a safe, secure, sterile environment, reducing risk of disease, making things easier to manage. The producers most successful at large scale are controlling those variables to the T.”

Ultimately, one segment of the market can’t prevent a bottleneck. It is a complex supply chain that requires systems thinking. Regulators need to work with producers, manufacturers, retailers, distributors, patients, consumers and laboratories to keep an eye on the overall supply chain flow.

Diane Czarkowski says the California market should prepare for this now if they haven’t already. “We have seen supply issues in every market going through a change. Other potential bottlenecks will occur because former distribution channels will be required to change,” says Czarkowski. “So yes, the rumors are true.”

Washington Lab Conducts Transparency Study

By Aaron G. Biros
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Earlier this week Capitol Analysis Group, a cannabis-testing laboratory based in Lacey, Washington, announced they are conducting a “data-driven Lab Transparency Project, an effort to improve accuracy of cannabis testing results in the state through transparency and a new third-party auditing process,” according to a press release. They plan to look through the state’s traceability data to find patterns of deviations and possible foul play.

The project launch comes after Straightline Analytics, a Washington cannabis industry data company, released a report indicating they found rampant laboratory shopping to be present in the state. Lab shopping is a less-than-ethical business practice where cannabis producers look for the lab that will give them the most favorable results, particularly with respect to higher potency figures and lower contamination fail rates.“Lab shopping shouldn’t exist, because it is a symptom of lab variability,”

According to the press release, their report “shows that businesses that pay for the highest number of lab tests achieve, on average, reported potency levels 2.71% higher than do those that pay for the lowest number of lab tests.” They also found labs that provide higher potency figures tend to have the largest market share.

The Lab Transparency Project logo
The Lab Transparency Project logo

The goal of The Lab Transparency Project is to provide summaries of lab data across the state, shining a light in particular on which labs provide the highest potency results. “Lab shopping shouldn’t exist, because it is a symptom of lab variability,” says Jeff Doughty, president of Capitol Analysis. “We already have standards that should prevent variations in lab results and proficiency testing that shows that the labs are capable of doing the testing.” The other piece to this project is independent third party auditing, where they hope other labs will collaborate in the name of transparency and honesty. “Problems arise when the auditors aren’t looking,” says Doughty. “Therefore, we’re creating the Lab Transparency Project to contribute to honesty and transparency in the testing industry.”

Dr. Jim McRae, founder of Straightline Analytics, and the author of that inflammatory report, has been a vocal critic of the Washington cannabis testing industry for years now. “I applaud Capitol Analysis for committing to this effort,” says McRae. “With the state’s new traceability system up and running following a 4-month breakdown, the time for openness and transparency is now.” Dr. McRae will be contributing to the summaries of lab data as part of the project.

According to Doughty, the project is designed to be a largely collaborative effort with other labs, dedicated to improving lab standards and transparency in the industry.

Cannabis Regulation Update, Terpene & Residual Solvent Analysis and Heavy Metals Analysis

This free webinar, hosted by Perkin Elmer and CIJ, will dive into the regulatory environments surrounding cannabis testing, as well as terpene, residual solvent and heavy metals testing. Are you finding it difficult to keep up with changing regulations for the cannbis industry? Are you looking to reduce hands-on analysis time? Speed up sample processing? Have less waste? Possibly reduce the number of individual tests running in your laboratory?
Learning Objectives:
• Understand and prepare for new state regulations in the US and pending federal regulations for Canada
• Learn how to run two regulated tests (terpenes and residual solvents) in one analysis, on one instrument, and with one SOP
• Overcome challenging cannabis matrices for heavy metal analysis with microwave digestion

Sign up for free today!

A2LA Accredits First Cannabis Lab in PA

By Aaron G. Biros
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According to a press release sent out this morning, the American Association for Laboratory Accreditation (A2LA) accredited their first Pennsylvania cannabis-testing laboratory. Located in Harrisburg, PA, Keystone State Testing finalized their accreditation for ISO/IEC 17025 on February 21, 2018.

KeystoneStateTestinglogoA2LA also accredited the laboratory to two cannabis-testing-specific programs, ISO/IEC 17025 – General Requirements for the Competence of Testing and Calibration Laboratories and A2LA R243 – Specific Requirements – Cannabis Testing Laboratory Accreditation Program. The R243 program is a collaboration with Americans for Safe Access (ASA) that takes some recommendation for regulators from the American Herbal Products Association (AHPA).

KellyGreenland
Dr. Kelly Greenland, owner and operator of Keystone State Testing

Keystone State Testing is now able to perform all of the tests for cannabis products under the state of Pennsylvania’s regulations. According to Dr. Kelly Greenland, owner and operator of Keystone State Testing, getting accredited is about safeguarding patient safety. “Keystone State Testing is proud to be the first Pennsylvania laboratory to earn A2LA ISO/IEC 17025 accreditation as well as ASA’s Patient Focused Certification,” says Dr. Greenland. “We regard these accreditations and certifications as the first steps in ensuring patient safety and will continue to do everything within our power to ensure medical marijuana patient safety.”

A2LA General Manager Adam Gouker says he wants to see more accreditations include the ASA requirements in R243. “A2LA is pleased to see the growing adoption of the combined assessment to include the ASA requirements,” says Gouker. “Our staff has worked tirelessly in conjunction with ASA staff to create this combined program and offer something that no other accreditation body in the world offers. We congratulate Keystone State Testing Labs on leading the charge in the state of Pennsylvania and laying the groundwork for future laboratories to follow.”

Amy-Ankrum-headshot
From The Lab

The Case for ISO/IEC 17025 Accreditation in Cannabis Testing Laboratories

By Amy Ankrum
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Amy-Ankrum-headshot

Government regulations keep millions of Americans safe every year by controlling what companies can put in their products and the standards those products must meet to be sold to consumers.

Enter the strange case of legal cannabis: In order for cannabis to be legally distributed by licensed medical professionals and businesses, it must be tested. But unlike other consumable goods, cannabis is not regulated by the FDA. Without an overarching federal policy requiring cannabis testing laboratory accreditation, the testing and laboratory requirements differ greatly across state lines.For medical cannabis specifically, accredited testing facilities are especially important. 

To be federally regulated, cannabis would first have to be federally legalized. It turns out that states and businesses alike are not willing to wait for a federal mandate. Many states have begun to adopt standards for cannabis testing and some, such as Ohio, have even announced mandatory ISO/IEC 17025 accreditation for all cannabis testing laboratories. As the industry evolves, increased compliance expectations are certain to evolve in tandem.

Some cannabis labs have even taken the initiative to seek ISO/IEC 17025 accreditation of their own volition. Seth Wong, President of TEQ Analytics Laboratories, shared in a press release:

“By achieving ISO/IEC 17025 accreditation, TEQ Analytical Labs believes that we can address the concerns throughout the cannabis industry regarding insufficient and unreliable scientific analysis by providing our clients with State required tests that are accredited by an international standard.”

Other laboratories, such as DB Labs in Las Vegas and EVIO Labs in Florida are also leading the accreditation charge in their respective states, ahead of any state mandates.

There are key reasons why accreditation in cannabis testing labs is important. First and foremost, cannabis is a consumable product. Like fruits and vegetables, cannabis is prone to pesticides, fungi and contaminants. The result of putting a potentially hazardous material on the market without proper and documented testing could lead to a public health crisis. An accredited testing lab, however, will ensure that the cannabis products they test are free from harmful contaminants.

By utilizing role-based trainings, labs can trust employees are receiving proper onboarding.

For medical cannabis specifically, accredited testing facilities are especially important. Because many consumers of medical cannabis are immuno-compromised (such as in the case of chemotherapy patients), ensuring that products are free from any and all contaminants is critical. Further, in order to accurately determine both short- and long-term effects of prescribed cannabis consumption, accredited and compliant laboratories are necessary.

Accreditation standards like ISO/IEC 17025 also provide confidence that testing is performed properly and to an internationally accepted standard. Rather than returning a “pass/fail” rating on products, the Cannabis Safety Institute reports that an ISO/IEC 17025 laboratory is required to produce numerical accuracy percentages in testing for “at a minimum, cannabinoids, pesticides, microbiology, residual solvents, and water activity.” Reliable data sets that can be reviewed by both accreditors and the public foster trust between producers and consumers.

Finally, ISO/IEC 17025 accreditation demonstrates that a laboratory is properly staffed and trained. The Cannabis Safety Institute’s “Standards for Cannabis Testing Laboratories” explains that conducting proper analytical chemistry on cannabinoids (the chemical compounds extracted from cannabis that alter the brain’s neurotransmitter release) requires personnel who have met specific academic and training credentials. A system to monitor, manage and demonstrate proficiency is necessary to achieve and maintain accreditation. With electronic systems in place, this management and documentation minimizes risk and also minimizes administrative time tracking and maintaining training records.

Following the proper steps of a standardized process is key to improving and growing the cannabis industry in coming yearsFor cannabis testing labs, utilizing a comprehensive software solution to achieve and maintain compliance to standards such as ISO/IEC 17025 is key. Absent of a software solution, the necessary compliance requirements can become a significant burden to the organization. Paper tracking systems and complex spreadsheets open up organizations to the likelihood of errors and ultimately risk.

Because ISO/IEC 17025 has clearly defined expectations for training, a software solution also streamlines the training process while simultaneously documenting proficiency. By utilizing role-based trainings, organizations can be confident employees are receiving proper onboarding and in-service training. Additionally, the effectiveness of training can be proven with reports, which results in smoother audits and assessments.

Following the proper steps of a standardized process is key to improving and growing the cannabis industry in coming years- which means utilizing technology tools such as electronic workflows to ensure proper process controls. Beyond adding critical visibility, workflows also create efficiencies that can eliminate the need to increase staffing as companies expand and grow.

For an industry that is changing at a rapid pace, ensuring traceability, efficient processes and visibility across organizations is paramount. Using a system that enables automation, process control, document management and documented training procedures is a step in the right direction. With the proper software tools in place, cannabis testing labs can achieve compliance goals, demonstrate reliable and relevant results and most importantly ensure consumer safety.

VinceSebald

Maintenance and Calibration: Your Customers Are Worth It!

By Vince Sebald
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VinceSebald

Ultimately, the goal of any good company is to take care of their customers by providing a quality product at a competitive price. You take the time to use good practices in sourcing raw materials, processing, testing and packaging to make sure you have a great final product. Yet in practice, sometimes the product can degrade over time, or you find yourself facing costly manufacturing stoppages and repairs due to downed equipment or instrumentation. This can harm your company’s reputation and result in real, negative effects on your bottom line.

One thing you can do to prevent this problem is to have a properly scaled calibration and maintenance program for your organization.

First, a short discussion of terms:

Balance Calibration
Figure 1– Periodic calibration of an electronic balance performed using traceable standard weights helps to ensure that the balance remains within acceptable operating ranges during use and helps identify problems.

Calibration, in the context of this article, refers to the comparison of the unit under test (your equipment) to a standard value that is known to be accurate. Equipment readings often drift over time due to various reasons and may also be affected by damage to the equipment. Periodic calibration allows the user to determine if the unit under test (UUT) is sufficiently accurate to continue using it. In some cases, the UUT may require adjustment or may not be adjustable and should no longer be used.

Maintenance, in the context of this article, refers to work performed to maximize the performance of equipment and support a long life span for the equipment. This may include lubrication, adjustments, replacement of worn parts, etc. This is intended to extend the usable life of the equipment and the consistency of the quality of the work performed by the equipment.

There are several elements to putting together such a program that can help you to direct your resources where they will have the greatest benefit. The following are some key ingredients for a solid program:

Keep it Simple: The key is to scale it to your operation. Focus on the most important items if resources are strained. A simple program that is followed and that you can defend is much better than a program where you can never catch up.

Written Program: Your calibration and maintenance programs should be written and they should be approved by quality assurance (QA). Any program should include the following: 

  • Equipment Assessment and Identification: Assess each piece of equipment or instrument to determine if it is important enough to be calibrated and/or requires maintenance. You will probably find much of your instrumentation is not used for a critical purpose and can be designated as non-calibrated. Each item should have an ID assigned to allow tracking of the maintenance and/or calibration status.
  • Scheduling System: There needs to be some way to schedule when equipment is due for calibration or maintenance. This way it is easy to stay on top of it. A good scheduling system will pay for itself over time and be easy to use and maintain. A web-based system is a good choice for small to mid-sized companies.
  • Calibration Tolerance Assignment: If you decide to calibrate an instrument, consider what kind of accuracy you actually need from the equipment/instrument. This is a separate discussion on its own, but common rule of thumb is that the instrument should be at least 4 times more accurate than your specification. For very important instruments, it may require spending the money to get a better device.
  • Calibration and Maintenance Interval Assignments: Consider what interval you are going to perform maintenance for each equipment item. Manufacturer recommendations are based on certain conditions. If you use the equipment more or less often than “normal” use, consider adjusting the interval between calibrations or maintenance. 
  • OOT Management: If you do get an Out of Tolerance (OOT) result during a calibration and you find that the instrument isn’t as accurate as you need. Congratulations! You just kept it from getting worse. Review the history and see if this may have had an effect since the last passing calibration, adjust or replace the instrument, take any other necessary corrective actions, and keep it up.

    Maintenance with Checklist
    Figure 2- Maintenance engineers help keep your systems running smoothly and within specification for a long, trouble-free life.
  • Training: Make sure personnel that use the equipment are trained on its use and not to use equipment that is not calibrated for critical measurements. Also, anyone performing calibration and/or maintenance should be qualified to do so. It is best to put a program in place as soon as you start acquiring significant equipment so that you can keep things running smoothly, avoid costly repairs and quality control problems. Don’t fall into the trap of assuming equipment will keep running just because it has run flawlessly for months or years. There are many bad results that can come of mismanaged calibration and/or maintenance including the following:
  • Unscheduled Downtime/Damage/Repairs: A critical piece of equipment goes down. Production stops, and you are forced to schedule repairs as soon as possible. You pay premium prices for parts and labor, because it is an urgent need. Some parts may have long lead times, or not be available. You may suffer reputational costs with customers waiting for delivery. Some calibration issues could potentially affect operator safety as well.
  • Out of Specification Product: Quality control may indicate that product is not maintaining its historically high quality. If you have no calibration and maintenance program in place, tracking down the problem is even more difficult because you don’t have confidence in the readings that may be indicating that there is a problem.
  • Root Cause Analysis: Suppose you find product that is out of specification and you are trying to determine the cause. If there is no calibration and maintenance program in place, it is far more difficult to pinpoint changes that may have affected your production system. This can cause a very significant impact on your ability to correct the problem and regain your historical quality standards of production.

A solid calibration and maintenance program can go a long way to keeping your production lines and quality testing “boring”, without any surprises or suspense, and can allow you to put more sophisticated quality control systems in place. Alternatively, an inappropriate system can bog you down with paperwork, delays, unpredictable performance, and a host of other problems. Take care of your equipment and relax, knowing your customers will be happy with the consistent quality that they have become accustomed to.

oregon

Turning the Oregon Outdoor Market into a Research Opportunity

By Dr. Zacariah Hildenbrand, Dr. Kevin A. Schug
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oregon

Much has been made about the plummeting market value of cannabis grown outdoors in Oregon. This certainly isn’t a reflection of the product quality within the marketplace, but more closely attributable to the oversaturation of producers in this space. This phenomenon has similarities to that of ‘Tulip Mania’ within the Dutch Golden Age, whereby tulip bulbs were highly coveted assets one day, and almost worthless the next. During times like these, it is very easy for industry professionals to become disheartened; however, from a scientific perspective, this current era in Oregon represents a tremendous opportunity for discovery and fundamental research.

Dr. Zacariah Hildenbrand
Dr. Zacariah Hildenbrand, chief technical officer at Inform Environmental.

As we have mentioned in previous presentations and commentaries, our research group is interested in exploring the breadth of chemical constituents expressed in cannabis to discover novel molecules, to ultimately develop targeted therapies for a wide range of illnesses. Intrinsically, this research has significant societal implications, in addition to the potential financial benefits that can result from scientific discovery and the development of intellectual property. While conducting our experiments out of Arlington, Texas, where the study of cannabis is highly restricted, we have resorted to the closet genetic relative of cannabis, hops (Humulus lupulus), as a surrogate model of many of our experiments (Leghissa et al., 2018a). In doing so, we have developed a number of unique methods for the characterization of various cannabinoids and their metabolites (Leghissa et al., 2018b; Leghissa et al., 2018c). These experiments have been interesting and insightful; however, they pale in comparison to the research that could be done if we had unimpeded access to diverse strains of cannabis, as are present in Oregon. For example, gas chromatography-vacuum ultraviolet spectroscopy (GC-VUV) is a relatively new tool that has recently been proven to be an analytical powerhouse for the differentiation of various classes of terpene molecules (Qiu et al., 2017). In Arlington, TX, we have three such GC-VUV instruments at our disposal, more than any other research institution in the world, but we do not have access to appropriate samples for application of this technology. Similarly, on-line supercritical fluid extraction – supercritical fluid chromatography – mass spectrometry (SFE-SFC-MS) is another capability currently almost unique to our research group. Such an instrument exhibits extreme sensitivity, supports in situ extraction and analysis, and has a wide application range for potential determination of terpenes, cannabinoids, pesticides and other chemical compounds of interest on a single analytical platform. Efforts are needed to explore the power and use of this technology, but they are impeded based on current regulations.

Dr Kevin Schug
Dr. Kevin A. Schug, Professor and the Shimadzu Distinguished Professor of Analytical Chemistry in the Department of Chemistry and Biochemistry at The University of Texas at Arlington (UTA)

Circling back, let’s consider the opportunities that lie within the abundance of available outdoor-grown cannabis in Oregon. Cannabis is extremely responsive to environmental conditions (i.e., lighting, water quality, nutrients, exposure to pest, etc.) with respect to cannabinoid and terpene expression. As such, outdoor-grown cannabis, despite the reduced market value, is incredibly unique from indoor-grown cannabis in terms of the spectrum of light to which it is exposed. Indoor lighting technologies have come a long way; full-spectrum LED systems can closely emulate the spectral distribution of photon usage in plants, also known as the McCree curve. Nonetheless, this is emulation and nothing is ever quite like the real thing (i.e., the Sun). This is to say that indoor lighting can certainly produce highly potent cannabis, which exhibits an incredibly robust cannabinoid/terpene profile; however, one also has to imagine that such lighting technologies are still missing numerous spectral wavelengths that, in a nascent field of study, could be triggering the expression of unknown molecules with unknown physiological functions in the human body. Herein lies the opportunity. If we can tap into the inherently collaborative nature of the cannabis industry, we can start analyzing unique plants, having been grown in unique environments, using unique instruments in a facilitative setting, to ultimately discover the medicine of the future. Who is with us?


References

Leghissa A, Hildenbrand ZL, Foss FW, Schug KA. Determination of cannabinoids from a surrogate hops matrix using multiple reaction monitoring gas chromatography with triple quadrupole mass spectrometry. J Sep Sci 2018a; 41: 459-468.

Leghissa A, Hildenbrand ZL, Schug KA. Determination of the metabolites of Δ9-Tetrahydrocannabinol using multiple reaction monitoring gas chromatography – triple quadrapole – mass spectrometry. Separation Science Plus 2018b; 1: 43-47.

Leghissa A, Smuts J, Changling Q, Hildenbrand ZL, Schug KA. Detection of cannabinoids and cannabinoid metabolites using gas chromatography-vacuum ultraviolet spectroscopy. Separation Science Plus 2018c; 1: 37-42.

Qiu C, Smuts J, Schug KA. Analysis of terpenes and turpentines using gas chromatography with vacuum ultraviolet detection. J Sep Sci 2017; 40: 869-877.

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.