Tag Archives: Testing

dSPE cleanups

The Grass Isn’t Always Greener: Removal of Purple Pigmentation from Cannabis

By Danielle Mackowsky
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dSPE cleanups
strains
Cannabis strains used (clockwise from top left): Agent Orange, Tahoe OG, Blue Skunk, Grand Daddy and Grape Drink

Cannabis-testing laboratories have the challenge of removing a variety of unwanted matrix components from plant material prior to running extracts on their LC-MS/MS or GC-MS. The complexity of the cannabis plant presents additional analytical challenges that do not need to be accounted for in other agricultural products. Up to a third of the overall mass of cannabis seed, half of usable flower and nearly all extracts can be contributed to essential oils such as terpenes, flavonoids and actual cannabinoid content1. The biodiversity of this plant is exhibited in the over 2,000 unique strains that have been identified, each with their own pigmentation, cannabinoid profile and overall suggested medicinal use2. While novel methods have been developed for the removal of chlorophyll, few, if any, sample preparation methods have been devoted to removal of other colored pigments from cannabis.

QuEChERS
Cannabis samples following QuEChERS extraction

Sample Preparation

Cannabis samples from four strains of plant (Purple Drink, Tahoe OG, Grand Daddy and Agent Orange) were hydrated using deionized water. Following the addition of 10 mL acetonitrile, samples were homogenized using a SPEX Geno/Grinder and stainless steel grinding balls. QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) non-buffered extraction salts were then added and samples were shaken. Following centrifugation, an aliquot of the supernatant was transferred to various blends of dispersive SPE (dSPE) salts packed into centrifugation tubes. All dSPE tubes were vortexed prior to being centrifuged. Resulting supernatant was transferred to clear auto sampler vials for visual analysis. Recoveries of 48 pesticides and four mycotoxins were determined for the two dSPE blends that provided the most pigmentation removal.

Seven dSPE blends were evaluated for their ability to remove both chlorophyll and purple pigmentation from cannabis plant material:

  • 150 mg MgSO4, 50 mg PSA, 50 mg C18, 50 mg Chlorofiltr®
  • 150 mg MgSO4, 50 mg C18, 50 mg Chlorofiltr®
  • 150 mg MgSO4, 50 mg PSA
  • 150 mg MgSO4, 25 mg C18
  • 150 mg MgSO4, 50 mg PSA, 50 mg C18
  • 150 mg MgSO4, 25 mg PSA, 7.5 mg GCB
  • 150 mg MgSO4, 50 mg PSA, 50 mg C18, 50 mg GCB

Based on the coloration of the resulting extracts, blends A, F and G were determined to be the most effective in removing both chlorophyll (all cannabis strains) and purple pigments (Purple Drink and Grand Daddy). Previous research regarding the ability of large quantities of GCB to retain planar pesticides allowed for the exclusion of blend G from further analyte quantitation3. The recoveries of the 48 selected pesticides and four mycotoxins for blends A and F were determined.

dSPE cleanups
Grand Daddy following various dSPE cleanups

Summary

A blend of MgSO4, C18, PSA and Chlorofiltr® allowed for the most sample clean up, without loss of pesticides and mycotoxins, for all cannabis samples tested. Average recovery of the 47 pesticides and five mycotoxins using the selected dSPE blend was 75.6% were as the average recovery when including GCB instead of Chlorofiltr® was 67.6%. Regardless of the sample’s original pigmentation, this blend successfully removed both chlorophyll and purple hues from all strains tested. The other six dSPE blends evaluated were unable to provide the sample clean up needed or had previously demonstrated to be detrimental to the recovery of pesticides routinely analyzed in cannabis.


References

(1)           Recommended methods for the identification and analysis of cannabis and cannabis products, United Nations Office of Drugs and Crime (2009)

(2)            W. Ross, Newsweek, (2016)

(3)            Koesukwiwat, Urairat, et al. “High Throughput Analysis of 150 Pesticides in Fruits and Vegetables Using QuEChERS and Low-Pressure Gas Chromatography Time-of-Flight Mass Spectrometry.” Journal of Chromatography A, vol. 1217, no. 43, 2010, pp. 6692–6703., doi:10.1016/j.chroma.2010.05.012.

HACCP

Hazard Analysis and Critical Control Points (HACCP) for the Cannabis Industry: Part 1

By Kathy Knutson, Ph.D.
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HACCP

Hazard Analysis and Critical Control Points (HACCP) Defined

Farm-to-fork is a concept to describe the control of food safety starting in the fields of a farm and ending with deliciousness in my mouth. The more that is optimized at every step, the more food safety and quality are realized. Farm-to-fork is not a concept reserved for foodies or “eat local” food campaigns and applies to all scales of food manufacture. HACCP is like putting the last piece of a huge puzzle in the middle and seeing the whole picture develop. HACCP is a program to control food safety at the step of food processing. In states where cannabis is legal, the state department of public health or state department of agriculture may require food manufacturers to have a HACCP plan. The HACCP plan is a written document identifying food safety hazards and how those hazards are controlled by the manufacturer. While there are many resources available for writing a HACCP plan, like solving that puzzle, it is a do-it-yourself project. You can’t use someone else’s “puzzle,” and you can’t put the box on a shelf and say you have a “puzzle.”

HACCP is pronounced “ha” as in “hat” plus “sip.”

(Say it aloud.)

3-2-1 We have liftoff.

The history of HACCP starts not with Adam eating in the garden of Eden but with the development of manned missions to the moon, the race to space in the 1950s. Sorry to be gross, but imagine an astronaut with vomiting and diarrhea as a result of foodborne illness. In the 1950s, the food industry relied on finished product testing to determine safety. Testing is destructive of product, and there is no amount of finished product testing that will determine food is safe enough for astronauts. Instead, the food industry built safety into the process. Temperature was monitored and recorded. Acidity measured by pH is an easy test. Rather than waiting to test the finished product in its sealed package, the food industry writes specifications for ingredients, ensures equipment is clean and sanitized, and monitors processing and packaging. HACCP was born first for astronauts and now for everyone.HACCP

HACCP is not the only food safety program.

If you are just learning about HACCP, it is a great place to start! There is a big world of food safety programs. HACCP is required by the United States Department of Agriculture for meat processors. The Food and Drug Administration (FDA) requires HACCP for seafood processing and 100% juice manufacture. For all foods beyond meat, seafood and juice, FDA has the Food Safety Modernization Act (FSMA) to enforce food safety. FSMA was signed in 2011 and became enforceable for companies with more than 500 employees in September of 2016; all food companies are under enforcement in September 2018. FSMA requires all food companies with an annual revenue greater than $1 million to follow a written food safety plan. Both FDA inspectors and industry professionals are working to meet the requirements of FSMA. There are also national and international guidelines for food safety with elements of HACCP which do not carry the letter of law.

The first step in HACCP is a hazard analysis.

Traditionally HACCP has focused on processing and packaging. Your organization may call that manufacturing or operations. In a large facility there is metering of ingredients by weight or volume and mixing. A recipe or batch sheet is followed. Most, but not all, products have a kill step where high heat is applied through roasting, baking, frying or canning. The food is sealed in packaging, labeled, boxed and heads out for distribution. For your hazard analysis, you identify the potential hazards that could cause injury or illness, if not controlled during processing. Think about all the potential hazards:

  • Biological: What pathogens are you killing in the kill step? What pathogens could get in to the product before packaging is sealed?
  • Chemical: Pesticides, industrial chemicals, mycotoxins and allergens are concerns.
  • Physical: Evaluate the potential for choking hazards and glass, wood, hard plastic and metal.

The hazards analysis drives everything you do for food safety.

I cannot emphasize too much the importance of the hazard analysis. Every food safety decision is grounded in the hazard analysis. Procedures will be developed and capital will be purchased based on the hazard analysis and control of food safety in your product. There is no one form for the completion of a hazard analysis.

HACCP risk matrix
A risk severity matrix. Many HACCP training programs have these.

So where do you start? Create a flow diagram naming all the steps in processing and packaging. If your flow diagram starts with Receiving of ingredients, then the next step is Storage of ingredients; include packaging with Receiving and Storage. From Storage, ingredients and packaging are gathered for a batch. Draw out the processing steps in order and through to Packaging. After Packaging, there is finished product Storage and Distribution. Remember HACCP focuses on the processing and packaging steps. It is not necessary to detail each step on the flow diagram, just name the step, e.g. Mixing, Filling, Baking, etc. Other supporting documents have the details of each step.

For every step on the flow diagram, identify hazards.

Transfer the name of the step to the hazard analysis form of your choice. Focus on one step at a time. Identify biological, chemical and physical hazards, if any, at that step. The next part is tricky. For each hazard identified, determine the probability of the hazard occurring and severity of illness or injury. Some hazards are easy like allergens. If you have an ingredient that contains an allergen, the probability is high. Because people can die from ingestion of allergens when allergic, the severity is high. Allergens are a hazard you must control. What about pesticides? What is the probability and severity? I can hear you say that you are going to control pesticides through your purchasing agreements. Great! Pesticides are still a hazard to identify in your hazard analysis. What you do about the hazard is up to you.

Multi-analyte Configuration for Cannabis Testing Services

Managing Cannabis Testing Lab Workflows using LIMS

By Dr. Susan Audino
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Multi-analyte Configuration for Cannabis Testing Services

With the state led legalization of both adult recreational and medical cannabis, there is a need for comprehensive and reliable analytical testing to ensure consumer safety and drug potency. Cannabis-testing laboratories receive high volumes of test requests from cannabis cultivators for testing quantitative and qualitative aspects of the plant. The testing market is growing as more states bring in stricter enforcement policies on testing. As the number of testing labs grow, it is anticipated that the laboratories that are now servicing other markets, including high throughput contract labs, will cross into cannabis testing as regulations free up. As the volume of tests each lab performs increases, the need for laboratories to make effective use of time and resource management, such as ensuring accurate and quick results, reports, regulatory compliance, quality assurance and many other aspects of data management becomes vital in staying competitive.

Cannabis Testing Workflows

To be commercially competitive, testing labs offer a comprehensive range of testing services. These services are available for both the medical and recreational cannabis markets, including:

  • Detection and quantification of both acid and neutral forms of cannabinoids
  • Screening for pesticide levels
  • Monitoring water activity to indicate the possibility of microbiological contamination
  • Moisture content measurements
  • Terpene profiling
  • Residual solvents and heavy metal testing
  • Fungi, molds, mycotoxin testing and many more

Although the testing workflows differ for each test, here is a basic overview of the operations carried out in a cannabis-testing lab:

  1. Cannabis samples are received.
  2. The samples are processed using techniques such as grinding and homogenization. This may be followed by extraction, filtration and evaporation.
  3. A few samples will be isolated and concentrated by dissolving in solvents, while others may be derivatized using HPLC or GC reagents
  4. The processed samples are then subjected to chromatographic separation using techniques such as HPLC, UHPLC, GC and GC-MS.
  5. The separated components are then analyzed and identified for qualitative and quantitative analysis based on specialized standards and certified reference materials.
  6. The quantified analytical data will be exported from the instruments and compiled with the corresponding sample data.
  7. The test results are organized and reviewed by the lab personnel.
  8. The finalized test results are reported in a compliant format and released to the client.

In order to ensure that cannabis testing laboratories function reliably, they are obliged to follow and execute certain organizational and regulatory protocols throughout the testing process. These involve critical factors that determine the accuracy of testing services of a laboratory.

Factors Critical to a Cannabis Testing Laboratory 

  • Accreditations & Regulatory Compliance: Cannabis testing laboratories are subject to regulatory compliance requirements, accreditation standards, laboratory practices and policies at the state level. A standard that most cannabis testing labs comply to is ISO 17025, which sets the requirements of quality standards in testing laboratories. Accreditation to this standard represents the determination of competence by an independent third party referred to as the “Accreditation Body”. Accreditation ensures that laboratories are adhering to their methods. These testing facilities have mandatory participation in proficiency tests regularly in order to maintain accreditation.
  • Quality Assurance, Standards & Proficiency Testing: Quality assurance is in part achieved by implementing standard test methods that have been thoroughly validated. When standard methods are not available, the laboratory must validate their own methods. In addition to using valid and appropriate methods, accredited laboratories are also required to participate in appropriate and commercially available Proficiency Test Program or Inter-Laboratory Comparison Study. Both PT and ILC Programs provide laboratories with some measure of their analytic performance and compare that performance with other participating laboratories.

    Multi-analyte Configuration for Cannabis Testing Services
    CloudLIMS Cannabis Testing LIMS: Multi-analyte Configuration for Cannabis Testing Services
  • Real-time Collaboration: Testing facilities generate metadata such as data derived from cannabis samples and infused products. The testing status and test results are best served for compliance and accessibility when integrated and stored on a centralized platform. This helps in timely data sharing and facilitates informed decision making, effective cooperation and relationships between cannabis testing facilities and growers. This platform is imperative for laboratories that have grown to high volume throughput where opportunities for errors exist. By matching test results to samples, this platform ensures consistent sample tracking and traceability. Finally, the platform is designed to provide immediate, real-time reporting to individual state or other regulatory bodies.
  • Personnel Management: Skilled scientific staff in cannabis-testing laboratories are required to oversee testing activities. Staff should have experience in analytical chromatography instruments such as HPLC and GC-MS. Since samples are often used for multi-analytes such as terpenes, cannabinoids, pesticides etc., the process often involves transferring samples and tests from one person to another within the testing facility. A chain of custody (CoC) is required to ensure traceability and ‘ownership’ for each person involved in the workflow.

LIMS for Laboratory Automation

Gathering, organizing and controlling laboratory-testing data can be time-consuming, labor-intensive and challenging for cannabis testing laboratories. Using spreadsheets and paper methods for this purpose is error-prone, makes data retrieval difficult and does not allow laboratories to easily adhere to regulatory guidelines. Manual systems are cumbersome, costly and lack efficiency. One way to meet this challenge is to switch to automated solutions that eliminate many of the mundane tasks that utilize valuable human resources.. Laboratory automation transforms the data management processes and as a result, improves the quality of services and provides faster turnaround time with significant cost savings. Automating the data management protocol will improve the quality of accountability, improve technical efficiency, and improve fiscal resources.

cloudlims screenshot
Real Time Test Status in CloudLIMS

A Laboratory Information Management System (LIMS) is a software tool for testing labs that aids efficient data management. A LIMS organizes, manages and communicates all laboratory test data and related information, such as sample and associated metadata, tests, Standard Operating Procedures (SOPs), test reports, and invoices. It also enables fully automated data exchange between instruments such as HPLCs, GC-FIDs, etc. to one consolidated location, thereby reducing transcription errors.

How LIMS Helps Cannabis Testing Labs

LIMS are much more capable than spreadsheets and paper-based tools for streamlining the analytical and operational lab activities and enhances the productivity and quality by eliminating manual data entry. Cloud-enabled LIMS systems such as CloudLIMS are often low in the total cost of acquisition, do not require IT staff and are scalable to help meet the ever changing business and regulatory compliance needs. Some of the key benefits of LIMS for automating a cannabis-testing laboratory are illustrated below [Table 1]:

Key Functionality Benefit
Barcode label designing and printing Enables proper labelling of samples and inventory

Follows GLP guidelines

Instant data capture by scanning barcodes Facilitates quick client registration and sample access
3600 data traceability Saves time and resources for locating samples and other records
Inventory and order management Supports proactive planning/budgeting and real time accuracy
Custodian management Promotes overall laboratory organization by assigning custodians for samples and tests

Maintains the Chain-of-custody (CoC)

Test management Accommodates pre-loaded test protocols to quickly assign tests for incoming samples
Accounting for sample and inventory quantity Automatically deducts sample and inventory quantities when consumed in tests
Package & shipment management Manages incoming samples and samples that have been subcontracted to other laboratories
Electronic data import Electronically imports test results and metadata from integrated instruments

Eliminates manual typographical errors

Report management Generates accurate, customizable, meaningful and test reports for clients

Allows user to include signatures and additional sections for professional use

21 CFR Part 11 compliant Authenticates laboratory activities with electronic signatures
ISO 17025 accreditation Provides traceable documentary evidence required to achieve ISO 17025 accreditation
Audit trail capabilities Adheres to regulatory standards by recording comprehensive audit logs for laboratory activities along with the date and time stamp
Centralized data management Stores all the data in a single, secure database facilitating quick data retrieval
Workflow management Promotes better data management and resource allocation
High-configurability Enables modification of screens using graphical configuration tools to mirror testing workflows
State compliance systems Integrates with state-required compliance reporting systems and communicates using API
Adheres to regulatory compliance Creates Certificates of Analysis (CoA) to prove regulatory compliance for each batch as well as batch-by-batch variance analysis and other reports as needed.
Data security & confidentiality Masks sensitive data from unauthorized user access

 

Cloud-based LIMS encrypts data at rest and in-transit while transmission between the client and the server

Global accessibility Cloud-based LIMS provides real-time access to laboratory data from anytime anywhere
Real-time collaboration Cloud-based LIMS enhances real-time communication within a laboratory, between a laboratory and its clients, and across a global organization with multiple sites

Table 1. Key functionality and benefits of LIMS for cannabis testing laboratories

Upon mapping the present day challenges faced by cannabis testing laboratories, adopting laboratory automation solutions becomes imperative. Cloud-based LIMS becomes a valuable tool for laboratory data management in cannabis testing laboratories. In addition to reducing manual workloads, and efficient resource management, it helps labs focus on productive lab operations while achieving compliance and regulatory goals with ease.

For more information on this, check out a webinar here: Webinar: How to Meet Cannabis Testing Standards and Regulatory Requirements with LIMS by Stephen Goldman, laboratory director at the State of Colorado certified Cannabis testing facility, PhytaTech.

EVIO Logo

EVIO Labs: The First Accredited Cannabis Lab in Florida

By Lauren Masko
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EVIO Logo

EVIO Labs recently became the first cannabis laboratory in Florida to obtain ISO 17025 accreditation. Perry Johnson Laboratory Accreditation, Inc. (PJLA), an organization that provides third-party assessments to ISO/IEC 17025, accredited EVIO Labs. The assessment process that lead to ISO 17025 accreditation for EVIO Labs included a thorough review of their quality management system, their capability to perform potency and contaminant testing for cannabis products.

Tracy Szerszen, president and operations manager at PJLA, encourages this international standard for laboratories to provide confidence to end-users that the test results they receive are reliable. She says laboratories that achieve this accreditation are showing they have the proper tools, equipment and staff to provide accurate testing. “It is a very critical component of the industry, and becoming accredited provides the assurance that laboratories are performing to the highest standard,” says Szerszen. “EVIO Labs has taken the right step in their commitment towards meeting this standard and providing clean and safe cannabis for the patients of Florida.”

PJLAEVIO Labs provides cannabis testing for cannabinoid and terpene profiles, microbiological and pesticides contamination, residual solvent, heavy metals, mycotoxins, water activity and moisture content. Chris Martinez, co-founder and president of EVIO Labs Florida explains that the Florida Department of Health mandates that an independent third-party laboratory tests medical cannabis to ensure that these products are safe for human consumption. Martinez says their first priority is the safety of their patients, and ensuring that EVIO Labs provides clean and safe cannabis for Florida.

Chris Martinez
Chris Martinez, co-founder and president of EVIO Labs Florida

Martinez launched their laboratory with some help from Shimadzu last year. “Our Broward lab is powered by Shimadzu with over $1.2M in the latest testing equipment utilizing LCMS technology with the world’s fastest polarity switching time of 5 m/sec and scan speeds of 30,000 u/sec with UF Qarray sensitivity 90 times that of previously available technologies,” says Martinez. According to Martinez, their licensing agreement with EVIO Labs (OTC:SGBYD) marked a first for the publicly traded company with exclusivity in the Florida market. The agreement includes proprietary testing methodologies, operating procedures, training and support.

Every certificate of analysis is reviewed by a lab director with over 20 years of experience operating in FDA regulated labs. Martinez says that EVIO has some of the most advanced technology in the industry, which provides them the opportunity to quickly provide results, frequently as fast as a 24-hour period. Martinez and his team are currently building a 3,300 square-foot laboratory in Gainesville, which is expected to be running by March of this year.

Growing Pains a Month Into California’s Market Launch

By Aaron G. Biros
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For about a month now, California’s adult use market has been open for business and the market is booming. About thirty days into the world’s largest adult use market launch, we are beginning to see side effects of the growing pains that come with adjusting the massive industry.

Consumers are also feeling sticker shock as the new taxes add up to a 40% increase in price.While the regulatory and licensing roll out has been relatively smooth, some municipalities are slower than others in welcoming the adult use cannabis industry. It took Los Angeles weeks longer than other counties to begin licensing dispensaries. Meanwhile, retailers in San Diego say the first month brought a huge influx of customers, challenging their abilities to meet higher-than-expected demand.

Businesses are struggling to deal with large amounts of cash, but California State Treasurer John Chiang may have a solution in store. Yesterday, his department announced they are planning to create a taxpayer-backed bank for cannabis businesses.

Reports of possible supply shortages are irking some businesses, fearing that the state hasn’t licensed enough growers and distributors to handle the high demand. Consumers are also feeling sticker shock as the new taxes add up to a 40% increase in price.

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

In the regulatory realm, some are concerned that a loophole in the rules allows bigger cultivation operations to squeeze out the competition from smaller businesses. The California Growers Association filed a lawsuit against the California Department of Food and Agriculture to try and close this loophole, hoping to give smaller cultivators a leg up before bigger companies can dominate the market.

The Bureau of Cannabis Control (known as just “The Bureau”) began holding meetings and workshops to help cannabis businesses get acquainted with the new rules. Public licensing workshops in Irvine and San Diego held last week were designed to focus on information required for licensing and resources for planning. The Bureau also held their first cannabis advisory committee meeting, as well as announcing new subcommittees and an input survey to help the Bureau better meet business needs.

On the lab-testing front, the state has phased in cannabinoids, moisture content, residual solvent, pesticide, microbial impurities and homogeneity testing. On July 1, the state will phase in additional residual solvent and pesticide testing in addition to foreign material testing. At the end of 2018, they plan on requiring terpenoids, mycotoxins, heavy metals and water activity testing as well.

autoclave

10 Treatment Methods to Reduce Mold in Cannabis

By Ketch DeGabrielle
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autoclave

As the operations manager at Los Sueños Farms, the largest outdoor cannabis farm in the country, I was tasked with the challenge of finding a yeast and mold remediation treatment method that would ensure safe and healthy cannabis for all of our customers while complying with stringent regulations.

While outdoor cannabis is not inherently moldy, outdoor farms are vulnerable to changing weather conditions. Wind transports spores, which can cause mold. Each spore is a colony forming unit if plated at a lab, even if not germinated in the final product. In other words, perfectly good cannabis can easily fail microbial testing with the presence of benign spores.

Fun Fact: one square centimeter of mold can produce over 2,065,000,000 spores.

If all of those landed on cannabis it would be enough to cause over 450 pounds of cannabis to fail testing, even if those spores remained ungerminated.

Photo credit: Steep Hill- a petri dish of mold growth from tested cannabis

It should also be known that almost every food item purchased in a store goes through some type of remediation method to be considered safe for sale. Cannabis is finally becoming a legitimized industry and we will see regulations that make cannabis production look more like food production each year.

Regulations in Colorado (as well as Nevada and Canada) require cannabis to have a total yeast and mold count (TYMC) of ≤ 10,000 colony forming units per gram. We needed a TYMC treatment method that was safe, reliable, efficient and suitable for a large-scale operation. Our main problem was the presence of fungal spores, not living, growing mold.

Below is a short list of the pros and cons of each treatment method I compiled after two years of research:

Autoclave: This is the same technology used to sterilize tattoo needles and medical equipment. Autoclave uses heat and pressure to kill living things. While extremely effective, readily available and fiscally reasonable, this method is time-consuming and cannot treat large batches. It also utilizes moisture, which increases mold risk. The final product may experience decarboxylation and a change in color, taste and smell.

Dry Heat: Placing cannabis in dry heat is a very inexpensive method that is effective at reducing mold and yeast. However, it totally ruins product unless you plan to extract it.

autoclave
An autoclave
Image: Tom Beatty, Flickr

Gamma Ray Radiation: By applying gamma ray radiation, microbial growth is reduced in plants without affecting potency. This is a very effective, fast and scalable method that doesn’t cause terpene loss or decarboxylation. However, it uses ionizing radiation that can create new chemical compounds not present before, some of which can be cancer-causing. The Department of Homeland Security will never allow U.S. cannabis farmers to use this method, as it relies on a radioactive isotope to create the gamma rays.

Gas Treatment: (Ozone, Propylene Oxide, Ethylene Oxide, Sulfur Dioxide) Treatment with gas is inexpensive, readily available and treats the entire product. Gas treatment is time consuming and must be handled carefully, as all of these gases are toxic to humans. Ozone is challenging to scale while PPO, EO and SO2 are very scalable. Gases require special facilities to apply and it’s important to note that gases such as PPO and EO are carcinogenic. These methods introduce chemicals to cannabis and can affect the end product by reducing terpenes, aroma and flavor.

Hydrogen Peroxide: Spraying cannabis plants with a hydrogen peroxide mixture can reduce yeast and mold. However, moisture is increased, which can cause otherwise benign spores to germinate. This method only treats the surface level of the plant and is not an effective remediation treatment. It also causes extreme oxidation, burning the cannabis and removing terpenes.

Microwave: This method is readily available for small-scale use and is non-chemical based and non-ionizing. However, it causes uneven heating, burning product, which is damaging to terpenes and greatly reduces quality. This method can also result in a loss of moisture. Microwave treatment is difficult to scale and is not optimal for large cultivators.

Radio Frequency: This method is organic, non-toxic, non-ionizing and non-chemical based. It is also scalable and effective; treatment time is very fast and it treats the entire product at once. There is no decarboxylation or potency loss with radio frequency treatment. Minimal moisture loss and terpene loss may result. This method has been proven by a decade of use in the food industry and will probably become the standard in large-scale treatment facilities.

Steam Treatment: Water vapor treatment is effective in other industries, scalable, organic and readily available. This method wets cannabis, introducing further mold risk, and only treats the product surface. It also uses heat, which can cause decarboxylation, and takes a long time to implement. This is not an effective method to reduce TYMC in cannabis, even though it works very well for other agricultural products

extraction equipment
Extraction can be an effective form of remediating contaminated cannabis

Extraction: Using supercritical gas such as butane, heptane, carbon dioxide or hexane in the cannabis extraction process is the only method of remediation approved by the Colorado Marijuana Enforcement Division and is guaranteed to kill almost everything. It’s also readily available and easy to access. However, this time-consuming method will change your final product into a concentrate instead of flower and usually constitutes a high profit loss.

UV Light: This is an inexpensive and readily available method that is limited in efficacy. UV light is only effective on certain organisms and does not work well for killing mold spores. It also only kills what the light is touching, unless ozone is captured from photolysis of oxygen near the UV lamp. It is time consuming and very difficult to scale.

After exhaustively testing and researching all treatment methods, we settled on radio frequency treatment as the best option. APEX, a radio frequency treatment machine created by Ziel, allowed us to treat 100 pounds of cannabis in an hour – a critical factor when harvesting 36,000 plants during the October harvest.

Nevada Testing Lab Licenses Suspended, Then Reinstated

By Aaron G. Biros
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When Nevada legalized adult use sales this past summer, the market exploded and undoubtedly flooded licensed testing labs with samples to get products on shelves. In August, roughly a month after the start of adult use sales, a Las Vegas cannabis-testing lab, G3 Labs, had their license suspended for an unknown compliance issue.

“We can’t disclose the details of the suspension, including anything about penalties,” said Klapstein. “Under NRS 360.255, the information is confidential.”Then in late December, the Nevada Department of Taxation, one of the bodies tasked with regulating the state’s industry, announced in an email they suspended two more cannabis testing lab licenses. Certified Ag Lab in Sparks, Nevada and Cannex Nevada, LLC, in Las Vegas (also known as RSR Analytical Laboratories) both had their licenses suspended on December 22 and December 26 respectively.

Stephanie Klapstein, spokeswoman for the Department of Taxation, told the Reno Gazette Journal that both of those labs were not following proper protocols. “During separate, routine inspections, Department inspectors discovered that these two labs were not following proper lab procedures and good laboratory practices,” says Klapstein. “Their licenses were suspended until those deficiencies were corrected.”

According to the Reno Gazette Journal, both of those labs had their licenses reinstated and have since resumed normal business. During their license suspension, the labs were not allowed to operate and the department directed licensed cannabis businesses to submit samples to other labs. The department also directed the suspended labs in the email to coordinate with their clients who had samples in for testing; to either have their samples transferred to a different lab or a new sample taken for another lab to test. They did note that no product recalls were deemed necessary because of the suspension.

In that same email, the department directed licensed cannabis businesses to state-licensed labs in good standing, including 374 Labs, ACE Analytical Laboratory, DB Labs, Digipath Labs, MM Lab and NV CANN Lab. But on the department’s website, it says there are 11 licensed testing labs.

Back in September when we reported on the first lab license suspension, Klapstein told CIJ that under state law they couldn’t discuss any reasons behind why they suspended licenses. “We can’t disclose the details of the suspension, including anything about penalties,” said Klapstein. “Under NRS 360.255, the information is confidential.”

Because of that confidentiality, there are a number of questions left unanswered: With three lab licenses suspended in the first six months of the Nevada’s adult use market being open, how are testing labs keeping up with the market’s pace? What did those suspended labs do wrong? Do the regulations adequately protect public health and safety?

Sunrise Genetics Partners With RPC, Begins Genetic Testing in Canada

By Aaron G. Biros
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Sunrise Genetics, Inc., the parent company of Marigene and Hempgene, announced their partnership with New Brunswick Research & Productivity Council (RPC) this week, according to a press release. The company has been working in the United States for a few years now doing genomic sequencing and genetic research with headquarters based in Fort Collins, CO. This new partnership, compliant with Health Canada sample submission requirements, allows Canadian growers to submit plants for DNA extraction and genomic sequencing.

Sunrise Genetics researches different cannabis cultivars in the areas of target improvement of desired traits, accelerated breeding and expanding the knowledge base of cannabis genetics. One area they have been working on is genetic plant identification, which uses the plant’s DNA and modern genomics to create authentic, reproducible, commercial-ready strains.

Matt Gibbs, president of Sunrise Genetics, says he is very excited to get working on cannabis DNA testing in Canada. “RPC has a long track record of leadership in analytical services, especially as it relates to DNA and forensic work, giving Canadian growers their first real option to submit their plant samples for DNA extraction through proper legal channels,” says Gibbs. “The option to pursue genomic research on cannabis is now at Canadian cultivator’s fingertips.”

Canada’s massive new cannabis industry, which now has legal recreational and medical use, sales and cultivation, previously has not had many options for genetic testing. Using their genetic testing capabilities, they hope this partnership will better help Canadian cultivators easily apply genomic testing for improved plant development. “I’m looking forward to working with more Canadian cultivators and breeders; the opportunity to apply genomics to plant improvement is a win-win for customers seeking transparency about their Cannabis product and producers seeking customer retention through ‘best-in-class’ cannabis and protectable plant varieties,” says Gibbs. The partnership also ensures samples will follow the required submission process for analytical testing, but adding the service option of genetic testing so growers can find out more about their plants beyond the regular gamut of tests.

RPC is a New Brunswick provincial research organization (PRO), a research and technology organization (RTO) that offers R&D testing and technical services. With 130 scientists, engineers and technologists, RPC offers a wide variety of testing services, including air quality, analytical chemistry of cannabis, material testing and a large variety of pilot facilities for manufacturing research and development.

They have over 100 accreditations and certifications including an ISO 17025 scope from the Standards Council of Canada (SCC) and is ISO 9001:2008 certified. This genetic testing service for cannabis plants is the latest development in their repertoire of services. “This service builds on RPC’s established genetic strengths and complements the services we are currently offering the cannabis industry,” says Eric Cook, chief executive officer of RPC.

Microbiology 101 Part Two

By Kathy Knutson, Ph.D.
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Microbiology 101 Part One introduced the reader to the science of microbiology and sources of microbes. In Part Two, we discuss the control of microorganisms in your products.

Part 2

The cannabis industry is probably more informed about patients and consumers of their products than the general food industry. In addition to routine illness and stress in the population, cannabis consumers are fighting cancer, HIV/AIDS and other immune disorders. Consumers who are already ill are immunocompromised. Transplant recipients purposely have their immune system suppressed in the process of a successful transplant. These consumers have pre-existing conditions where the immune system is weakened. If the immunocompromised consumer is exposed to viral or bacterial pathogens through cannabis products, the consumer is more likely to suffer from a viral infection or foodborne illness as a secondary illness to the primary illness. In the case of consumers with weakened immune systems, it could literally kill them.Bacteria, yeast, and mold are present in all environments.

The cannabis industry shoulders great responsibility in both the medical and adult use markets. In addition to avoiding chemical hazards and determining the potency of the product, the cannabis industry must manufacture products safe for consumption. There are three ways to control pathogens and ensure a safe product: prevent them from entering, kill them and control their growth.

Prevent microorganisms from getting in

Think about everything that is outdoors that will physically come in a door to your facility. Control the quality of ingredients, packaging, equipment lubricants, cleaning agents and sanitizers. Monitor employee hygiene. Next, you control everything within your walls: employees, materials, supplies, equipment and the environment. You control receiving, employee entrance, storage, manufacturing, packaging and distribution. At every step in the process, your job is to prevent the transfer of pathogens into the product from these sources.

Kill microorganisms

Colorized low-temperature electron micrograph of a cluster of E. coli bacteria.
Image courtesy of USDA ARS & Eric Erbe

The combination of raw materials to manufacture your product is likely to include naturally occurring pathogens. Traditional heat methods like roasting and baking will kill most pathogens. Remember, sterility is not the goal. The concern is that a manufacturer uses heat to achieve organoleptic qualities like color and texture, but the combination of time and temperature may not achieve safety. It is only with a validated process that safety is confirmed. If we model safety after what is required of food manufacturers by the Food and Drug Administration, validation of processes that control pathogens is required. In addition to traditional heat methods, non-thermal methods for control of pathogens includes irradiation and high pressure processing and are appropriate for highly priced goods, e.g. juice. Killing is achieved in the manufacturing environment and on processing equipment surfaces after cleaning and by sanitizing.

If you have done everything reasonable to stop microorganisms from getting in the product and you have a validated step to kill pathogens, you may still have spoilage microorganisms in the product. It is important that all pathogens have been eliminated. Examples of pathogens include Salmonella, pathogenic Escherichia coli, also called Shiga toxin-producing E. coli (STEC) and Listeria monocytogenes. These three common pathogens are easily destroyed by proper heat methods. Despite steps taken to kill pathogens, it is theoretically possible a pathogen is reintroduced after the kill step and before packaging is sealed at very low numbers in the product. Doctors do not know how many cells are required for a consumer to get ill, and the immunocompromised consumer is more susceptible to illness. Lab methods for the three pathogens mentioned are designed to detect very low cell numbers. Packaging and control of growth factors will stop pathogens from growing in the product, if present.

Control the growth of microorganisms

These growth factors will control the growth of pathogens, and you can use the factors to control spoilage microbes as well. To grow, microbes need the same things we do: a comfortable temperature, water, nutrients (food), oxygen, and a comfortable level of acid. In the lab, we want to find the pathogen, so we optimize these factors for growth. When you control growth in your product, one hurdle may be enough to stop growth; sometimes multiple hurdles are needed in combination. Bacteria, yeast, and mold are present in all environments. They are at the bottom of the ocean under pressure. They are in hot springs at the temperature of boiling water. The diversity is immense. Luckily, we can focus on the growth factors for human pathogens, like Salmonella, pathogenic E. coli, and Listeria monocytogenes.

The petri dishes show sterilization effects of negative air ionization on a chamber aerosolized with Salmonella enteritidis. The left sample is untreated; the right, treated. Photo courtesy of USDA ARS & Ken Hammond

Temperature. Human pathogens prefer to grow at the temperature of the human body. In manufacture, keep the time a product is in the range of 40oF to 140oF as short as possible. You control pathogens when your product is at very hot or very cold temperatures. Once the product cools after a kill step in manufacturing, it is critical to not reintroduce a pathogen from the environment or personnel. Clean equipment and packaging play key roles in preventing re-contamination of the product.

Water. At high temperatures as in baking or roasting, there is killing, but there is also the removal of water. In the drying process that is not at high temperature, water is removed to stop the growth of mold. This one hurdle is all that is needed. Even before mold is controlled, bacterial and yeast growth will stop. Many cannabis candies are safe, because water is not available for pathogen growth. Packaging is key to keep moisture out of the product.

Nutrients. In general, nutrients are going to be available for pathogen growth and cannot be controlled. In most products nutrients cannot be removed, however, recipes can be adjusted. Recipes for processed food add preservatives to control growth. In cannabis as in many plants, there may be natural compounds which act as preservatives.

Oxygen. With the great diversity of bacteria, there are bacteria that require the same oxygen we breathe, and mold only grows in oxygen. There are bacteria that only grow in the absence of oxygen, e.g. the bacteria responsible for botulism. And then there are the bacteria and yeast in between, growing with or without oxygen. Unfortunately, most human pathogens will grow with or without oxygen, but slowly without oxygen. The latter describes the growth of Salmonella, E. coli, and Listeria. While a package seals out air, the growth is very slow. Once a package is opened and the product is exposed to air, growth accelerates.

Acid. Fermented or acidified products have a higher level of acid than non-acid products; the acid acts as a natural preservative. The more acid, the more growth is inhibited. Generally, acid is a hurdle to growth, however and because of diversity, some bacteria prefer acid, like probiotics which are non-pathogenic. Some pathogens, like E. coli, have been found to grow in low acid foods, e.g. juice, even though the preference is for non-acidic environments.

Each facility is unique to its materials, people, equipment and product. A safe product is made by following Good Agricultural Practices for the cannabis, by following Good Manufacturing Practices and by suppressing pathogens by preventing them coming in, killing them and controlling their growth factors. Future articles will cover Hazard Analysis and Critical Control Points (HACCP) and food safety in more detail.

California Rolls Out Licensing For Cannabis Businesses

By Aaron G. Biros
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Last week, the Bureau of Cannabis Control issued the first licenses for California’s new market. The first license went to Moxie, a cannabis distribution company out of Lynwood.

The search feature for the list of licenses issued so far

As of the publication of this article, the Bureau, the state authority tasked with leading the regulation of the industry, has issued 43 temporary licenses. So far, four laboratories have received licenses, along with a number of retailers, distributors, microbusinesses in both medical and adult-use markets.

The labs to receive their temporary licenses so far are pH Solutions, Steep Hill Labs, Pure Analytics and ORCA Cannalytics. Judging by the number of temporary medical and adult-use licenses awarded so far, it appears the Bureau is trying to issue a similar amount for each sector, distributing the number of licenses between the two equitably.

You can find the list of licensees here, and search between the dates of 12/15/17 to 1/2/18 to get the most up-to-date list of licenses awarded. “Last week, we officially launched our online licensing system, and today we’re pleased to issue the first group of temporary licenses to cannabis businesses that fall under the Bureau’s jurisdiction,” says Lori Ajax, Bureau of Cannabis Control Chief. “We plan to issue many more before January 1.”

According to the press release, temporary licenses are only issued to applicants with prior local authorization in the form of a license or permit from the jurisdiction where the business is. Those licenses will become effective on January 1, 2018. The temporary licenses will work for 120 days, or May 1, 2018, after which businesses will need to have a permanent license to continue operating.

More than 1,900 users have registered with the Bureau’s online system, and more than 200 applications have been submitted, according to the press release.

The various regulatory bodies in California have worked diligently for months now to roll out proposed emergency regulations, setting strict requirements for manufacturers, growers, retailers and testing labs. Manufacturing regulations, including specific labeling, packaging and processing requirements, give a good snapshot of how regulators plan to move forward. Testing requirements could also be significantly firmer, with rules for documentation, sample sizes, sampling procedures, storage and transportation.

Yet when the adult-use sales become fully legal on January 1, 2018, those regulations will not be fully implemented.

Donald Land, a UC Davis chemistry professor and chief scientific consultant at Steep Hill Labs Inc., told The Associated Press, “Buyer beware.” There will be a six-month range where existing inventory will be allowed on the shelves, products that might not meet the standards of the new rules. So dispensaries will get half a year of sales before all products have to meet the new, stricter testing requirements.