You’ve survived seasons of cannabis cultivations, bringing in quality plants in spite of mold, mites, drought and other challenges that had to be conquered. Extraction methods are sometimes challenging, but you are proud to have a cannabinoid extract that can be added into your own products for sale. Edibles are just waiting to be infused with the cannabinoids, for consumers demanding brownies, gummies, tinctures and almost any food and beverage imaginable. You’ve been through the fire, and now the rest is easy peasy, right?
Actually, producing edibles may not be so seamless as you think. Just as in the rest of the food industry, food safety practices have to be considered when you’re producing edibles for public consumption, regardless of the THC, CBD, terpene or cannabinoid profile. Once you’ve acquired the extract (a “food grade ingredient”) containing the active compounds, there are three types of hazards that could still contribute to foodborne illness from your final product if you’re not careful- Biological, Chemical and Physical.
Biological hazards include pathogenic bacteria, viruses, mold, mildew (and the toxins that they can produce) that can come in ingredients naturally or contaminate foods from an outside source. Chemical hazards are often present in the kitchen environment, including detergents, floor cleaners, disinfectants and caustic chemicals, which can be harmful if ingested- they are not destroyed through cooking. Physical objects abound in food production facilities, including plastic bits, metal fragments from equipment, staples or twist ties from ingredient packages, and personal objects (e.g., buttons, jewelry, hair, nails.)
There are three main safety precautions that can help control these hazards during all the stages of food production, from receiving ingredients to packaging your final products:
1. Avoid Cross Contamination
Prevent biological, chemical or physical hazards from coming into contact with foods
Keep equipment, utensils and work surfaces clean and sanitized.
Prevent raw foods (as they usually carry bacteria) from coming into contact with “Ready-to-eat” foods (foods that will not be cooked further before consuming).
Keep chemicals away from food areas.
2. Personal Hygiene
Don’t work around foods if you’re sick with fever, vomiting or diarrhea. These could be signs of contagious illness and can contaminate foods or other staff, and contribute to an outbreak.
Do not handle ready-to-eat foods with bare hands, but use a barrier such as utensils, tissues or gloves when handling final products such as pastries or candies.
Wash hands and change gloves when soiled or contaminated.
Wear hair restraints and clean uniforms, and remove jewelry from hands and arms.
3. Time & Temperature control
Prevent bacterial growth in perishable foods such as eggs, dairy, meats, chicken (TCS “Time and Temperature Control for Safety” foods according to the FDA Model Food Code) by keeping cold foods cold and hot foods hot.
Refrigerate TCS foods at 41˚ F or below, and cook TCS foods to proper internal temperatures to kill bacteria to safe levels, per state regulations for retail food establishments.
If TCS foods have been exposed to room temperature for longer than four hours (Temperature Danger Zone 41˚ F – 135˚ F,) these foods should be discarded, as bacteria could have grown to dangerous levels during this time.
As cannabis companies strive for acceptance and legalization on a federal level, adopting these food safety practices and staff training is a major step in the right direction, on par with standards maintained by the rest of the retail food industry. The only difference is your one specially extracted cannabinoid ingredient that separates you from the rest of the crowd… with safe and healthy edibles for all.
Across the country and across the world, governments that legalize cannabis implement increasingly rigorous requirements for laboratory testing. Helping to protect patients and consumers from contaminants, these requirements involve a slew of lab tests, including quantifying the levels of microbial contaminants, pathogens, mold and heavy metals.
Cannabis and hemp have a unique ability to accumulate elements found in soil, which is why these plants can be used as effective tools for bioremediation. Because cannabis plants have the ability to absorb potentially toxic and dangerous elements found in the soil they grow in, lab testing regulations often include the requirement for heavy metals testing, such as Cadmium, Lead, Mercury, Arsenic and others.
In addition to legal cannabis markets across the country, the USDA announced the establishment of the U.S. Domestic Hemp Production Program, following the enactment of the 2018 Farm Bill, essentially legalizing hemp. This announcement comes with information for hemp testing labs, including testing and sampling guidelines. While the information available on the USDA’s website only touches on testing for THC, required to be no greater than 0.3% dry weight concentration, more testing guidelines in the future are sure to include a discussion of heavy metals testing.
In an application note produced by Agilent Technologies, Inc., the Agilent 7800 ICP-MS was used to analyze 25 elements in a variety of cannabis and hemp-derived products. The study was conducted using that Agilent 7800 ICP-MS, which includes Agilent’s proprietary High Matrix Introduction (HMI) system. The analysis was automated by using the Agilent SPS 4 autosampler.
The instrument operating conditions can be found in Table 1. In this study, the HMI dilution factor was 4x and the analytes were all acquired in the Helium collision mode. Using this methodology, the Helium collision mode consistently reduces or completely eliminates all common polyatomic interferences using kinetic energy discrimination (KED).
As a comparison, Arsenic and Selenium were also acquired via the MassHunter Software using half-mass correction, which corrects for overlaps due to doubly charged rare earth elements. This software also collects semiquantitative or screening data across the entire mass region, called Quick Scan, showing data for elements that may not be present in the original calibration standards.
SRMs and Samples
Standard reference materials (SRMs) analyzed from the National Institute of Standards and Technology (NIST) were used to verify the sample prep digestion process. Those included NIST 1547 Peach Leaves, NIST 1573a Tomato Leaves and NIST 1575 Pine Needles. NIST 1640a Natural Water was also used to verify the calibration.
Samples used in the study include cannabis flower, cannabis tablets, a cannabidiol (CBD) tincture, chewable candies and hemp-derived cream.
Calibration standards were prepared using a mix of 1% HNO3 and 0.5% HCl. Sodium, Magnesium, Potassium, Calcium and Iron were calibrated from 0.5 to 10 ppm. Mercury was calibrated from 0.05 to 2 ppb. All the other elements were calibrated from 0.5 to 100 ppb.
After weighing the samples (roughly 0.15 g of cannabis plant and between 0.3 to 0.5 g of cannabis product) into quartz vessels, 4 mL HNO3 and 1 mL HCl were added and the samples were microwave digested using the program found in Table 2.
HCI was included to ensure the stability of Mercury and Silver in solution. They diluted the digested samples in the same acid mix as the standards. SRMs were prepared using the same method to verify sample digestion and to confirm the recovery of analytes.
Four samples were prepared in triplicate and fortified with the Agilent Environmental Mix Spike solution prior to the analysis. All samples, spikes and SRMs were diluted 5x before testing to reduce the acid concentration.
The calibration curves for Arsenic, Cadmium, Lead and Mercury can be found in Figure 1 and a summary of the calibration data is in Table 3. For quality control, the SRM NIST 1645a Natural Water was used for the initial calibration verification standard. Recoveries found in Table 4 are for all the certified elements present in SRM NIST 1640a. The mean recoveries and concentration range can also be found in Table 4. All the continuing calibration solution recoveries were within 10% of the expected value.
Internal Standard Stability
Figure 2 highlights the ISTD signal stability for the sequence of 58 samples analyzed over roughly four hours. The recoveries for all samples were well within 20 % of the value in the initial calibration standard.
In Table 5, you’ll find that three SRMs were tested to verify the digestion process. The mean results for most elements agreed with the certified concentrations, however the results for Arsenic in NIST 1547 and Selenium in both NIST 1547 and 1573a did not show good agreement due to interreferences formed from the presence of doubly-charged ions
Some plant materials can contain high levels of rare earth elements, which have low second ionization potentials, so they tend to form doubly-charged ions. As the quadrupole Mass Spec separates ions based on their mass-to-charge ratio, the doubly-charged ions appear at half of their true mass. Because of that, a handful of those doubly-charged ions caused overlaps leading to bias in the results for Arsenic and Selenium in samples that have high levels of rare earth elements. Using half mass correction, the ICP-MS corrects for these interferences, which can be automatically set up in the MassHunter software. The shaded cells in Table 5 highlight the half mass corrected results for Arsenic and Selenium, demonstrating recoveries in agreement with the certified concentrations.
In Table 6, you’ll find the quantitative results for cannabis tablets and the CBD tincture. Although the concentrations of Arsenic, Cadmium, Lead and Cobalt are well below current regulations’ maximum levels, they do show up relatively high in the cannabis tablets sample. Both Lead and Cadmium also had notably higher levels in the CBD tincture as well.
A spike recovery test was utilized to check the accuracy of the method for sample analysis. The spike results are in Table 6.
Using the 7800 ICP-MS instrument and the High Matrix Introduction system, labs can routinely analyze samples that contain high and very variable matrix levels. Using the automated HMI system, labs can reduce the need to manually handle samples, which can reduce the potential for contamination during sample prep. The MassHunter Quick Scan function shows a complete analysis of the heavy metals in the sample, including data reported for elements not included in the calibration standards.
The half mass correction for Arsenic and Selenium allows a lab to accurately determine the correct concentrations. The study showed the validity of the microwave sample prep method with good recovery results for the SRMs. Using the Agilent 7800 ICP-MS in a cannabis or hemp testing lab can be an effective and efficient way to test cannabis products for heavy metals. This test can be used in various stages of the supply chain as a tool for quality controls in the cannabis and hemp markets.
Disclaimer: Agilent products and solutions are intended to be used for cannabis quality control and safety testing in laboratories where such use is permitted under state/country law.
Microbial contamination on cannabis products represents one of the most significant threats to cannabis consumers, particularly immunocompromised patients who are at risk of developing harmful and potentially fatal infections.
As a result, regulatory bodies in the United States and Canada mandate testing cannabis products for certain microbes. The two most popular methods for microbial safety testing in the cannabis industry are culture-based testing and quantitative polymerase chain reaction (qPCR).
When considering patient safety, labs should choose a method that provides an accurate account of what is living on the sample and can specifically target the most harmful microbes, regardless of the matrix.
1. The Method’s Results Must Accurately Reflect the Microbial Population on the Sample
The main objective of any microbial safety test is to give the operator an indication of the microbial population present on the sample.
Culture-based methods measure contamination by observing how many organisms grow in a given medium. However, not all microbial organisms grow at the same rate. In some cases, certain organisms will out-compete others and as a result, the population in a post-culture environment is radically different than what was on the original sample.
One study analyzed fifteen medicinal cannabis samples using two commercially available culture-based methods. To enumerate and differentiate bacteria and fungi present before and after growth on culture-based media, all samples were further subjected to next-generation sequencing (NGS) and metagenomic analyses (MA). Figure 1 illustrates MA data collected directly from plant material before and after culture on 3M petrifilm and culture-based platforms.
The results demonstrate substantial shifts in bacterial and fungal growth after culturing on the 3M petrifilm and culture-based platforms. Thus, the final composition of microbes after culturing is markedly different from the starting sample. Most concerning is the frequent identification of bacterial species in systems designed for the exclusive quantification of yeast and mold, as quantified by elevated total aerobic count (TAC) Cq values after culture in the total yeast and mold (TYM) medium. The presence of bacterial colonies on TYM growth plates or cartridges may falsely increase the rejection rate of cannabis samples for fungal contamination. These observations call into question the specificity claims of these platforms.
The Live Dead Problem
One of the common objections to using qPCR for microbial safety testing is the fact that the method does not distinguish between live and dead DNA. PCR primers and probes will amplify any DNA in the sample that matches the target sequence, regardless of viability. Critics claim that this can lead to false positives because DNA from non-viable organisms can inflate results. This is often called the Live-Dead problem. However, scientists have developed multiple solutions to this problem. Most recently, Medicinal Genomics developed the Grim Reefer Free DNA Removal Kit, which eliminates free DNA contained in a sample by simply adding an enzyme and buffer and incubating for 10 minutes. The enzyme is instantaneously inactivated when lysis buffer is added, which prevents the Grim Reefer Enzyme from eliminating DNA when the viable cells are lysed (see Figure 2).
2. Method Must Be Able to Detect Specific Harmful Species
Conversely, qPCR assays, such as the PathoSEEK, are designed to target DNA sequences that are unique to pathogenic Aspergillus species, and they can be run using standard qPCR instruments such as the Agilent AriaMx. The primers are so specific that a single DNA base difference in the sequence can determine whether binding occurs. This specificity reduces the frequency of false positives in pathogen detection, a frequent problem with culture-based cannabis testing methods.
Additionally, Medicinal Genomics has developed a multiplex assay that can detect the four pathogenic species of Aspergillus (A. flavus, A. fumigatus, A. niger, and A. terreus) in a single reaction.
3. The Method Must Work on Multiple Matrices
Marijuana infused products (MIPs) are a very diverse class of matrices that behave very differently than cannabis flowers. Gummy bears, chocolates, oils and tinctures all present different challenges to culture-based techniques as the sugars and carbohydrates can radically alter the carbon sources available for growth. To assess the impact of MIPs on colony-forming units per gram of sample (CFU/g) enumeration, The Medicinal Genomics team spiked a known amount of live E. coli into three different environments: tryptic soy broth (TSB), hemp oil and hard candy. The team then homogenized the samples, pipetted amounts from each onto 3M™ Petrifilm E. coli / Coliform Count (EC) Plates, and incubated for 96 hours. The team also placed TSB without any E. coli onto a petrifilm to serve as a control. Figures 3 and 4 show the results in 24-hour intervals.
This implies the MIPs are interfering with the reporter assay on the films or that the MIPs are antiseptic in nature.
Many MIPs use citric acid as a flavoring ingredient which may interfere with 3M reporter chemistry. In contrast, the qPCR signal from the Agilent AriaMx was constant, implying there is microbial contamination present on the films, but the colony formation or reporting is inhibited.
This is not an issue with DNA-based methods, so long as the DNA extraction method has been validated on these matrices. For example, the SenSATIVAx DNA extraction method is efficient in different matrices, DNA was spiked into various MIPs as shown in Table 1, and at different numbers of DNA copies into chocolate (Table 2). The SenSATIVAx DNA extraction kit successfully captures the varying levels of DNA, and the PathoSEEK detection assay can successfully detect that range of DNA. Table 3 demonstrates that SenSATIVAx DNA extraction can successfully lyse the cells of the microbes that may be present on cannabis for a variety of organisms spiked onto cannabis flower samples.
AOAC INTERNATIONAL is an independent, third party, not-for-profit association and voluntary consensus standards developing organization. Founded in 1884, AOAC INTERNATIONAL was originally coined the Association of Official Agricultural Chemists. Later on, they changed their name to the Association of Official Analytical Chemists. Now that their members include microbiologists, food scientists as well as chemists, the organization officially changed its name to just AOAC INTERNATIONAL.
Much of AOAC’s work surrounds promoting food safety, food security and public health. Their work generally encompasses setting scientific standards for testing methodology, evaluating and adopting test methods and evaluating laboratory proficiency of test methods. The organization provides a forum for scientists to develop microbiological and chemical standards.
In December of 2018, they appointed Dr. Palmer Orlandi as deputy executive director and chief science officer. Dr. Orlandi has an extensive background at the U.S. Food and Drug Administration (FDA), serving the regulatory agency for more than 20 years. Most recently, he was the CSO and research director in the Office of Food and Veterinary Medicine at the FDA. He earned the rank of Rear Admiral and Assistant Surgeon General in 2017.
Where It All Began With Cannabis
As recently as three years ago, AOAC began getting involved in the cannabis laboratory testing community, with a working group dedicated to developing standard method performance requirements for AOAC Official MethodsSM for cannabis testing. We sat down with Dr. Palmer Orlandi and a number of AOAC’s leaders to get an update on their progress working with cannabis testing as well as food security and food fraud.
According to Scott Coates, senior director of the AOAC Research Institute, they were approached three years ago to set up a working group for cannabis testing. “We created standards that we call the standard method performance requirements (SMPR®), which are detailed descriptions of what analytical methods should be able to do,” says Coates. “Using SMPRs, we issued a series of calls for methods and looked for methods that meet our standards. So far, we’ve completed four SMPRs- cannabinoids in plant material, cannabinoids in plant extracts, cannabinoids in chocolate (edibles), and one for pesticides in cannabis plant material.” AOAC doesn’t develop methods themselves, but they perform a comprehensive review of the methods and if they deem them acceptable, then the methods can be adopted and published in the AOAC compendium of methods, the Official Methods of Analysis of AOAC INTERNATIONAL.
Deborah McKenzie, senior director of Standards and Official MethodsSM at AOAC, says the initial working group set the stage for really sinking their teeth into cannabis testing. “It started with methods for testing cannabinoids in plant dried material and plant extract,” says McKenzie. “That’s where our previous work has started to mold into the current effort we are launching.” McKenzie says they are looking forward to getting more involved with methods regarding chemical contaminants in cannabis, cannabinoids in various foods and consumables, as well as microbial organisms in cannabis. “We are pretty focused on testing labs having reliable and validated analytical solutions as our broad goal right now.”
Moving Forward, Expanding Their Programs
Coates says the work they’ve done over the past few years was more of a singular project, developed strictly for creating standards and to review methods. Now they are currently developing their Cannabis Analytical Science Program (CASP), which is expected to be an ongoing program. “We are looking to fully support the cannabis analytical community as best we can, which will potentially include working on reference materials, proficiency testing, education, training and ISO 17025 accreditation, all particularly as it applies to lab testing in the cannabis industry,” says Coates. “So, this CASP work is a much bigger and broader effort to cover more and to provide more support for labs doing the analysis of cannabis and its constituents, as well as hemp.”
According to Dr. Orlandi, they want this program to have a broad reach in the cannabis testing community. “As Scott pointed out, it’s not just strictly developing standards and methods,” says Dr. Orlandi. “It is going to be as all-encompassing as possible and will lead to training programs, a proficiency testing program and other areas.” Arlene Fox, senior director of AOAC’s Laboratory Proficiency Testing Program, says they are actively engaging in proficiency testing. “We are in the process of evaluating what is out there, what is possible and what’s needed as far as expanding proficiency testing for cannabis labs,” says Fox.
Regulatory Challenges & Obstacles
The obvious roadblock to much of AOAC’s work is that cannabis is still considered a controlled substance. “That creates some challenges for the work that we do in certain areas,” says Dr. Orlandi. “That is why this isn’t just a one-year project. We will work with these challenges and our stakeholders to address them.” AOAC had to put some limits on participation- for example, they had to decide that they cannot look for contributions or collaborations with producers and distributors, so long as cannabis is still a Schedule I controlled substance in the US.
Muddying the waters even further, the recent signing of the Farm Bill puts a clear distinction between most types of cannabis and industrial hemp. David Schmidt, executive director of AOAC realizes they need to be realistic with their stakeholders and in the eye of federal law.
While scientifically speaking, it’s pretty much the same plant just with slightly different chemical constituents, AOAC INTERNATIONAL has to draw a line in the sand somewhere. “As Palmer suggests, because of the Farm Bill being implemented and hemp being defined now as a legal substance from a controlled substance standpoint, industrial hemp has been given this exclusion,” says Schmidt. “So, we are trying to be realistic now, working with our stakeholders that work with hemp, trying to understand the reality of the federal law. We want to make clear that we can meet stakeholder needs and we want to distinguish hemp from cannabis to remain confident in the legality of it.” Schmidt says this is one of a number of topics they plan on addressing in detail at their upcoming 9thannual 2019 Midyear Meeting, held March 11-14 in Gaithersburg, Maryland.
Uniformity in Methodology: The Future of Cannabis Testing
Dr. Orlandi says his experience at the FDA has prepared him well for the work being done at AOAC. “The role that I served at the FDA prior to joining my colleagues here at AOAC was very similar: And that is to bring together stakeholders to accomplish or to solve a common problem.” Some of their stakeholders in the CASP program include BC Testing, Inc., the Association of Food and Drug Officials (AFDO), Bia Diagnostics, Bio-Rad, Industrial Laboratories, Materia Medica Labs, PerkinElmer, R-Biopharm AG, Supra R & D, TEQ Analytical Laboratories, Titan Analytical and Trilogy Analytical, among others.
“The underlying reason behind this effort is to create some level of harmonization for standards and methods,” says Dr. Orlandi. “They can be used in the near future to stay ahead of the curve for when regulatory agencies become involved. The idea is that these standards for analytical methods will already be established and as uniform as possible.”
When comparing cannabis to other industries in the US, Scott Coates mentions that most standards are signed off by the federal government. “When we started looking at pesticides in cannabis, it became really clear that we have a number of states doing things differently with different limits of quantification,” says Coates. “Each state, generally speaking, is setting their own standards. As Palmer was saying, one thing we are trying to do with this CASP program eventually will be to have some harmonization, instead of 30 different states having 30 different standards and methods.” So, on a much broader level, their goal for the CASP program is to develop a common set of standard methods, including hemp testing and even the Canadian market. “Hopefully this will be an international collaboration for standards for the methodology,” says Coates. They want to create a common set of standards, setting limits of quantification that will be accepted internationally, that will be accurate and repeatable and for the entire cannabis industry, not just state by state.
Food Authenticity & Fraud
One of the other activities that AOAC just launched recently is the food authenticity and fraud program. As the name implies, the goal is to start developing standards and methods and materials to look at economically adulterated foods, says Dr. Orlandi. That includes non-targeted analyses looking at matrices of food products that may be adulterated with an unknown target, as well as targeted analytes, identifying common adulterants in a variety of food products. “One example in the food industry is fraudulent olive oil,” says Dr. Orlandi. “Honey is another commodity that has experienced adulteration.” He says that in most cases these are economically motivated instances of fraud.
By Ravi Kanipayor, Christian Bax, Dr. George Anastasopoulos No Comments
As state cannabis regulatory frameworks across the country continue to evolve, accreditation is becoming increasingly important. Because it provides consistent, turnkey standards and third-party verification, accreditation is quickly emerging as an important tool for regulators. For cannabis testing laboratories, this trend has been especially pronounced with the increasing number of states that require accreditation to ISO/IEC 17025.
As of 2017 there were nearly 68,000 laboratories accredited to ISO/IEC 17025, making it the single most important benchmark for testing laboratories around the world. ISO/IEC 17025:2005 specifies the general requirements for the competence to carry out tests including sampling. It covers testing performed using standard methods, non-standard methods and laboratory-developed methods. It is applicable to all organizations performing tests including cannabis labs. The standard is applicable to all labs regardless of the number of personnel or the extent of the scope of testing activities. Developed to promote confidence in the operation of laboratories, the standard is now being used as a key prerequisite to operate as a cannabis lab in many states.
There are currently 26 states in the United States (also Canada) that require medical or adult-use cannabis to be tested as of February 2019. Of those states, 18 require cannabis testing laboratories to be accredited – with the vast majority requiring ISO/IEC 17025 accreditation. States that require testing laboratories to attain ISO/IEC 17025 accreditation represent some of the largest and most sophisticated cannabis regulatory structures in the country, including California, Colorado, Maryland, Massachusetts, Michigan, Nevada and Ohio. As a consequence, many cannabis testing laboratories are taking note of recent changes to ISO/IEC 17025 standards.
ISO/IEC 17025 was first issued in 1999 by the International Organization for Standardization. The standard was updated in 2005, and again in 2017. The most recent update keeps many of the legacy standards from 2005, but adds several components – specifically requirements for impartiality, risk assessment and assessing measurement uncertainty. The remainder of this article takes a deeper dive into these three areas of ISO/IEC 17025, and what that means for cannabis testing laboratories.Objectivity is the absence or resolution of conflicts of interest to prevent adverse influence on laboratory activities.
ISO/IEC 17025:2005 touched on an impartiality requirement, but only briefly. The previous standard required laboratories that belonged to organizations performing activities other than testing and/or calibration to identify potential conflicts of interest for personnel involved with testing or calibration. It further required that laboratories had policies and procedures to avoid impartiality, though that requirement was quite vague.
ISO/IEC17025:2017 emphasizes the importance of impartiality and establishes strict requirements. Under the new standard, labs are responsible for conducting laboratory activities impartially and must structure and manage all laboratory activities to prevent commercial, financial or other operational pressures from undermining impartiality. The definitions section of the standard defines impartiality as the “presence of objectivity.” Objectivity is the absence or resolution of conflicts of interest to prevent adverse influence on laboratory activities. For further elaboration, the standard provides similar terms that also convey the meaning of impartiality: lack of prejudice, neutrality, balance, fairness, open-mindedness, even-handedness, detachment, freedom from conflicts of interest and freedom from bias.
To comply with the new standard, all personnel that could influence laboratory activities must act impartially. ISO/IEC 17025:2017 also requires that laboratory management demonstrate a commitment to impartiality. However, the standard is silent on how labs must demonstrate such commitment. As a starting point, some cannabis laboratories have incorporated statements emphasizing impartiality into their employee handbooks and requiring management and employee training on identifying and avoiding conflicts of interest.
Both the 2005 and 2017 versions contain management system requirements. A major update to this is the requirement in ISO/IEC 17025:2017 that laboratory management systems incorporate actions to address risks and opportunities. The new risk-based thinking in the 2017 version reduces prescriptive requirements and incorporates performance-based requirements.
Under ISO/IEC 17025:2017, laboratories must consider risks and opportunities associated with conducting laboratory activities. This analysis includes measures that ensure that:
The lab’s management system is successful;
The lab has policies to increase opportunities to achieve its goals and purpose;
The lab has taken steps to prevent or reduce undesired consequences and potential failures; and
The lab is achieving overall improvement.
Labs must be able to demonstrate how they prevent or mitigate any risks to impartiality that they identify.To comply with ISO/IEC 17025:2017, labs must plan and implement actions to address identified risks and opportunities into management systems. They must also measure the effectiveness of such actions. Importantly, the standard requires that the extent of risk assessments must be proportional to the impact a given risk may have on the validity of the laboratory’s test results.
ISO/IEC 17025:2017 does not require that labs document a formal risk management process, though labs have discretion to develop more extensive methods and processes if desired. To meet the requirements of the standard, actions to address risks can include sharing the risk, retaining the risk by informed decision, eliminating the risk source, pinpointing and avoiding threats, taking risks in order to pursue an opportunity, and changing the likelihood or consequence of the risk.
ISO/IEC 17025:2017 references “risks” generally throughout most of the standard. However, it specifically addresses risks to a laboratory’s impartiality in section 4.1. Note, the new standard requires that labs must not only conduct activities impartially, but also actively identify risks to their impartiality. This requirement is on-going, not annually or bi-annually. Risks to impartiality include risks arising from laboratory activities, from laboratory relationships, or from relationships of laboratory personnel. Relationships based on ownership, governance, shared resources, contracts, finances, marketing, management, personnel and payment of a sales commission or other inducements to perform under pressure can threaten a laboratory’s impartiality. Labs must be able to demonstrate how they prevent or mitigate any risks to impartiality that they identify.
Assessing Measurement Uncertainty With Decision Rules
ISO/IEC 17025:2005 required (only where necessary and relevant) test result reports to include a statement of compliance/non-compliance with specifications and to identify which clauses of the specification were met or not met. Such statements were required to take into account measurement uncertainty and if measurement results and uncertainties were omitted from the statement, the lab was required to record and maintain the results for future reference.
ISO/IEC 17025:2017 requires similar statements of conformity with an added “decision rule” element. When statements of conformity to a specification or standard are provided, labs must record the decision rule it uses and consider the level of risk the decision rule will have on recording false positive or negative test results. Like the 2005 version, labs must include statements of conformity in test result reports (only if necessary and relevant- see 126.96.36.199 (b)). Now, test result reports on statements of conformity must include the decision rule that was employed.
Because many states require ISO/IEC 17025 accreditation for licensing, cannabis testing labs across the country would be well advised to closely monitor the implications of changes in ISO/IEC 17025:2017 related to impartiality, risk assessment and measurement uncertainty. If you run a cannabis testing lab, the best way to ensure compliance is education, and the best place to learn more about the new requirements is from a globally recognized accreditation body, especially if it is a signatory to the International Laboratory Accreditation Cooperation (ILAC) for testing laboratories, calibration laboratories and inspection agencies.
For all the success of the cannabis market in the United States, there are two big issues that still confound the industry because of a lack of federal reform. The first, of course, is national recognition of an industry that still struggles with banking, insurance and selling products across state lines. The other is international trade.
However, it appears that one Colorado-based company, United Cannabis, has now successfully begun to navigate the complex regulatory and standards puzzle, and further, has set up trade and import agreements in both France and Switzerland. Even more interesting? It managed to do the same before the passage of the Farm Bill.
At present they are exporting to Europe from Florida – but the fact that they are exporting in the European direction at all is a feat still unmatched by many other American firms all looking to do the same thing.
In Switzerland, they are also partnering with an equally intriguing firm called Cloud 9 Switzerland. We sat down with Francis Scanlan, founder of Cloud 9 Switzerland, to talk about what they are doing and how they are doing it- and from the European perspective.
The First Compliant Swiss Chocolate Maker
Cloud 9 is a start-up that is going head to head with the larger Canadian firms in innovative ways and in several directions. That includes the creation of food and beverage products. It also includes pharmaceuticals.
As of January 22, 2019, Cloud 9 also received approval from Swiss authorities to proceed with production of what will be, as Scanlan describes it, “the first EU-compliant hemp chocolate bar.” The hemp they are using contains a full spectrum hemp extract, which does not fall under the rubric of a so-called “novel food” because hemp has been a product in the consumer market here for a long time.
The product will be on Italian shelves as of the end of Q1 this year. Beyond the regulatory approvals necessary to get to market, it also took him about a year to find and convince a chocolate manufacturer in Switzerland to work with him.
Scanlan describes his year and a half old firm as the “value added” between suppliers, manufacturers and distributors. With a background in the corporate food and beverage industry including a stint at Nestlé, he and his team create the formulations and commercialize new products. And they keep a sharp eye on the regulatory bottom line in Europe.
Cloud 9’s corporate mission, Scanlan says, is to improve the quality of life and wellness of their customers. “We are not in the opportunistic marketing business” he says. “We want to create products that really benefit people. Our motto has always been Win-Win for both our partners and consumers.”
Bringing A Glaucoma Drug To The EU Market
However do not mistake Cloud 9 or even Scanlan himself as a kind of cannabis Willy Wonka one hit wonder. Or a firm that is solely operating in the wellness space. They are also now working to bring a Glaucoma drug into the EU where they will begin with medical trials to start the approval process. That said, Scanlan is confident about the success of this product as well. “It has a great dossier in its home country,” he says. “And that has also already caught the interest of doctors in Italy and Switzerland.”
Beyond that, there are other plans in the works, including the introduction of a transdermal patch that delivers cannabinoids through the skin. “The great thing about this kind of approach,” Scanlan says, “is that it allows people to get over their fear of orally ingested drugs. They don’t like the effect, they can just take it off.” He also noted that the patch uses a patented technology that allows a far more efficient delivery mechanism, which creates a time-delayed medication approach and allows for a 90% transfer of cannabinoids.
In other words, this small, privately funded start-up, using innovative approaches to a market Scanlan knows well, is absolutely in the ring and going to market. And further doing so with a European mindset and operating philosophy that incorporates not only hemp exported from the American hemisphere, but is mixed with a large dollop of good old “American” entrepreneurial gusto and inclinations.
Disclaimer: Cloud9 is a sponsor of the MedPayRx pilot to market program in the EU.
Editor’s Note: The following is based on research and studies performed in their Santa Cruz Lab, with contributions from Mikhail Gadomski, Lab Manager, Ryan Maus, Technical Services Analyst, Dr. Laurie Post, Director of Food Safety & Compliance, Andy Sechler, Lab Director, Toby Astill, Senior Business Development Leader at Perkin Elmer and Charles Deibel, President of Deibel Cannabis Labs.
Pesticides represent the leading cause of batch failures in the cannabis industry. They are also the hardest tests to run in the laboratory, even one equipped with state-of-the-art equipment. The best instruments on the market are HPLC and GC dual mass spectrometer detectors, called “HPLC-qqq”, “GC-qqq,” or just triple quads.
As non-lab people, we envision a laboratory that can take a cannabis sample, inject it into a triple quad and have the machine quickly and effortlessly print out a report of pesticide values. Unfortunately, this is far from reality. The process is much more hands on and complex.In the current chemistry lab, trained analysts have to first program the triple quads to look for the pesticides of concern; in cannabis pesticide testing, this is done by programming the first of two mass spectrometers to identify a single (precursor) mass that is characteristic of the pesticide in question. For BCC requirements in California, this has to be done for all 66 pesticides, one at a time.
Next, these precursor ions are degraded into secondary chemicals called the “product” ions, also called transition ions. The second of the two mass spectrometers is used to analyze these transition ions. This process is graphed and the resulting spectrum is analyzed by trained chemists in the lab, pesticide by pesticide, for all the samples processed that day. If the lab analyzes 10 samples, that translates to 660 spectra to analyze (66 pesticides x 10 samples). When looking at the spectra for each pesticide, the analysts must compare the ratios of the precursor ions to the product ions.
If these spectra indicate a given pesticide may be present, the chemists must then compare the ratios between the precursor and the products. If these ratios are not what is expected, then the analyst must perform confirmation testing to prove the precursor mass either is or is not the pesticide of concern. If the ratios are not what is expected, it means the molecule is similar to the pesticide in question, but may not be that pesticide. This confirmatory testing is key to producing accurate results and not failing batches when dealing with closely related chemicals. This process of analyzing spectra is done in all labs that are performing pesticide testing. In this fledgling industry, there are few published cannabis pesticide methods.
The need for this type of confirmation testing doesn’t happen all of the time, but when it does, it will take longer than our targeted three-day turn-around time. In the picture above, one precursor mass is ionized into several product masses; but only two are large enough to be used for comparison. In this hypothetical situation, two product masses are produced for every one precursor, the expected ion abundance ratio should be less than 30%. When performing any confirmatory testing, if the ion abundance ratio is >30%, it means the original precursor molecule was not the pesticide of concern. For example, if the ion abundance ratio was 50%, then the original molecule broke down into too many parts; it was not the pesticide we were looking for. This ion abundance ratio threshold was established by FANCO, the international organization that sets guidelines for all pesticide testing.
Methodology: In this fledgling industry, there are few published cannabis pesticide methods. The identification of the precursor mass and product ions are not always published, leaving labs to research which ions should be used. This adds to the potential for differences between lab results. Once selected, labs should validate their research, through a series of experiments to ensure the correct precursor and transition (product) ions are being used in the method.
Sample Preparation: Beyond the time-consuming work that is required to develop sound pesticide methods, the extraction step is absolutely critical for credible results. If the pesticides aren’t fully extracted from the cannabis product, then the results will be lower than expected. Sample preparations are often not standardized between labs, so unless a given extraction technique is validated for accuracy, there is the possibility for differences between labs.
Getting a Representative Sample
The current California recommended amount of sample is one gram of product per batch. Batch sizes can vary greatly and it is entirely likely that two different one gram samples can have two different results for pesticides. Has the entire plant been evenly coated with exactly the same amount of pesticide onto every square inch of its leaves? No, probably not. That is why it is imperative to take a “random” sample, by taking several smaller samples from different areas of the entire batch.
Sampling Plans: We can learn a lot from the manufacturing and sampling best practices developed by the food industry through the years. If a food manufacturer is concerned with the possibility of having a bacteria pathogen, like Salmonella, in their finished product, they test the samples coming off their production lines at a statistically relevant level. This practice (theory) is called the sampling plan and it can easily be adapted to the cannabis industry. The basic premise is that the more you test, the higher your likelihood of catching a contaminate. Envision a rectangular swimming pool, but instead of water, it’s filled with jello. In this gelatinous small pool, 100 pennies are suspended at varying levels. The pennies represent the contaminates.
Is the pool homogenized? Is jello evenly represented in the entire pool? Yes.
Is your concentrate evenly distributed in the extraction vessel? Yes. The question is, where are the pennies in that extraction vessel? The heavy metals, the microbial impurities and the pesticides should be evenly distributed in the extraction vessel but they may not be evenly represented in each sample that is collected. Unfortunately, this is the bane of the manufacturing industry and it’s the unfortunate reality in the food industry. If you take one random cup of jello, will you find the penny? Probably not. But it you take numerous 1 cup samples from random areas within the batch, you increase your chances of finding the contaminate. This is the best approach for sampling any cannabis product.
The best way to approve a batch of cannabis product is to take several random samples and composite them. But you may need to run several samples from this composite to truly understand what is in the batch. In the swimming pool example, if you take one teaspoon scoop, will you find one of the pennies? The best way to find one of the pennies is to take numerous random samples, composite them and increase the number of tests you perform at the lab. This should be done on any new vendor/cultivator you work with, in order to help establish the safety of the product.
Editor’s Note: The following is based on research and studies performed in their Santa Cruz Lab, with contributions from Mikhail Gadomski, Lab Manager, Ryan Maus Technical Services Analyst, Laurie Post, Director of Food Safety & Compliance, and Charles Deibel, President Deibel Cannabis Labs.
Heavy metals are common environmental contaminants resulting from human industrial activities such as mining operations, industrial waste, automotive emissions, coal fired power plants and farm/house hold water run-off. They affect the water and soil, and become concentrated in plants, animals, pesticides and the sediments used to make fertilizers. They can also be present in low quality glass or plastic packaging materials that can leach into the final cannabis product upon contact. The inputs used by cultivators that can be contaminated with heavy metals include fertilizers, growing media, air, water and even the clone/plant itself.
The four heavy metals tested in the cannabis industry are lead, arsenic, mercury and cadmium. The California Bureau of Cannabis Control (BCC) mandates heavy metals testing for all three categories of cannabis products (inhalable cannabis, inhalable cannabis products and other cannabis and cannabis products) starting December 31, 2018. On an ongoing basis, we recommend cultivators test for the regulated heavy metals in R&D samples any time there are changes in a growing process including changes to growing media, cannabis strains, a water system or source, packaging materials and fertilizers or pesticides. Cultivators should test the soil, nutrient medium, water and any new clones or plants for heavy metals. Pre-qualifying a new packaging material supplier or a water source prior to use is a proactive approach that could bypass issues with finished product.
The best approach to heavy metal detection is the use of an instrument called an Inductively Coupled Plasma Mass Spectrometry (ICP-MS). There are many other instruments that can test for heavy metals, but in order to achieve the very low detection limits imposed by most states including California, the detector must be the ICP-MS. Prior to detection using ICP-MS, cannabis and cannabis related products go through a sample preparation stage consisting of some form of digestion to completely break down the complex matrix and extract the heavy metals for analysis. This two-step process is relatively fast and can be done in a single day, however, the instruments used to perform the digestion are usually the limiting step as the digesters run in a batch of 8-16 samples over a 2-hour period.
Only trace amounts of heavy metals are allowed by California’s BCC in cannabis and cannabis products. A highly sensitive detection system finds these trace amounts and also allows troubleshooting when a product is found to be out of specification.
For example, during the course of testing, we have seen lead levels exceed the BCC’s allowable limit of 0.5 ppm in resin from plastic vape cartridges. An investigation determined that the plastic used to make the vape cartridge was the source of the excessive lead levels. Even if a concentrate passes the limits at the time of sampling, the concern is that over time, the lead leached from the plastic into the resin, increasing the concentration of heavy metals to unsafe levels.
Getting a Representative Sample
The ability to detect trace levels of heavy metals is based on the sample size and how well the sample represents the entire batch. The current California recommended amount of sample is 1 gram of product per batch. Batch sizes can vary but cannot be larger than 50 pounds of flower. There is no upper limit to the batch sizes for other inhalable cannabis products (Category II).
It is entirely likely that two different 1 gram samples of flower can have two different results for heavy metals because of how small a sample is collected compared to an entire batch. In addition, has the entire plant evenly collected and concentrated the heavy metals into every square inch of it’s leaves? No, probably not. In fact, preliminary research in leafy greens shows that heavy metals are not evenly distributed in a plant. Results from soil testing can also be inconsistent due to clumping or granularity. Heavy metals are not equally distributed within a lot of soil and the one small sample that is taken may not represent the entire batch. That is why it is imperative to take a “random” sample by collecting several smaller samples from different areas of the entire batch, combining them, and taking a 1 g sample from this composite for analysis.
Linger, P. J. Mussig, H. Fischer, J. Kobert. 2002. Industrial hemp (Cannabis sativa L.) growing on heavy metal contaminated soil: fibre quality and phytoremediation potential. Ind. Crops Prod. 11, 73–84.
The combination of gas chromatography and infrared spectroscopy (GC/IR) is a powerful tool for the characterization of compounds in complex mixtures. (1-5) Gas chromatography with mass spectroscopy detection (GC/MS) is a similar technique, but GC/MS is a destructive technique that tears apart the sample molecules during the ionization process and then these fragments are used to characterize the molecule. In GC/IR the molecules are not destroyed but the IR light produced by molecular vibrations are used to characterize the molecule. IR spectrum yields information about the whole molecule which allows the characterization of specific isomers and functional groups. GC/IR is complementary to GC/MS and the combination results in a powerful tool for the analytical chemist.
A good example of the utility of GC/IR vs GC/MS is the characterization of stereo isomers. Stereo isomers are mirror images such as a left hand and a right hand. In nature, stereo isomers are very important as one isomers will be more active then its mirror image. Stereo isomers are critical to medicinal application of cannabis and also a factor in the flavor components of cannabis.
GC/MS is good at identifying basic structure, where GC/IR can identify subtle differences in structure. GC/MS could identify a hand, GC/IR could tell you if it is a left hand or right hand. GC/MS can identify a general class of compounds, GC/IR can identify the specific isomer present.
Gas chromatography interfaced with infrared detection (GC/IR), combines the separation ability of GC and the structural information from IR spectroscopy. GC/IR gives the analyst the ability to obtain information complementary to GC/MS. GC/IR gives the analyst the power to perform functional group detection and differentiate between similar molecular isomers that is difficult with GC/MS. Isomer specificity can be very important in flavor and medical applications.
Gas chromatography with mass spectrometry detection (GC/MS) is the state-of-the-art method for the identification of unknown compounds. GC/MS, however, is not infallible and many compounds are difficult to identify with 100 % certainty. The problem with GC/MS is that it is a destructive method that tears apart a molecule. In infrared spectrometry (IR), molecular identification is based upon the IR absorptions of the whole molecule. This technique allows differentiation among isomers and yields information about functional groups and the position of such groups in a molecule. GC/IR complements the information obtained by GC/MS.
Initial attempts to couple GC with IR were made using high capacity GC columns and stopped flow techniques. As GC columns and IR technology advanced, the GC/IR method became more applicable. The advent of fused silica capillary GC columns and the availability of Fourier transform infrared spectrometry made GC/IR available commercially in several forms. GC/IR using a flow cell to capture the IR spectrum in real time is known as the “Light Pipe”. This is the most common form of GC/IR and the easiest to use. GC/IR can also be done by capturing or “trapping” the analytes of interest eluting from a GC and then measuring the IR spectrum. This can be done by cryogenically trapping the analyte in the solid phase. A third possibility is to trap the analyte in a matrix of inert material causing “Matrix Isolation” of the analyte followed by measuring the IR spectrum.
The physical state of the sample has a large effect upon the IR spectrum produced. Molecular interactions (especially hydrogen bonding) broadens absorption peaks. Solid and liquid samples produce IR spectra with broadened peaks that loses much of the potential information obtained in the spectra. Surrounding the sample molecule with gas molecules or in an inert matrix greatly sharpens the peaks in the spectrum, revealing more of the information and producing a “cleaner” spectrum. These spectra lend themselves better to computer searches of spectral libraries similar to the computer searching done in mass spectroscopy. IR spectral computer searching requires the standard spectra in the library be of the same physical state as the sample. So, a spectrum taken in a gaseous state should be searched against a library of spectra of standards in the gaseous state.
Gas Phase – Lack of molecular interactions sharpen absorption peaks.
Matrix Isolation – Lack of molecular interactions sharpen absorption peaks.
GC/IR yields chromatograms of infrared absorbance over time. These can be total infrared absorbance which is similar to the total ion chromatogram (TIC) in GC/MS or the infrared absorbance over a narrow band or bands analogous to selected ion chromatogram. This is a very powerful ability, because it gives the user the ability to focus on selected functional groups in a mixture of compounds.
Gas chromatography with infrared detection is a powerful tool for the elucidation of the structure of organic compounds in a mixture. It is complementary to GC/MS and is used to identify specific isomers and congeners of organic compounds. This method is greatly needed in the Cannabis industry to monitor the compounds that determine the flavor and the medicinal value of its products.
GC–MS and GC–IR Analyses of the Methoxy-1-n-pentyl-3-(1-naphthoyl)-Indoles: Regioisomeric Designer Cannabinoids, Amber Thaxton-Weissenfluh, Tarek S. Belal, Jack DeRuiter, Forrest Smith, Younis Abiedalla, Logan Neel, Karim M. Abdel-Hay, and C. Randall Clark, Journal of Chromatographic Science, 56: 779-788, 2018
Simultaneous Orthogonal Drug Detection Using Fully Integrated Gas Chromatography with Fourier Transform Infrared Detection and Mass Spectrometric Detection , Adam Lanzarotta, Travis Falconer, Heather McCauley, Lisa Lorenz, Douglas Albright, John Crowe, and JaCinta Batson, Applied Spectroscopy Vol. 71, 5, pp. 1050-1059, 2017
High Resolution Gas Chromatography/Matrix Isolation Infrared Spectrometry, Gerald T. Reedy, Deon G. Ettinger, John F. Schneider, and Sid Bourne, Analytical Chemistry, 57: 1602-1609, 1985
GC/Matrix Isolation/FTIR Applications: Analysis of PCBs, John F. Schneider, Gerald T. Reedy, and Deon G. Ettinger, Journal of Chromatographic Science, 23: 49-53, 1985
A Comparison of GC/IR Interfaces: The Light Pipe Vs. Matrix Isolation, John F. Schneider, Jack C. Demirgian, and Joseph C. Stickler, Journal of Chromatographic Science, 24: 330- 335, 1986
Gas Chromatography/Infrared Spectroscopy, Jean ‐ Luc Le Qu é r é , Encyclopedia of Analytical Chemistry, John Wiley & Sons, 2006
The track will have presentations discussing food safety planning in cannabis manufacturing, HACCP, GMPs, regulatory compliance and supply chain issues among other areas. One particular topic of interest in the quality and safety of cannabis products is laboratory testing. At the event this year, leading laboratory accreditation bodies in the country will sit together on a panel titled Accreditation, Regulation & Certification: Cannabis Labs and Production.
Laboratories that are new to the industry and looking to get accredited should be aware of the new ISO/IEC 17025:2017 standard, which was released last year. According to Tracy Szerszen, labs that have already been accredited to the 2005 version will be required to transition to the 2017 version by November 29, 2020. “This can be done in conjunction with routine assessments scheduled in 2019 and 2020,” says Szerszen. “However, laboratories are cautioned to transition within a reasonable timeframe to avoid their 17025: 2005 certificate from lapsing prior to the transition deadline. Some of the changes to the standard include but are not limited to: the re-alignment of clauses similar to ISO 9001:2015 and other ISO industry standards, modifications to reporting and decision rules, the addition of risked based thinking and a new approach to managing complaints.” Szerszen, along with the other panelists, will go much more in-depth on changes to the new ISO 17025 and other topics during the panel at the Food Safety Consortium.
Some of the other topics the panel will discuss include:
ISO/IEC 17025 –what’s expected, benefits of accreditation, common deficiencies, updates to the new 17025 standard
Standards available for production facilities-GMPs & GFSI standards
How standards can be used to safeguard the quality of production and safety requirements
An open discussion with panelists from leading accreditation bodies on the state of cannabis lab testing
According to Chris Gunning, many states are requiring accreditation to ISO/IEC 17025, the standard used throughout the world in many other high-profile industries such as the testing of food and pharmaceuticals, environmental testing, and biosafety testing. “In an industry where there are few standard methods, where one hears that you can ‘pay to play,’ and where there are ‘novice’ laboratories popping up with little experience in operating a testing laboratory, it is extremely important to have an experienced, independent, 3rd party accrediting body evaluating the laboratory,” says Gunning. “This process confirms their adherence to appropriate quality management system standards, standard methods or their own internally developed methods, and can verify that those methods produce valid results. Ultimately, the process of accreditation gives the public confidence that a testing laboratory is meeting their state’s requirements and therefore consumers have access to a quality product.” He says most states with legal cannabis recognize the need for product testing by a credentialed laboratory.
Another important topic that the panel will address is the role of food safety standards in the cannabis industry. Lauren Maloney says cannabis product manufacturers should consider GMP and HACCP certifications for their businesses. “Food safety is important to the cannabis industry because although individual states have mandated several food safety requirements there still considerable risks involved in the production of cannabis products,” says Lauren Maloney. “Consumers want the assurance that the cannabis products are safe and therefore should be treated like a food product. Because FDA does not have oversight of these production facilities, third party certification is essential to ensure these facilities implement a robust food safety system.”
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