RefWork:LIMS Buyer’s Guide for Cannabis Testing Laboratories/Laboratory testing of cannabis and its derivative products
2. Laboratory testing of cannabis and its derivative products
The Cannabis plant, when chemically broken down, has some 750 or more constituents, with as many as 140 of them being identified as cannabinoids. However, analyzing the chemical constituents of cannabis is a difficult task due to its matrix, and the task becomes even more difficult when it's added to food and other matrix types, requiring established and consistent methods for testing. Regulators, users, and the testing industry have over the years made calls for standardizing both the production and testing of medical and recreational marijuana. Without proper standardized testing, several issues are bound to arise:
- label claims may not match actual contents;
- contaminants may linger, causing illness or even death;
- chemical properties and medicinal benefits of specific strains and their unique cannabinoid-terpene profiles can't be isolated; and
- research on potential therapeutic qualities can't be replicated, hindering scientific progress.
As legalization efforts continue at the federal level in the U.S., it's more important than ever that the standards and practices that make up laboratory testing of cannabis continue to solidify under a more consensus-based approach.
But what do we know so far about the Cannabis plant and its constituents? What are the current standards and practices used in various U.S. states (and other parts of the world)? What does the workflow of laboratory testing of cannabis look like today? And what are the requirements for certifying and reporting results? This chapter addresses these questions.
2.1 Analytical aspects
Cannabis has many constituents, and, being a plant, can absorb or come in contact with a variety of contaminants. From a regulatory perspective, cannabinoids, terpenes, and contaminants are most likely to be analyzed by a cannabis testing lab. Therefore, this section will focus on those.
Somewhere between 104 and upwards of more than 140 of the over 750 constituents of Cannabis sativa have been identified as cannabinoids, active chemical compounds that act in a similar way to compounds our body naturally produces, and new cannabinoids continue to be identified during cannabis research. Many of our body's cells have cannabinoid receptors capable of modulating neurotransmitter release in the brain and other areas. The plant's cannabinoids vary, with each bonding to specific receptors in our body, providing differing effects. From a theoretical and medical standpoint, crafting a strain of cannabis that has specific cannabinoids that can aid with a particular malady, while also carefully reproducing the grow conditions to consistently make that strain in the future, is a desirable but difficult goal to achieve. However, even as new strains are developed, identifying an existing strain effectively has its own set of challenges, as Mudge et al. point out: "the total [tetrahydrocannabinol] and [cannabidiol] content is not sufficient to distinguish strains [though] a combination of targeted and untargeted chemometric approaches can be used to predict cannabinoid composition and to better understand the impact of informal breeding program and selection on the phytochemical diversity of cannabis."
Lab testing of cannabinoids is done primarily as a measure of psychoactive "potency," though cannabinoids have many other potential therapeutic uses. Current laboratory testing looks at only a handful of cannabinoids; more research and development of analytical techniques that can quickly and accurately detect and separate the rest is required. Some of the major cannabinoids tested for include:
- THC (∆9-Tetrahydrocannabinol): This is the most commonly known cannabinoid found in cannabis, notable for its strong psychoactive effects and ability to aid with pain, sleep, and appetite issues. Included is its analogue ∆8-Tetrahydrocannabinol (which shows notably less strong psychoactive effects than ∆9) and its homologue THCV (Tetrahydrocannabivarin), which tends to appear in trace amounts and has a more pronounced psychoactive effect, but for a shorter duration. THCV shows promise in fighting anxiety, tremors from neurological disorders, appetite issues, and special cases of bone loss. Also notable is ∆9-THCA (∆9-Tetrahydrocannabinolic acid), a non-psychoactive biosynthetic precursor to THC.
- CBC (Cannabichromene): This non-psychoactive cannabinoid is found in trace amounts; however, it tends to be markedly more effective at treating anxiety and stress than CBD (see next). It's also notable for its anti-inflammatory properties and potential use for bone deficiencies.
- CBD (Cannabidiol): CBD is a non-psychoactive component of cannabis, typically accounting for up to 35 to 40 percent of cannabis extracts. It acts as a counter-balance to THC, regulating its psychoactivity. It's been researched as a treatment for anxiety, sleep loss, inflammation, stress, pain, and epilepsy, among other afflictions. Included is its homologue CBDV (Cannabidivarin), which is also non-psychoactive and demonstrates promise as a treatment for epileptic seizures. Also notable is CBDA (cannabidiolic acid), a non-psychoactive biosynthetic precursor to CBD.
- CBG (Cannabigerol): This cannabinoid is also non-psychoactive but only appears in trace amounts of cannabis. It has potential as a sleep aid, anti-bacterial, and cell growth stimulant. Also notable is CBGA (cannabigerolic acid), a non-psychoactive biosynthetic precursor to CBG.
- CBN (Cannabinol): CBN is mildly psychoactive at best and appears only in trace amounts in Cannabis sativa and Cannabis indica. It occurs largely as a metabolite of THC and tends to have one of the strongest sedative effects among cannabinoids. It shows promise as a treatment for insomnia, glaucoma, and certain types of pain.
Mandated lab testing of terpenes—volatile organic compounds that distinctly affect cannabis aroma and taste—is done primarily as a way to ensure proper labeling of cannabis and related products, including extracts and concentrates, so buyers have confidence in what they are purchasing. However, additional lab research goes into terpenes as they also show potentially useful pharmacological properties, and they demonstrate synergies (referred to at times as the "entourage effect") with cannabinoids, requiring further research. Testing for specific terpenes (discussed later) has histoically been less of a standardized practice, though it's rapidly improving. Commonly tested terpenes by third-party testing labs include:
Generally speaking, a contaminant is an unwanted substance that may show up in the final product, be it recreational marijuana or a pharmaceutical company's therapeutic tincture. The following are examples of contaminants that laboratories may test for in cannabis products.
Pesticides: Pesticides represent an oft-discussed aspect not only of growing cannabis but also performing analytical testing on it. One of the core issues, again, is the fact that on the federal level marijuana is illegal. Because it's illegal, government agencies such as the Environmental Protection Agency (EPA) have historically failed to develop standards or guidelines for what's safe when it comes to residual pesticides in cannabis, let alone how to best test for them. Additionally, researchers have faced their fair share of difficulties obtaining product to test over the years. The end result is we're only now barely understanding how inhalation of pesticide-coated marijuana smoke affects long-term health, and standard methods for pesticide application and testing have been slow to develop. With numerous pesticide products and little oversight on what growers apply to their plants, combined with the technical difficulty of testing for pesticides in the lab, pesticides remain one of the most difficult contaminants to test for. That said, several classes of of pesticides are commonly applied during cannabis cultivation and can be tested for by labs:
- avermectins: function as an insecticide that is useful against mites, which are a common problem for cultivators
- carbamates: function as an insecticide, similar to organophosphates, but with decreased dermal toxicity and higher degradation
- heterocyclics: function as a broad set of compounds with many industrial uses, including as pesticides
- organochlorides: function as a broadly useful chemical in applications such as plastics, cleaning agents, insulators, and pesticides
- organophosphates: function as the base of many insecticides and herbicides, valued for its easy organic bonding
- pyrethroids: function as the base of most household insecticides and exhibits insect repellent properties
Solvents: In 2003, Canadian Rick Simpson published a recipe of sorts for preparing cannabis extract via the use of solvents such as naphtha or petroleum ether. Claiming the resulting oil helped cure his skin cancer, others hoping for a cure tried it, and the solvent method of preparation grew in popularity. Dubious healing claims aside, the solvent extraction method remains viable today, though it has evolved over the years to include less harmful solvents such as supercritical carbon dioxide, which has low toxicity, low environmental impact, and beneficial extraction properties. However, chemical solvents are still used, and if not evaporated out properly, the remaining solvents can be particularly harmful to sick patients using the extract. As for what solvents should be tested for, it gets a bit trickier, though Chapter 467 of United States Pharmacopeia and The National Formulary, the Oregon Health Authority's December 2015 technical report on contaminant testing of cannabis, and the Massachusetts Department of Public Health's response to public comments on cannabis testing provide helpful guidance. Listed solvents include benzene, butane, cumene, dimethoxyethane, ethanol, hexane, pentane and propane, among others.
Heavy metals: 2013 research on contaminant testing on the behalf of Washington State provides insights into heavy metals and why they're looked for in cannabis testing. That research, as well as other more recent sources, tell us:
- Heavy metals contribute to several health problems, including those of a neurological nature.
- Cannabis can "hyperaccumulate metals from contaminated soils."
- Research parallels can be found in tobacco research and how the FDA regulates heavy metal content in foods.
- The most prominently tested heavy metals include arsenic (As), cadmium (Cd), chromium (Cr), copper (Cu), lead (Pb), mercury (Hg), and nickel (Ni).
Mycotoxins and microorganisms: "The ideal conditions for cannabis growth are also ideal for the growth of potentially harmful bacteria and fungi, including yeast and molds," say Shimadzu's Scott Kuzdzal and William Lipps, "therefore microbial contamination poses health risks to consumers and immunocompromised individuals." In truth, these concerns have already borne out. In fact, the University of California, Davis reported in February 2017 one of its patients had contracted an incurable fungal infection from inhaling aerosolized marijuana. They later tested 20 marijuana samples from Northern California dispensaries—using specialized techniques—and found a wide variety of potentially hazardous microorganisms.
The degree to which such contaminants commonly appear in grown and stored cannabis material and to which microbiological contaminants should be tested is not clear, however. As mentioned previously, the U.S. EPA has historically had little in the way of significant guidance on cannabis testing, including microbiological contaminants. Like heavy metal testing, parallels are drawn from microbial testing guidelines and standards relating to tobacco and food, where they exist. As warm, moist environments are conducive to microorganism growth, maintaining stable moisture levels during cultivation and storage is essential. Regularly measuring water activity—how moist something is—is particularly useful as a front-line preventative tool to better ensure microbial growth is limited. Regardless, testing of some kind is still required by many U.S. states, including for organisms such as:
- E. coli
2.2 Analytical methods and tools
A great number of approaches to analyzing cannabis constituents and contaminants have been developed and prescribed over the years. The following section examines the most common of these approaches.
Random, representative sampling is encouraged. When dealing with solid cannabis, BOTEC Analysis recommends a "quartering" method that divides the sample into four equal parts and takes portions from opposite sections of a square-shaped arrangement of the sample. For liquid cannabis products, remembering to stir before sample collection is advised. Sampling techniques may also vary depending on the constituent being tested, as with terpene testing, which may favor full evaporative technique (FET) headspace-based (HS) sampling for reducing certain sampling biases. Another consideration may be the matrix being tested, as when deriving a sample from a cannabis-laden edible; the QuEChERS approach used by food safety labs for pesticide testing may have practical use. In fact, a variety of parallels have been drawn from the food and herbal medicine industries' sampling guidelines, including from the Codex Alimentarius Commission's CAC/GL 50-2004 General Guidelines on Sampling as well as various chapters of the United States Pharmacopeia and The National Formulary. As the Association of Public Health Laboratories (APHL) points out, "[g]ood sampling is key to improving analytical data equivalency among organizations," and it provides a solid base for any future testing and standardization efforts.
Additional sampling insight can be found by examining other states' guidelines, e.g., Massachusetts' Protocol for Sampling and Analysis of Finished Medical Marijuana Products and Marijuana-Infused Products for Massachusetts Registered Medical Marijuana Dispensaries, as well as ASTM D8334/D8334M-20 Standard Practice for Sampling of Cannabis/Hemp Post-Harvest Batches for Laboratory Analyses.
Quantifying cannabinoids for label accuracy is a major goal of testing, though calculation and testing processes may vary slightly from state to state. Despite any differences, laboratorians generally agree that when testing for cannabinoids such as THC and CBD, as well as their respective biosynthetic precursors THCA and CBDA, the methodology used must be scrutinized. The naturally occurring THCA of cannabis isn't psychoactive; it requires decarboxylation (a chemical reaction induced by drying/heating that releases carbon dioxide) to convert itself into the psychoactive cannabinoid THC. Chemical calculations show that the process of decarboxylation results in approximately 87.7 percent of the THCA's mass converting to THC, with the other 12.3 percent bubbling off as CO2 gas. The problem with this in the testing domain is gas chromatography (GC) involves heating the sample solution. If you, the lab technician, require precise numbers of both THCA and THC, then GC analysis poses the risk of under-reporting THC total values. As such, liquid chromatography-diode array detection (LC-DAD) may be required if a concise profile of all cannabinoids must be made, primarily because it provides environmental stability for them all during analysis. If GC is used, the analysis requires extra considerations such as sample derivatization.
The APHL briefly describes analysis methods of cannabinoids using both LC and GC on pages 31–32 of their May 2016 Guidance for State Medical Cannabis Testing Programs. They also point to New York Department of Health - Wadsworth Center's various guidance documents (MML-300, -301, and -303) for methodologies when testing sample types other than solids, particularly using high-performance liquid chromatography photodiode array detection (HPLC-PAD). Also worth noting is that ASTM's Subcommittee D37.03 has been working on various standard methods for determining cannabinoid concentrations using different chromatography techniques, while the Association of Official Agricultural Chemists (AOAC) has already developed a Standard Method Performance Requirement (SMPR) for analyzing cannabinoids in hemp (i.e., low THC cannabis varieties).
- Fourier-transform infrared spectroscopy (FTIR; has limitations, such as requiring standard samples tested w/ other methods)
- Gas chromatography-flame ionization detection (GC-FID; requires sample derivatization for both acid and neutral compounds; good with standards like 5α-cholestane, docosane, and tetracosane)
- Gas chromatography–mass spectrometry (GC-MS; requires sample derivatization for both acid and neutral compounds; good with standards like deuterated cannabinoids)
- Gas chromatography–vacuum ultraviolet spectroscopy (GC-VUV)
- High-performance liquid chromatography photodiode array detection (HPLC-PAD; stable for all forms of cannabinoids)
- High-performance liquid chromatography UV detection (HPLC-UV)
- Liquid chromatography–mass spectrometry (LC-MS; can reduce or eliminate selectivity challenges and speed up testing, though at an increased price cost and need for greater laboratory expertise)
- Supercritical fluid chromatography (SFC; newer technology w/ added benefits)
- Thin-layer chromatography (TLC; older, less common technology)
- Ultra-performance chromatography (UPC; newer technology w/ added benefits)
Also worthy of note is recent investigation of viably using nuclear magnetic resonance (NMR) spectroscopy as a more affordable and rapid solution to identifying cannabinoid contents and profiles of samples. Conferences, research, and articles over the last four or five years have advanced the use of NMR spectroscopy for cannabinoid analysis.
Identifying and quantifying terpenes is one of the more difficult tasks facing laboratorians, according to Cassidy:
Terpenes present an analytical challenge because they are nonpolar and structurally similar, and many structural isomers exist. Mass spectrometry (MS) cannot distinguish terpenes that co-elute from a GC column because many have the same molecular weight and share fragment ions.
Goldman et al. share Cassidy's view about MS, though reminding that it has some benefits over flame ionization detection (FID). They note that recent MS methods add another level of confirmation for terpene identification using retention indexing and electron impact mass spectral matching.
Of course, types of gas chromatography work; but like cannabinoids, terpenes can degrade with the high heat of gas chromatography. Combined with the problems mentioned above, highly specialized gas chromatography processes that include additional steps, such as full evaporation technique headspace gas chromatography flame ionization detection (FET-HS-GC-FID), can be used to produce cleaner results, particularly for volatile components. It's less clear if high-performance liquid chromatography (HPLC) is used frequently; some entities such as Eurofins Experchem Laboratories claim HPLC works best for them, while others such as Restek Corporation claim the method is problematic at best.
- Full evaporation technique–headspace–gas chromatography–flame ionization detection (FET-HS-GC-FID; tends to be semi-quantitative)
- Gas chromatography–flame ionization detection (GC-FID)
- Gas chromatography–mass spectrometry (GC-MS)
- Gas chromatography–tandem-mass spectrometry (GC-MS/MS)
- Gas chromatography–vacuum ultraviolet spectroscopy (GC-VUV)
- Headspace–gas chromatography–mass spectrometry (HS-GC-MS)
- Headspace–solid-phase microextraction (HS-SPME)
- High-performance liquid chromatography (HPLC; may have limitations due to coelution of terpenes and cannabinoids at certain ranges)
Pesticides: Gas and liquid chromatography methods are by and large the staple of testing methods for pesticides, which remain "the hardest analyses that are going to be done in the cannabis industry." Goldman et al. echo the sentiment: "pesticide testing is difficult and requires advanced analytical instrumentation and highly skilled staff to meet regulatory demands with a robust, accurate, and precise test method." Notably, high-performance liquid chromatography–tandem-mass spectrometry (HPLC-MS/MS) tends to be one of the most thorough methods says Emerald Scientific's CTO Amanda Rigdon. "Ninety-five percent of the pesticides out there can be analyzed by HPLC-MS/MS, although there are some that you would need a GC-MS/MS for," she says. A popular sample extraction method for detecting multiple pesticide residues in cannabis is the QuEChERS (quick, easy, cheap, effective, rugged, and safe) method, which shows "acceptable recoveries and relative standard deviations" for almost all known pesticides, though the release of heat and increase in pH of QuECHERS may degrade particularly sensitive pesticides in the sample. QuECHERS may also not be ideal for some labs due to its organic solvents having a tendency of extracting hydrophobic compounds like cannabinoids. However, other methods such as solvent extraction (such as with acetonitrile) with dispersive solid-phase extraction (dSPE) cleanup and energized dispersive guided extraction (EDGE) may also been used. Common testing methods that have historically been used, after sample preparation, include:
- Gas chromatography–electron capture detection (GC-ECD)
- Gas chromatography–mass spectrometry (GC-MS)
- Gas chromatography–tandem-mass spectrometry (GC-MS/MS)
- Liquid chromatography–mass spectrometry (LC-MS; also high-performance or HPLC-MS)
- Liquid chromatography–tandem-mass spectrometry (LC-MS/MS; also high-performance or HPLC-MS/MS)
Solvents: Testing for solvents is largely standardized into a few options, which have parallels to existing pharmaceutical testing standards outlined in Chapter 467 of United States Pharmacopeia and The National Formulary (USP <467>):
- Headspace–gas chromatography/mass spectrometry (HS-GC/MS)
- Headspace–gas chromatography/tandem-mass spectrometry (HS-GC-MS/MS; may be required when high concentrations of terpenes are present)
- Headspace–gas chromatography–flame ionization detection–mass spectrometry (HS-GC-FID-MS)
- Full evaporation technique–headspace–gas chromatography–flame ionization detection (FET-HS-GC-FID)
Massachusetts and Oregon—and likely other states—have used a variety of guidance documents such as USP <467>, reports from the Commission of the European Communities' Scientific Committee on Food (now the European Food Safety Authority), and the International Conference on Harmonization's (ICH) Q3C(R5) to set their action level testing values for particular solvents. The AOAC provides another standardized option in the form of their SMPR 2019.002.
Heavy metals: The methods used for quantifying levels of highly toxic metals in plants depend on ease-of-use, level of accuracy, and overall cost. Sample preparation typically includes the use of closed-vessel microwave digestion to get the sample into solution for analysis. Once prepared, the following methods are most common for testing cannabis and other plants for heavy metals:
- Atomic absorption spectrometry (AAS; may struggle to meet detection limits)
- Inductively coupled plasma–atomic emission spectroscopy (ICP-AES), sometimes called inductively coupled plasma optical emission spectrometry (ICP-OES) (at times coupled with an ultrasonic nebulizer)
- Inductively coupled plasma–mass spectrometry (ICP-MS; a preferred technique)
- Inductively coupled plasma–tandem-mass spectroscopy (ICP-MS/MS)
Mycotoxins and microorganisms: A standard method of testing for the existence of microorganisms is through the process of culturing a sample in a Petri dish, a common diagnostic method in microbiology. Enzyme-linked immunosorbent assay (ELISA) is also used, particularly to identify mycotoxins. However, Petri culture analysis isn't rigorous, and ELISA can at times be time-consuming, as it's limited to one mycotoxin per test. The following are other, more precise techniques that are improving laboratorians' analyses, particularly using DNA snippets of microbiological contaminants:
- Quantitative polymerase chain reaction (qPCR)
- Whole metagenome shotgun (WMGS) sequencing
- Matrix-assisted laser desorption/ionization (MALDI)
- High-performance liquid chromatography (HPLC)
- High-performance liquid chromatography–fluorescence spectroscopy (HPLC–FS)
- Liquid chromatography–tandem-mass spectrometry (LC-MS/MS)
- Liquid chromatography–electrospray ionization–tandem-mass spectrometry (LC-ESI-MS/MS)
- Liquid chromatography–atmospheric pressure chemical ionization–tandem-mass spectrometry (LC-APCI-MS/MS)
The extent of mycotoxin testing required remains in question by several entities. The APHL claims "[t]here is no readily available evidence to support the contention that cannabis harbors significant levels of mycotoxins." The Oregon Health Authority takes a more middle-ground approach, noting that testing for E. coli and Salmonella will "protect public health," though Aspergillus only deserves a warning for people with suppressed immune systems due to its prevalence in the environment. USP <561> recommendations largely limit mycotoxin testing of botanical products to those borne from root or rhizome material, "which THC-containing cannabis products presumably do not possess," emphasizes the APHL.
Regardless, U.S. Pharmacopeia's Chapter 561 remains a useful document for testing guidelines and limits regarding microbials, as does the AOAC's SMPR 2019.001 for the detection of Aspergillus. In the less common case of dealing with powdered cannabis—a relatively new THC extract form—Chapter 2023 provides at least some testing parallels, though Dr. Tony Cundell, a microbiologist consulting for the pharmaceutical industry, suggests USP <2023> doesn't go far enough for immunocompromised patients.
Somewhat related and worth mentioning is moisture content testing. As previously mentioned, warm, moist environments are conducive to microorganism growth, and regularly measuring water activity is useful for the prevention of microbial growth. The APHL references specifications from the Dutch Office of Medical Cannabis that recommend water content be between five to ten percent in cannabis.
2.3 Cannabis testing laboratory workflow
The analytical methods of testing cannabis constituents and contaminants, as well as their associated workflows, depend on the type of laboratory conducting testing. For example, an extraction-specific lab's workflow will look a bit different from the workflow of a commercial production lab or a state-mandated, independent quality testing lab. And medical cannabis testing labs may ignore terpenes entirely, for instance. Broadly speaking, however, non-extraction cannabis testing lab workflows will have some aspects in common. Those workflow similarities, from beginning to end, include:
- reception of test orders—often through a secure web portal—and samples, as well as the start of sample tracking with RFID and barcodes for chain-of-custody purposes;
- assignment of tests to analysts and instruments;
- processing of samples—including any required quality control (QC) samples—as well as any necessary grinding, homogenization, extraction, filtration, and evaporation processes;
- chromatographic separation, or any other non-chromatographic preparative methods, for samples, based upon the target constituent or contaminant;
- actual qualitative and/or quantitative analysis, based on standards and reference materials, with appropriate notification of out-of-range or -specification results;
- exporting of instrument data, preferably to an information management system like a LIMS, where the data are processed and recorded with the associated existing sample data;
- organization and review of results by designated laboratory personnel, with results either getting approved or not approved; and
- reporting of approved results in a compliant format, e.g., a certificate of analysis (COA), and distributed to appropriate stakeholders (often through a secure web portal).
Of course, the specific details of the methods you choose to employ will slightly modify your workflows, as will your lab's own process and procedure (P&P) documentation. For example, your workflow for testing heavy metals may differ slightly from the U.S. FDA's ICP-MS methodology. Ultimately, your workflow will be based upon many factors, including the analyses you decided to perform, methods you choose, the equipment you use, the way your lab is laid out, the P&P you follow, and the data management systems and automation you choose. Some components of your workflow will remain the same, however, regardless of the mentioned factors. Sample tracking and accurate weight reconciliation, while maintaining complete chain-of-custody, remain vital throughout the entire process, from test ordering to after the results are reported. Additionally, any calculations performed must be steadfastly accurate for every type of workflow, at every step. These aspects are practically non-negotiable given the regulatory requirements for cannabis track-and-trace mechanisms (see the next section on reporting and certification for more).
That said, workflows can usually be optimized in any laboratory, saving time and money while increasing productivity. Keeping P&P documents, methods, and training documentation aligned with a rapidly changing industry like cannabis testing is vital to ensuring smooth workflows. Other minor considerations for smoothing out workflow problems in the cannabis testing laboratory include adding additional automation elements, optimizing workspaces (e.g., well-spaced lab tables, sufficient cabinets and storage), and staggering shifts (e.g., for improving social distancing success during a pandemic). It's best to address these and other such issues early on to ensure the best outcomes from your workflows.
2.3.1 Differences beyond analyzing plant material
Workflows can also differ slightly based upon the substrate/matrix being tested, as well as the product's intended use. Here are a few differences to consider.
Medical vs. recreational
Speaking in broad terms, medical cannabis tends to differ from recreational cannabis such that medical cannabis has lower THC and higher CBD than recreational cannabis, and vice versa. However, even this broad generalization isn't all that accurate. Another way to generalize this is to say that medical cannabis patients prefer a wider variety of cannabis products with varying formulated balances in THC, CBD, and other cannabinoids and terpenes, whereas recreational users may—perhaps incorrectly—put more emphasis on high-THC content, caring less about some other aspects of the product. Some medical cannabis patients may require high amounts of THC to manage pain, while others may find that a much lower THC dose manages their pain better. And recreational cannabis users who are knowledgeable don't always want to go for the highest THC content in their acquisitions. Given these facts, it's difficult to make generalizations about the content of medical cannabis vs. recreational cannabis. Complicating the matter even further is a recent study in PLOS One that found "more than 90 percent of the legal cannabis products offered in medical dispensaries vastly exceed the THC levels recommended for chronic pain relief." The results of that study—which showed little difference between advertised medical and recreational cannabis THC concentrations across various states—further highlight not only the lack of unified regulations across the U.S. concerning THC quantities in medical cannabis, but also the difficulty—analytically—in making assumptions about the difference in testing medical vs. recreational cannabis.
That aside, one significant workflow difference between the two is that, at least for now, terpenes testing isn't generally mandated for medical cannabis, unless a specific terpene type and/or amount is to be shown on a product label (as is the case in, e.g., Washington and California). This may change at some future point as researchers learn more about the "entourage effect" and the role terpenes play in medical cannabis administered to patients.
Marijuana vs. hemp
On November 29, 2018, lawmakers "struck a deal in principle" to finalize the 2018 Farm Bill, which, if passed, would remove industrial hemp from the Controlled Substance Act's definition of "marijuana" as well as strike it from Schedule I. On December 20, 2018, President Trump signed the Farm Bill into law, legalizing at the federal level the cultivation and sale of hemp, defined as "the plant Cannabis sativa L. and any part of the plant with a ∆9-THC concentration of not more than 0.3 percent by dry weight."
Of course, this arbitrary THC cutoff comes with its own set of challenges to growers, transporters, forensic labs, and cannabis testing labs. The 0.3% number appears to derive from a a 1976 research paper by Small and Cronquist that provided definitions of Cannabis sativa and Cannabis indica, which set 0.3% ∆9-THC as a cutoff distinction between the two. This apparently "was not intended to be a prescriptive measure of the plant’s capacity to get users high." As Brian Smith of Big Sur Scientific notes in a 2019 Cannabis Science and Technology article, other problems arise as well, given that a Cannabis strain could potentially have less than 0.3% THC yet contain more than 20% THCA, the acid precursor to THC. Since THCA decarboxylates to THC when heated, a person smoking such a low-THC strain could still get high:
A strict interpretation of the law means this material may be legally considered hemp and can be sold and transported throughout the U.S. Of course, when smoked, material that contains more than 20% THCA will be highly intoxicating. I doubt it was the intention of the U.S. Congress to allow high THCA marijuana to become legal nationwide.
If the intent of the U.S. Congress was to prevent people from getting high when smoking industrial hemp, total available THC should be calculated. Smith noted in 2019 that, for example, Kentucky used only THC, whereas Oregon uses total available THC for their calculations, further highlighting discrepancies between federal and state law, and as such, more testing headaches. Add in other problems such as inconsistent cannabinoid extraction methods, variations in representative sampling, differences in agricultural variability, a too-short testing timeframe, insufficient USDA sampling protocol, and the time required to take accurate analyses down to the 0.3% level (as found with forensic and law enforcement labs attempting to enforce laws, and the end result is the whole hemp laboratory testing regime appears to be shaky. And hemp farmers only end up increasingly frustrated at the potential loss of their crops, which must be destroyed if they test above the 0.3% THC threshold. At the end of 2020—and again in early 2021—Senator Rand Paul introduced The Hemp Economic Mobilization Plan (HEMP) Act to make changes to the 2018 Farm Bill, chief among them raising the THC limit from 0.3% to 1.0%. Other aspects such as increasing the timeframe for analytical testing and making a margin of error transparent are also included. It remains to be seen what the end result will be for laboratories testing industrial hemp.
That said, laboratory workflows for analytical testing of industrial hemp will be different from those working with cannabis-derived products with higher THC counts. On the plus side, only ∆9-THC need be analyzed in submitted samples. More challenging is the requirement that labs wanting to perform hemp compliance testing under the U.S. Domestic Hemp Production Program must be registered with the DEA by, at the latest, October 31, 2021. This may or may not be a burdensome process for some labs. Also of consideration is whether the state where the lab is located has their own state-licensed program with its own testing guidelines, or if a state-licensed program isn't set up yet, requiring the use of USDA testing guidelines. In many cases, those states with their own programs will utilize the USDA testing guidelines, but a few states may have slightly more stringent guidelines. Lab managers must be clear on what ∆9-THC testing guidelines must be used for their lab to better shape their workflow.
Supplements, cosmetics, and topical cannabis products
As of July 2021[update], the FDA still prohibits THC and CBD products from being sold as a "dietary supplement." This positions appears to largely rest on the FDA's belief that "provisions in the Food, Drug and Cosmetic Act (FDCA) related to the use of dietary supplement and food ingredients that have been previously studied as drug ingredients" forces the FDA to continue to treat CBD like a new, regulated drug. Efforts were made in 2020 with proposed legislation like H.R. 8179 (Hemp and Hemp-Derived CBD Consumer Protection and Market Stabilization Act of 2020), but the FDA's input on the proposal revealed a significant gap of understanding and agreement on the topic. That gap may be shrinking, with the FDA signaling some concessions in December 2020. However, it would seem that laboratory testing of cannabis dietary supplements won't be more widespread until the FDA and lawmakers can formally find middle ground.
Cosmetics and topical creams or lotions containing THC and CBD are not exempt from FDA scrutiny either, though the FDA tends to be more forgiving. Cosmetics containing cannabis components are fine as long as the added cannabis components do not cause the end product to be "injurious to users under the conditions of use prescribed in the labeling, or under such conditions of use as are customary or usual." The FDA adds that if a cosmetic or topical cream product "is intended to affect the structure or function of the body, or to diagnose, cure, mitigate, treat or prevent disease, it is a drug, or possibly both a cosmetic and a drug, even if it affects the appearance." As such, FDA approval as a drug would be required, as occurred with GW Pharmaceuticals' Epidiolex in 2018. (Note that Canada has slightly different regulations on cannabis topicals.)
From a workflow standpoint, lotions, creams, and balms provide their own set of challenges. Dr. Jamie York of Restek notes:
These matrices contain many components such as fatty acids, plant extracts, fragrances, and esters of fatty acids. When creating a workflow, we knew it was important to develop a method that is robust, produces accurate data, and prevents instrument downtime, which was especially challenging for lotions, balms, and creams since we intended to include these three different, albeit similar, types of matrices in one method.
Restek's cannabinoid testing method appears to involve the use of liquid chromatography paired with ultraviolet detection (LC-UV), with specific sample preparation and recovery experiments. This loosely aligns (fine details not given) with an LC-UV method employed by ACS Laboratory and an HPLC-UV method used by analytical lab BSCG.
Edibles and drinkables
Edibles can be problematic. Like the fatty components referenced by Dr. York in regards to cosmetic matrices, so too do fatty components seem to cause problems for edibles. Take for example chocolate, which is full of fat. THC is known to dissolve quite well in lipids, which can cause problems, as found by David Dawson of CW Analytical Laboratories. In 2019, he found that varying a method testing for cannabinoids in chocolate only by the sample amount caused variable values across two otherwise identical analyses. Infused gummy candies and other substrates also cause problems with depositing unwanted residues in mass spectrometry instruments. All of that is to say that there is a highly diverse array of food matrices out there, adding their own complications to isolating and analyzing cannabinoids and other substances; more often than not, a single method won't suffice for all edible matrices.
When choosing a methodology and workflow for edibles, several questions must be asked. What are the expected challenges with sample preparation and analysis, based on the ingredients? What constitutes a serving size for the matrix? Is the edible homogenously or non-homogeneously mixed in manufacturing? Ultimately, the workflow will likely be unique based upon each matrix tested. However, there is at least some growing similarity in preparation and analysis of complex matrices such as edibles. Take for example a THC analysis method described by ModernCanna Labs:
...the product is frozen with dry ice or liquid nitrogen and then placed in a high speed blender with diatomaceous earth, an abrasive substance that enables solvents to draw out the THC. The solvent is then placed in a flash chromatography column that separates various chemical components. When the THC is extracted from the column, it can be tested in the usual manner, with high-pressure liquid chromatography.
ACS Laboratory and CW Analytical Laboratories describe a somewhat similar process for their edibles testing, lending credence to a slowly growing consensus on at least sample preparation methods. Millipore Sigma describes a brownie preparation and analysis method that differs slightly, using shakers, centrifuges, salts, and refrigeration for prep, and then HPLC-UV for the subsequent analysis. Dr. Rob O'Brien of Supra Research and Development states that their lab has developed a preparation method for dealing with the challenges of chocolate where they "extract it hot with acetyl-nitro in an ultrasonic bath. And then when we move the cannabinoids out, we also then draw those waxes and other fats out." In the end, your lab will have to do due diligence in finding a consensus standard workflow methodology for the matrix being tested. In some cases, a standardized method and workflow may not be openly established, requiring more research by your laboratorians.
On the beverage side, it's worth noting that cannabinoids are not soluble in water, requiring special formulations to stabilize the beverage over time. This issue, as well as larger sample volume requirements, and the brewing processes not being precise in what cannabinoids are transferred into hot water, bring extra complications to testing and comparing to label claims. PerkinElmer's Toby Astill notes that producers of "cannabeverages" turn to HPLC for accurate cannabinoid measurement, LC-MS/MS for pesticide residues and mycotoxins, headspace GC-MS for residual solvents, and ICP-MS for heavy metals. Flavor and aroma profiles can also be managed with headspace GC-MS.
The FDA currently prohibits the use of hemp and CBD in animal food, based off Association of American Feed Control Officials (AAFCO) findings. Despite this, some manufacturers have continued to illegally produce and sell CBD- and hemp-containing pet products. And like human products "intended to affect the structure or function of the body, or to diagnose, cure, mitigate, treat or prevent disease," animal products with the same claims must be treated as drugs, requiring FDA approval.
Federal law, as well as many states' laws, also prohibit veterinarians "from possessing, administering, dispensing, or prescribing cannabis and related products" not approved by the FDA. Extralabel use of CBD- or THC-infused drug approved for human use is allowed with animals under specific conditions.
What does that mean for veterinarians? The American Veterinary Medical Association (AVMA) said in early 2019:
Under existing federal and state law, veterinarians who administer, dispense, prescribe, or recommend ‘hemp’ or other cannabis-derived products that are not approved for use in animals, or approved for animals or people in accord with FDA extralabel drug use regulations, face increased potential legal risk if there is an adverse event. Adverse events can include unintended effects (side effects) of a drug or it could be that the drug doesn’t deliver the intended therapeutic effect.
That said, unless something changes, laboratory testing of CBD and other cannabinoids is for now largely limited to the realm of approved research groups and manufacturers skirting largely unenforced law. Still, a handful of methods for testing pet-marketed CBD products are floating out there. In a December 2019 piece for Cannabis Science and Technology, Curtis et al. describe a study of six commercially available CBD oils marketed as pet supplements, analyzed with gas chromatography–quadrupole time-of-flight mass spectrometry (GC-QTOF) in discovery (untargeted) mode. A broader study performed by Wakshlag et al. in 2020 used a cannabinoid methodology which included ultra-performance liquid chromatography (UPLC) with a diode array detector (DAD) and quadrupole mass spectrometer. Like edibles, methods and workflows may vary based on the matrix being examined.
2.4 Certification and reporting of results
Providing accurate and timely results is an important part of any laboratory operation. Given the expanding regulatory atmosphere surrounding cannabis testing, and the need for cannabis consumers—especially medical marijuana users—to have a safe product to use, consistent and accurate testing is especially vital. By extension, the results must be certified as accurate and rapidly reported in a clear and concise fashion, not only to appease clients but also lawmakers and regulators. This means compliant certificates of analysis (COAs) and mandated results reporting to state and local bodies and their track-and-trace software platforms.
2.4.1 Certificates of analysis
As far as what precisely must appear on a COA or lab report, there's little in the way of standardization, though some U.S. states have outlined requirements for what must be included in such reports. The Oregon Health Authority's Oregon Administrative Rules, Chapter 333, Division 64, Section 0100: Marijuana Item Sampling Procedures and Testing stipulates that any report must include total THC and total CBD (by dry weight) and, if discovered, "up to five tentatively identified compounds (TICS) that have the greatest apparent concentration." It also lays out requirements for pesticides, failed tests, limits of quantification, and specimen identifiers such as test batch number. California dictates reported values for cannabinoids and contaminates be shown on the COA with three significant figures and water-activity level at two significant digits, as well as "pass" and "fail" statuses, demographics, sample history, test methods used, and more. Pennsylvania provides another example with its medical marijuana program (28 Pa. Code Chapter 1171), which includes a section on test results and reporting (1171.31). The regulations stipulate reporting by electronic tracking system, with stipulations on using certificates of analysis which include lot/batch number and the specific compounds and contaminants tested.
Regulations aside, it's largely up to the laboratory—and often by extension, the software they're using—to decide how a report is formatted. Some labs like Seattle-based Analytical 360 offer clean, color-based certificates of analysis, with high-magnification photographs, the chromatogram, potency, cannabinoid content, contaminant content, and explanation of limits, with the name of the approving analyst. Others may simply generate a computer printout with the basic data and a legend. Reports may originate from the measuring device itself (e.g., an integrator in a chromatography device), a middleware or data station attached to the instrument, or a LIMS that accepted data from the instrument.
Be sure to consult your state and local regulations to confirm what aspects are mandatory to include in your COAs.
2.4.2 Track-and-trace and chain of custody
At least in the U.S., given the federal status of recreational and medicinal marijuana, labs operating in cannabis-legal states still have to be particularly mindful of their operations for fear of breaking even a state or local regulation, potentially putting the lab out of business. Samples are tracked internally from receipt to distribution or destruction. However, it's often not enough to issue certificates of analysis and keep careful track of the cannabis samples that move in and out of the laboratory; sample activity must be tracked every single step of the way through laboratory workflows. This is particularly true in states that mandate track-and-trace (sometimes called "seed-to-sale") monitoring and reporting. In that case, keeping data siloed in the lab isn't an easy option to work with. States mandating the use of a particular track-and-trace software platform means either manually transferring data from the lab's systems—or, worst case, from the lab's paper documentation—to the mandated track-and-trace software. This is where integration between the lab's data management platform and the state's system proves useful.
Below are representative examples of the most commonly used track-and-trace software systems that cannabis testing laboratories are required to use and integrate with:
- BioTrackTHC: As both a track-and-trace system and an enterprise resource planning (ERP) solution, BioTrackTHC streamlines data management and workflows from cultivation and processing to laboratory testing and dispensation. Compliance features include customized reporting to meet government-specific needs, tracking of destruction and waste activities, transport manifests, recall tracking, regulation labels, workflow management, and more. The software has also been adopted by state governments such as Illinois, Hawaii, New Mexico, and New York.
- Leaf Data Systems: Similar to BioTrackTHC, Leaf Data Systems is used by both industry operators and government agencies trying to regulate the cannabis industry. The system can manage data at all points along the cannabis lifecycle, from cultivation and processing to distribution, testing, and sale. Leaf can handle customized reporting depending on state or municipality, as well as customizable alerting to ensure enforcement activities are effective. The software has been adopted by the governments of Pennsylvania and Washington.
- Metrc: Developed by Franwell, Metrc represents another major solution used by not only businesses in the cannabis supply chain but also state and local governments. Special features include trend analysis, employee activity tracking, credentialing, and process metrics. States using it include California, Colorado, Massachusetts, Montana, and the District of Colombia, among others.
- SICPATRACE: Perhaps less known in the U.S., the Swiss company SICPA has been involved in security inks and financial security for many decades. It introduced its SICPATRACE software in 2007 for governments to better "fight counterfeiting, illicit trade and tax evasion." It has since been adopted for regulatory activities involving tobacco, alcohol, and now cannabis. Among its technological features is the use of multi-layer label security that incorporates multiple ways to track and trace products, batches, and samples. In the U.S., SICPATRACE has been adopted by several California counties.
Also of note is the somewhat new concept of "tag-and-trace," the molecular application of DNA markers in a plant to allow for forensic tracking across the supply chain. Products like ETCH Biotrace may eventually also be part of the integrated workflow for cannabis testing laboratories.
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Citation information for this chapter
Chapter: 2. Laboratory testing of cannabis
Edition: Summer 2021
Title: LIMS Buyer’s Guide for Cannabis Testing Laboratories
Author for citation: Shawn E. Douglas
License for content: Creative Commons Attribution-ShareAlike 4.0 International
Publication date: August 2021