Developing a Standard for Cannabis Sample Prep in Quality Control Testing

From 1937 to 1996, cannabis was just weed. Maybe you knew a kid in high school who would break free on occasion or sell it out of his locker. Since its legalization as a botanical medicinal substance in California and elsewhere, then as a recreational drug in Colorado, a stateby-state patchwork of medicinal and recreational legalizations followed. Cannabis has now become a legitimized global retail marketplace, projected to reach $146 billion by 2025. Even though the federal Drug Enforcement Agency still recognizes cannabis as a Schedule I substance (illegal and with high potential for abuse), the National Institutes of Health now devotes $100 million annually to cannabis medical research.

The state of the market is a murky gumbo consisting of a handful of legitimate FDA-approved pharmaceuticals; hundreds of vaporizable, edible, and topical concoctions; and a slew of modern-day patent medicines based on the consensus understanding that CBD (cannabidiol) is what makes you feel good, and THC (tetrahydrocannabinol) is what makes you feel funny. This is complicated further by the dozens of terpenes that can be extracted from cannabis to help fuel the essential oil wellness craze, or added back to recreate a skunky smell and impart brand identity and loyalty. In fact, both THC and CBD are phytochemicals called cannabinoids that mirror structurally and functionally analogous endocannabinoid compounds in our brains with their own cognate receptors. They are only the two most famous of at least 113 cannabinoids identified from the marijuana plant, Cannabis sativa, a specialized cultivar of the same species as industrial hemp, which has been used to make clothing and rope for centuries. While THC is the psychoactive component of cannabis, the defined effects of CBD are more elusive; however, the biological effects of each can be harnessed to make medicine, and they can synergize more anecdotally along with other cannabinoids and terpenes in an “entourage effect.” There are currently four approved therapeutic drugs, including CBD-derived Epidiolex, which reduces epileptic seizures, and three antiemetics derived from natural or synthetic THC to assuage the burden of chemotherapy for cancer patients. There is additionally a field of research that is examining potential benefits of CBD treatments to dopaminerelated deficits in Parkinson’s disease patients. Beyond this patch of clarity, legalized cannabis use resides on constantly shifting and growing sands, with millions of people feeling good, sometimes feeling funny, and sometimes feeling really bad.

Presumably, the consumer doesn’t want to feel bad, so if you buy a cannabis product, then what can you expect, beyond caveat emptor? For starters, hopefully you aren’t getting a product compromised by haphazard practices somewhere along the production line between cultivation, harvest, drying and curing, extraction, formulation, and distribution. Moreover, because of the lack of FDA regulation, consensus, or consistency from state to state on recommended dosages, and discrepancies between the United States, Canada, and European countries on allowable impurity levels, the amounts people are ingesting or applying can vary widely and unpredictably. There is consequently an evolving quality control industry to support the cannabis market, and the Bureau of Cannabis Control in California recently implemented comprehensive regulations requiring cannabis derivatives to be QC-tested before they can be qualified for sale in dispensaries. Therefore, California has led a national trend in the establishment of fully certified cannabis QC testing labs. Realistically, however, QC testing is a major bottleneck in the production and distribution stream, with many more accredited laboratories needed, and a demand for producers themselves to develop in-house QC capabilities through the purchase of instrumentation, and recruitment of qualified experts.

The QC bottleneck can be exacerbated by inadequately reproducible sample preparation. Sample prep is a crucial consideration that has struggled with credibility within the wider testing industry because of 1) competition between testing labs that results in the proprietary sequestering of important information; 2) federal laws and regulations that severely curtail movement across state lines of samples for testing, combined with the countercultural history of cannabis that manifests in a recalcitrant attitude toward many regulations; and 3) the heterogeneous nature of plant material, in which seeds, stems, leaves, and flowers produce varying levels of desired phytochemicals and take up varying levels and types of contaminants. The current gold standard for sample prep comes from QC analysis of fruits and vegetables to meet FDA standards, and is called QuEChERS (Quick, Easy, Cheap, Rugged, and Safe). Recently, a working group within the Association of Official Analytical Chemists (AOAC), including stalwart scientific supply companies such as Sigma and Restek, recommended the adoption of QuEChERS as a cannabis industry standard.

QuEChERS for cannabis is analogous to the procedure of salting out and dispersive solid phase extraction used for produce, but is complicated by two interrelated factors: sample size and heterogeneity. The recommended sample size in California is one gram per batch of plant, and a single batch may be up to 50 pounds. A batch may therefore contain material from different microenvironments within adjacent plots of soil, and consequently incorporate widely different cannabinoid, pesticide, and heavy metal levels and identities. The recommended solution is to randomize samples by taking small amounts from different parts of a batch, and combining them to make up a total of one gram. This can be accomplished by quartering, or dividing a mixture diagonally into fourths and remixing it. After this step, QuEChERS becomes a straightforward preparation for quantitative, analytical gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS), in which cannabis is 1) ground into a free-flowing powder; 2) rehydrated with water (unlike most produce, it is dried before analysis or extraction); 3) subject to precipitation with acetonitrile and salting out in a manner appropriate to fatty acid content; 4) centrifuged to resolve cannabinoids into the acetonitrile layer, to which PSA (primary secondary amine) and magnesium sulfate are added to remove sugars and fatty acids; and 5) aliquoted to compare to analytical standards using whatever quantification method is dictated by regulatory compliance. Even within the disparate and ever-changing state-dependent regulatory environment, instrument supply companies are coordinating with cannabis producers and testing labs to develop standards and streamline sample prep workflow.