Cannabis – Analytical Developments Beyond Potency

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Thursday, March 17, 2022 1:00 pm EST
Organizer: Eberhardt Kuhn, Shimadzu Scientific Instruments

Historically, cannabis testing has focused on cannabinoids, i.e. potency. Recently, considerable interest has developed in other constituents of the cannabis plant, like terpenes and flavonoids, as well as undesirable contaminants like pesticides, heavy metals, and mycotoxins. This symposium highlights the latest advances in chromatographic and spectroscopic technologies and method developments to measure these critical compounds.

Presentation 1
Characterization of Terpene Synthase Family Members
Anthony Torres, Front Range Biosciences
Cannabis Sativa, a diecious angiosperm, has long history of domestication by humans tracing back to Neolithic times (Ren2021, Tihelka 2021). The plant has been highly sought for its medicinal metabolites and is most well-known for its bioaccumulation of specialized compounds called cannabinoids and terpenoids. Monoterpenes such as Terpinolene, alpha-pinene, and beta myrcene undergo enzymatic synthesis from the precursor Geranyl pyrophosphate (GPP), while sesquiterpenes such as beta caryophyllene, beta farnescene, and alpha bisabolol are synthesized from the precursor farnesyl pyrophosphate (FPP)(Bohlmann 2011). To assess the activity of specific terpene synthase genes among Cannabis varieties in our germplasm, we utilized molecular cloning and functional gene characterization techniques to clone and characterize dominantly expressed terpene synthase genes TPS37 and TPS20 genes from Front Range Biosciences varieties using a bacterial expression system. We performed downstream enzymatic characterization after supplementing the enzyme with FPP/GPP substrate in vitro. Synthesized products were analyzed by selective ion monitoring (SIM) on a Shimadzu TQ8040 to characterize the enzymatic activity. We found that TPS37 produced terpinolene from GPP as a dominant product with several terpenes (alpha-cedrene, alpha-phellandrene, alpha-pinene, alpha-terpinene, alpha-terpineol, beta-myrcene, beta-pinene, E-beta-ocimene, and gamma-terpinene) present in lower quantities. Many of the lower abundance terpenes synthesized by TPS37 may not have been detected without selective ion monitoring, highlighting the need for more sensitive detection methodologies in characterizing terpene synthases. We also found that TPS20 produced alpha/beta farnescene and valencene in similar quantities from FPP.

Presentation 2
Analyzing for Adulterants and Contaminants: Pesticide Residues and Mycotoxins
Volker Bornemann, Avazyme, Inc.
Not only are consumers increasingly demanding more complex and detailed information about the quality and safety of the products they consume, but their expectations are to have the enormous aggregation of information compiled and delivered with speed in real-time, with accuracy and completeness. The previous way of answering concerns in dedicated, complex laboratory tests with slow turn around time have been replaced with integrated studies to enable benefits and strategies for Product Safety and Product Quality. With the advancements of current analytical instrumentation, in particular chromatographs and mass spectrometers, and automated testing technologies, actionable strategies are possible in a much shorter time frame. These technological advances combine a subset of the tests into a Full Spectrum Analysis. For example, in Pesticide Residues and Mycotoxin investigations, the analysis covers a wide variety of very different chemical compound families, whether man made or naturally occurring, which heretofore had been fragmented in multiple separate testing methods. With integrated complex and complete testing, quick turn-around times, traceability, and the pressure from consumers and regulators, a holistic approach is mandated for Product Safety and Product Quality benefits.

Presentation 3
Development of an LC-MS/MS and GC-MS/MS Method for the Quantitation of 104 Pesticides in Cannabis to Meet AOAC Standard Method Performance Requirements
Thomas Barkley, Trichome Analytical
Cannabis falls into a number of categories such as medicine, recreational drug, food source, and fiber, but it is first and foremost an agricultural crop. As such, Cannabis is subject to treatment with pesticides to improve quality and yield, and may also be unintentionally contaminated with pesticides. Regulatory agencies in various US states have enacted rules about pesticide use on Cannabis. These are typically found in states that have legalized high THC Cannabis for medicinal and recreational use, but pesticide regulations have recently been adopted by state hemp regulatory agencies such as the New York Cannabinoid Hemp Program. Other standards organizations have proposed testing limits and requirements for pesticides in Cannabis, such as the AOAC Standard Method Performance Requirements for Identification and Quantitation of Selected Pesticide Residues in Dried Cannabis Materials (AOAC SMPR 2018.011). In this presentation, the validation of an LC-MS/MS and GC-MS/MS method using a single extraction step for quantitation of 104 pesticides in Cannabis in accordance with AOAC SMPR 2018.011 will be discussed. The method has been developed for high throughput with minimal cleanup steps. Method performance such as recovery, RSD, and LOQ will be discussed, as well as the extraction efficiency of various solvents tested during the method development.

Presentation 4
Analysis of Flavonoids in Hemp by High Performance Liquid Chromatography
Caleb King, Front Range Biosciences
Flavonoids are one of the many secondary metabolites produced by hemp (Cannabis spp.), with over 16 compounds to-date positively identified in the pollen, roots, flowers, and leaves. Research on lipophilic flavonoids in Cannabis has been limited in comparison to terpenes and cannabinoids despite their potent antioxidant and anti-inflammatory properties. In this study, the concentrations of 15 flavonoids, flavones, and flavanols in hemp inflorescence were evaluated using a Shimadzu HPLC equipped with photodiode array detection. In addition to the challenges of chromatographic separation, special attention was given to the efficiency of various extraction techniques to ensure accuracy and scalability for Cannabis testing laboratories. This work will also address spatial, temporal, and varietal variations in flavonoid concentrations that need to be understood for large-scale extractions and commercialization of products.

Presentation 5
Regulating Heavy Metals in Cannabis and Hemp Consumer Products: What Can We Learn From the Pharmaceutical industry?
Robert Thomas, Scientific Solutions
The lack of federal oversight with regard to medicinal cannabis and hemp products in the US has meant that it has been left to the individual states to regulate its use. Medical marijuana is legal in 36 states, while 16 of those states allow its use for adult recreational consumption. The sale of these products is strictly regulated by their THC and CBD content, depending on their use. However, it’s also critical to monitor levels of contaminants such heavy metals, as the cannabis plant is known to be a hyper-accumulator of heavy metals in the soil. Unfortunately, there are many inconsistencies with heavy metal limits in different states where medical cannabis is legal. Some states define four heavy metals while others specify up to eight. Some are based on limits directly in the cannabis, while others are based on consumption per day. Others take into consideration the body weight of the consumer, while some states do not even have heavy metal limits. So clearly there is a need for consistency across state lines, in order that consumers know they are using products which are safe to use. This presentation will take a closer look at how the pharmaceutical industry replaced its 100-year-old colorimetric test for a small group of heavy metals with plasma spectrochemical techniques. Moreover, they expanded that panel to 24 elemental impurities categorized by a risk analysis study that identified sources of potential impurities in the drug product manufacturing process. The cannabis industry can learn a great deal from this process to not only understand the many potential sources of heavy metals but also how the final cannabis products can be contaminated by the manufacturing equipment, the extraction process and the delivery systems used.

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