Determination of Cannabinoid Content and Pesticide Residues in Cannabis Edibles and Beverages

February 26, 2018
Figure 1
Click to enlarge, Figure 1: Photographs of a hard candy (a) before and (b) after freezer mill grinding; (c) after QuEChERS extraction; and (d) the extracts before (left) and after (right) dSPE cleanup.
Abstract / Synopsis: 

As a result of the rapid growth of the cannabis industry, many testing laboratories are looking for efficient, reliable, and cost-effective analytical methods to analyze chemical residues, such as pesticides, mycotoxins, solvent residues, terpenes, and heavy metals, as well as cannabinoid concentration in cannabis-infused edibles and beverages. In this article, QuEChERS (quick, easy, cheap, effective, rugged, and safe), a sample preparation technique widely adopted in the food testing industry, is introduced to the discipline of forensic testing as a viable method for the extraction of pesticides and cannabinoids in various complex sample matrices. The claimed amounts of cannabinoids versus the actual amounts are compared, as well as the pesticide residue levels in edible and beverage samples.

To date, 25 states and the District of Columbia in the United States have legalized the medical use of marijuana, while four states and the District of Columbia have also legalized the recreational use of marijuana (1). Although the federal government still classifies any use or possession of the drug as illegal, all 50 states are starting to see an increase in the number of edible marijuana samples within their borders. As a result, many testing laboratories are looking for fast, reliable, and cost-effective methods to determine cannabis potency and chemical residues in cannabis edibles and beverages. The pros and cons of legalization are still heavily debated throughout the country, but all scientists agree that uniform testing policies and procedures need to be established as soon as possible and that overall sample cleanup is the main issue within these analyses. Many states legalized the use of recreational and medicinal marijuana without establishing any analytical protocols that are commonplace and routine in other scientific industries. Without any sort of regulatory control, laboratories in these states can analyze samples and report results without any fear of repercussion.

For states where any consumption or possession of marijuana is still illegal, forensic laboratories are saddled with the task of analyzing this contraband. Even though it is extremely common for drug chemistry laboratories to directly test plant material and even oil-based products for the presence of tetrahydrocannabinol (THC) and related compounds, the use of edibles and beverages recreationally has brought along a new set of analytical challenges. Drug chemistry departments are not necessarily equipped to handle this type of analysis. In spite of forensic toxicology laboratories being capable of handling extractions from various biological matrices, baked goods and hard candies have few similarities to their everyday case load.

One concept that does appear to be readily universal in the edible marijuana community is the establishment that 10 mg of THC is equal to one dose for this drug (2). While this standard works in theory, many edible manufacturers produce products that range from multiple doses in one package to just containing one individual dose. If proper interpretation of dosing instructions is not noted, some consumers may be put at risk for either undesirable effects from consuming too much in one sitting or no relief of symptoms at all for medicinal users. An imperative concept that novice edible users need to keep in mind is that digesting marijuana is not the same as smoking it when discussing the plant’s pharmacology. Those who have prior experience with smoking marijuana may assume that one brownie or one package of gummy bears equals one marijuana cigarette, though that is far from the case. Employees at marijuana shops may not be educated enough on this issue or be aware that edibles do not necessarily contain the THC amount as advertised on their packaging. This fact alone makes it even more critical that effective testing measures are put in place to ultimately protect the end users.

The development of an extraction method that can be used in a wide variety of laboratory settings is critical to the emerging fields of recreational and medicinal marijuana testing. Within environmental and food testing laboratories, the QuEChERS (quick, easy, cheap, effective, rugged, and safe) sample preparation method has been widely used for the past 13 years. In 2003, Anastassiades and Lehotay published the first QuEChERS application, which discussed the determination of pesticide residues in produce (3). Since then, QuEChERS has become the analytical gold standard for the testing and analysis of a wide variety of edible matrices, including oil, egg, meat, fish, wine, and beverage samples (4–9). Using disposable consumables, hundreds of pesticides can be analyzed in a single extraction with the QuEChERS approach. In addition to pesticide residues, other chemical residues such as antibiotics, veterinary drugs, mycotoxins, polycyclic aromatic hydrocarbons (PAHs), bisphenol A, and phthalates are routinely monitored using this technique (10–14). The solvent waste generated is much less than what is typically associated with complex organic extractions. This technique is a relatively easy analytical method for technicians to learn, allowing for laboratories to effortlessly adopt this system of sample preparation.

Neither traditional solid-phase extraction (SPE) columns nor liquid–liquid extraction techniques can successfully provide laboratories with the reproducible and fast results needed for cannabis food analysis. Unlike biological matrices, edible products do not easily pass through the porous frits and sorbent of an SPE column. In addition, they do not contain the same endogenous matrix interferences found in biological samples that ultimately need to be removed for accurate quantitation. Lastly, when analyzing the cannabinoid content in edible samples, the final extract often needs to be diluted rather than concentrated before instrumental analysis. This is in stark contrast to forensic samples, which often require concentration of target analytes at trace levels. Liquid-liquid extraction often requires large amounts of undesirable and toxic solvents to be used. The above limiting factors allow for QuEChERS to make a desirable transition to the forensic community.

Experimental

Reagents and Standards
High performance liquid chromatography (HPLC)-grade acetonitrile, HPLC-grade methanol, and American Chemical Society (ACS)-grade acetic acid were purchased from Spectrum. Also, 35 neat pesticide standards were purchased from Sigma-Aldrich, Chem Service, or Ultra Scientific. A 2-ppm working solution containing 35 pesticides was prepared in acetonitrile. Three cannabinoids including THC, cannabidiol (CBD), and cannabinol (CBN) were purchased from Cerilliant in 1-mg/mL solutions. A 10-ppm mixture of the three cannabinoids was prepared in acetonitrile. Neat triphenyl phosphate was purchased from Cerilliant and diluted to 10 ppm in acetonitrile.

Sample Preparation
Baked goods, chocolate bars, and hard candies were ground into a fine powder using a SPEX 6770 freezer mill before extraction. A hard candy sample before and after grinding is shown in Figures 1a and 1b. (see upper right for Figure 1, click to enlarge, caption: Figure 1: Photographs of a hard candy (a) before and (b) after freezer mill grinding; (c) after QuEChERS extraction; and (d) the extracts before (left) and after (right) dSPE cleanup.) Gummy-based candies can be ground into powder with the presence of liquid nitrogen; however, the powder will return to its elastic gel state when the temperature rises. Thus, gummy-based samples were cut into fine pieces instead of using a freezer mill. Carbonated beverages, such as sodas, were degassed for 30 min before analysis, while oil samples were extracted without any sample pretreatment.

Homogenized 1-g samples (baked goods, chocolate bars, hard candies, gummy bears, or oil samples) were weighed into a 50-mL centrifuge tube. To each of these samples, 10 mL of reagent water was added. Samples were hydrated for 1 h using a horizontal shaker. For beverage samples, 10 mL of degassed sample was added to 50-mL tubes without the water addition and the 1-h hydration step. Internal standard and 10 mL of acetonitrile containing 1% acetic acid were added to all samples, which were then shaken for 1 min using the SPEX Geno/Grinder homogenizer. A proprietary blend of QuEChERS extraction salts supplied by UCT was added to each tube, and the tubes were shaken vigorously to break up any salt agglomerates. The extraction salts help to facilitate phase separation and partition target analytes from the aqueous layer into the acetonitrile layer. After shaking, the samples were centrifuged for 5 min at 3000 rcf. Three distinct layers are formed after centrifugation as demonstrated in Figure 1c. The top layer is the organic phase (acetonitrile) containing pesticide residues, cannabinoids, and the organic-soluble matrix coextractives; the middle layer is the insoluble matrix components and water containing the water-soluble matrix components, such as sugars; and the bottom layer is the undissolved excess extraction salts.

For pesticide residue analysis, 1 mL of the supernatant was transferred to a 2-mL dispersive solid-phase extraction (dSPE) tube containing a proprietary blend of sorbents (UCT), and shaken for 1 min using the Geno/Grinder, then centrifuged for 5 min at 3000 rcf. This process removes chlorophyll, sugars, organic acids, and fatty compounds from the sample extracts by retaining them onto the sorbents. The resulting clean extract, illustrated in Figure 1d, is then diluted 2x with reagent water and analyzed by LC–MS/MS.

For cannabinoid content analysis, dSPE was not necessary because of the high cannabinoid concentration in the acetonitrile extract. Instead, serial dilutions ranging from 200x to 20,000x were carried out to obtain a concentration (a few hundred parts per billion) that is suitable for LC–MS/MS analysis.

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