The differentiation of hemp and marijuana using colorimetric testing
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Abstract
Cannabis is one of the most popular drugs consumed recreationally and medically all over the world for thousands of years. Cannabis is well known as being the most popular hallucinogen. The main psychoactive component, delta-9-tetrahydrocannabinol (D9-THC), is responsible for the drug’s effects. The 2018 Farm Bill impacted the farming community by removing hemp from the definition of marijuana in the Controlled Substances Act (CSA), further removing it from being a schedule I drug, and giving hemp its own definition. Hemp is now defined as a derivative of Cannabis Sativa with a concentration less than 0.3% THC dry weight. The new definition makes it possible for more research opportunities to grow and expand the knowledge of what hemp is, and the power it can have to impact people’s everyday lives. The 2018 Farm Bill was extended to be in effect until the end of 2024.The Cannabis plant contains over 550 chemicals called cannabinoids. Discovering and synthesizing THC and cannabidiol (CBD), the two major compounds in cannabis, set the stage for all future research and allowed scientists to have enough of the product to determine its effects. The major psychoactive component of marijuana is THC, and the major compound found in hemp is CBD. Cannabis is known for its dark green colored leaves, palmate shaped leaves, serrated leaf edges, leaves six times longer than broad, and an odd number of leaflets connected to the stem. What truly makes cannabis stand out from all the other drugs is the distinct “skunk like” odor. Marijuana and hemp both share these qualities, creating a challenge in distinguishing between the two, macroscopically and microscopically. The highest abundance of the cannabinoids is found in the buds of the flowering plants, where the glandular hairs, also known as glandular trichomes, are located, holding the THC in their bulbous head.
Colorimetric, presumptive testing using a test kit is the first destructive analysis performed on drug samples. Using the visuals from macroscopic and microscopic examination, a speculation of the possible plant identification can be determined. Spot tests and presumptive screening kits assist in helping make conclusions as to what the drug evidence is. Upon conducting the presumptive test, a color change occurs resulting from a chemical reaction and indicates a positive result. The test kits have evolved to increase specificity of the drug they are looking for. When it comes to testing for marijuana, there are five commercially available kits. When used in conjunction with one another, the analyst can determine more information about the sample. The objective of the research was to use three different kits to distinguish if the results were similar or different when comparing the known strains of hemp and marijuana to each other. Three different companies manufacture the D-L reagent. Results of each kit were also compared to determine if there were any similarities or differences amongst the hemp or marijuana results to determine if one kit was better than the others.
The Duquenois-Levine (D-L) test for cannabis is the oldest known test to detect the presence of THC. However, it does not have the ability to differentiate between hemp and marijuana. The D-L reagent is composed of three components: vanillin and acetaldehyde added to ethanol. The addition of two more reagents helps complete the test. Hydrochloric acid (HCl) is added to create an acidic environment for the blue/violet color change, followed by the addition of chloroform. The chloroform creates a bilayer formation and if the violet color transitions to the bottom of the bilayer, the result is a presumptive positive.
Similarly to the D-L test, the KN reagent tests for the presence of THC. Fast blue BB salts (FBBBS) form a red color in the presence of THC, an orange color in the presence of CBD, and a purple color in the presence of cannabinol (CBN). The last presumptive test is the 4-aminophenol (4-AP) test also known as the Hemp/CBD test. This test is unique from the others because it can presumptively differentiate between hemp and marijuana. A pink color change is a positive result for CBD-rich/THC-poor samples (hemp) and a blue color change is a positive result for THC-rich/CBD-poor samples (marijuana).
The D-L reagent is manufactured as a test kit used in the field, but is also easily formulated in the laboratory to test possible marijuana evidence. This research included seven different formulations of the D-L test to determine the results of changing the ratios of the different reagents, to see if any conclusions could be drawn when testing hemp and marijuana. The formulations included, the original literature formulation (Fotchman and Winek) (45), halving the amount of acetaldehyde, doubling the amount of acetaldehyde, halving the amount of vanillin, doubling the amount of vanillin, halving the amount of ethanol, and doubling the amount of ethanol. Each marijuana strain produced different color results for each of the formulations. Consistently among the four marijuana strains the formulation with double the amount of ethanol was distinctive from the other samples. The formulations doubling the amount of vanillin and halving the amount of ethanol, were consistently similar to each other in overall appearance, colors of their bilayers, and appearance of demarcation layers. Two hemp strains produced unique results among the formulations. The other two strains had nearly identical reactions to the formulations, with a few shade differences among the bilayers. Doubling the amount of vanillin and halving the amount of ethanol produced similar results for all four strains of hemp. Halving the acetaldehyde reacted similarly for three out of the four strains. Doubling the acetaldehyde and halving the vanillin produced similar results but had differences in shades of violet and magenta in the bottom layers of two strains. Similar to the marijuana strains, the vials with similar appearances did not have the same variable changed for the two formulations. The hemp strains had darker appearances than the marijuana samples and took more time to change color after each step.
Description
2024
License
Attribution 4.0 International