CBD is also known for these beneficial properties but is non-psychoactive, so it does not give a person a “high” when used. Typically, hemp is CBD-rich containing low concentrations of THC causing its THC:CBD ratio to be below 1. Cannabis is considered marijuana when it has a concertation of total Δ9 – THC ≥ 0.3% , but usually has a THC:CBD ratio above 1. Elsohly et. al. reported that from 2009 to 2019 marijuana in the U.S. increased in THC potency across the decade from an average of 10% THC in 2009 to 14% THC in 2019. In 2019, the average CBD concentration in marijuana was found to be 0.6% , and that the THC:CBD ratio was above 20 across the decade. This difference in THC:CBD ratios can be used in the design of an effective field test for the identification of marijuana-type cannabis. The most common field tests performed for the presumptive identification of unknown drugs are colorimetric assays. These tests are considered presumptive as they only indicate the possibility of the analyte being present in the substance. Until the Agricultural Improvement Act of 2018, the modified Duquenois-Levine test was the color test used to presumptively identify a suspicious plant material as cannabis. Although used for many years, the D-L test is known to produce false positives with reaction of molecules containing a resorcinol backbone and an aliphatic chain. Therefore, the D-L test isknown to produce false positive results from plants such as patchouli, spearmint, and eucalyptus. Furthermore, THC, CBD, and many other cannabinoids contain both a resorcinol group and an aliphatic chain, resulting in a D-L test that is not selective enough to differentiate between the cannabinoids. This shortcoming is the reason that the D-L test is no longer a suitable field test for the identification of marijuana-type cannabis.
One colorimetric test that is currently being used to differentiate between hemp and marijuana is the 4-aminophenol test developed by the Swiss Forensic Institute in Zurich. A recent validation study has shown that a pink color forms when the THC:CBD ratio is below 0.3 and a blue color forms when the plant has a THC: CBD ratio above 3. A confirmatory chemical test such as GC-FID or GC–MS is still required after a positive 4-AP test. The test requires the use of at least 1 mL of one of its reagents, 4-aminophenol, to produce a color result. Although the 4-AP test has demonstrated capability as a presumptive test for cannabis,trim tray for weed it has also been reported that it may not be selective for THC. False positive results have been obtained with sage, oregano, and several cannabinoids, such as cannabinol. A more selective and smaller-scale alternative presumptive test could improve the presumptive confirmation for marijuana in the field. A colorimetric reagent that has been used for many years as a visualization reagent for cannabinoids when analyzing cannabis extracts through thin layer chromatography is the Fast Blue BB reagent. The FBBB test is selective among major cannabinoids, providing a red color for THC, an orange color for CBD, and a purple color for CBN. Ultraviolet-Visible Spectroscopy has shown that the FBBB + THC chromophore has an absorption band at 471 nm, which is responsible for its red color. The Almirall lab previously reported the structure of the FBBB + THC chromophore using results from high resolution mass spectrometry and Hydrogen Nuclear Magnetic Resonance . It was determined that, in basic conditions THC becomes a phenolate anion and that this anion attacks the diazo group in FBBB at the para position to form the chromophore. A bathochromic shift results from the extended conjugation in the chromophore and the n→π* transition caused by the electrons in the diazo group of FBBB. In addition to characterizing the chromophore, the previous study evaluated the selectivity of FBBB for THC detection. Eight different types of tea, 3 hop products, and 3 authentic hemp buds were extracted and tested using FBBB. This test was performed by adding 10 µL of the extract to a filter paper, followed by 10 µL of 0.1% FBBB and 0.1 N NaOH. Extracts that were made from methylene chloride produced only 1 false positive with one of the teas. Of note, none of the hemp samples produced a false positive result, displaying an orange color indicative of CBD.
These results support the selective nature of the FBBB test for use as a presumptive field test to distinguish between hemp, marijuana, and other plant materials. In the previous study, filter paper, a Capillary Microextraction of Volatiles device, and CMV strips were used as possible substrates to perform the FBBB test. A CMV device is an open-ended 2 cm glass capillary tube that contains seven 2 cm by 2 mm glass filter strips have been coated with vinyl-terminated polydimethylsiloxane that was developed by the Almirall lab as an alternative to Solid Phase Microextraction. The modified glass filters that make up the CMV, known as Planar SPME , have excellent absorption/ adsorption capabilities and can withstand high temperatures. It was found that when the FBBB test is performed on one of the PSPME strips the LOD for THC was 100 ng, which is significantly lower than the known LOD for the D-L test, 5000 ng of THC. Using PSPME as a substrate is advantageous over regular filter paper since it can withstand high temperatures allowing the chromophores formed to be detected using DART-MS with very little background. In this current study, the capabilities of using FBBB as a presumptive field test to differentiate between hemp and marijuana are presented. We also report a fast and easy extraction method for plant material that can be used in the field. A previously reported substrate known as PSPME support was used for the FBBB reaction . Six cannabinoids, 5 retail hemp samples, 20 authentic cannabis samples, tobacco, hops, herbs, and essential oils were tested with the FBBB reagent. RGB numerical codes were obtained for each color result to confirm the color produced by the reaction in an objective manner. The fluorescence results of the FBBB + THC fluorophore is reported for the first time. The fluorescence spectra of the FBBB + THC product are distinguishable from the spectra of FBBB + CBD chromophores. The RGB score combined with the fluorescence of the FBBB + THC chromophore/fluorophore enhances the selectivity of the FBBB test for marijuana. Linear Discriminant Analysis was performed to determine whether FBBB could be used to classify cannabis correctly as hemp-type and marijuana type. Methanol and chloroform were purchased from Sigma Aldrich . Methanolic solutions of THC, CBD, cannabinol , cannabigerol , delta-9- tetrahydrocannabolic acid , and cannabidiolic acid were all purchased from Sigma-Aldrich. Standard working solutions of these cannabinoids were made from 1000 ppm stock solutions.
A 1 mL mixture containing THC, CBD, CBG, CBN, THCA, and CBDA in acetonitrile at 500 µg/mL was purchased from Cayman Chemical . Terpene mixture 1 and terpene mixture 2, each containing 21 different terpenes commonly found in the Cannabis plant, trimmming tray weed were also purchased from Cayman Chemical. Fast Blue BB Salt hemi was purchased from Sigma Aldrich and NaOH was purchased from Macron Fine Chemicals . Spec 7 strain hemp, Purple emperor strain hemp, Eighty-Eight strain hemp, Painted Lady strain hemp, and Elektra Strain Hemp were all purchased from Blue Ridge Hemp Co. The certificates of analysis of each of the strains was reported by Blue Ridge Hemp Co.’s confirming that the cannabis purchased contained <0.3% total THC. Cigars, apollo hop pellets, citra whole leaf hops, oregano, sage, parsley, red pepper flakes, black pepper, lavender, and eucalyptus leaves were all purchased from commercial retailers. Two herb spice tobacco grinders were purchased from commercial retailers. The Cannabis research program at the National Institute of Standards and Technology provided 20 cannabis samples, all of which had the % total THC and % total CBD previously determined through Liquid Chromatography-Photodiode Array . A notable difference can be observed when FBBB is reacted with 1000 ng/µL of CBD and an extract of “Painted Lady” hemp containing 1263 ng/µL of CBD and no THC present compared to a reaction with 1000 ng/µL THC. When reacted with FBBB, the CBD solution and hemp extract both produce an orange color and THC produces a deep red color . The difference in color can be observed immediately after the NaOH is added to the reaction and is later confirmed through the chromophore’s RGB code. A Dino-Lite digital microscope capable of fluorescence imaging shows that FBBB + THC fluoresces brightly under a 480 nm light source while FBBB + CBD and FBBB + Hemp do not fluoresce significantly . The fluorescence spectra obtained from the VSC2000 show a distinct difference in the fluorescence intensity and λmax of FBBB + THC and FBBB + CBD and FBBB + Hemp . FBBB + THC has a fluorescence intensity near 70% and λmax: 655 nm. FBBB + CBD has a fluorescence intensity below 13% and has a λmax: 661 nm. Hemp + FBBB has a fluorescence intensity at 20% and a broader band with λmax: 695 nm. This band at 695 nm interferes with the FBBB + CBD peak.
A possible explanation for the band at 695 nm is that is chlorophyll from the plant is also extracted during the extraction and may be causing fluorescence at this wavelength. Chlorophyll is also known to absorb blue and red light and fluoresces in the red region of the visible spectrum. Despite the wavelength difference between FBBB + CBD and FBBB + hemp, the intensity of the fluorescence bands 661 nm and 695 nm are significantly lower than that of FBBB + THC at 655 nm. The difference in visual color and fluorescence provides two ways to observe the chromophore/fluorophore formed and determine whether the plant material being observed is hemp or marijuana. FBBB and NaOH were placed into amber vials and stored at room temperature . A separate pair of vials containing the reagent were placed in a refrigerator . Both sets of reagents were left at that temperature for one week and then evaluated using 1000 ppm THC and 1000 ppm CBD. After a week at room temperature, the FBBB reagent had gone from a yellow color to a clear color, indicating that it had become unstable at room temperature within the week. When used to test THC and CBD, it produced feint red and orange chromophores that could not be easily visualized. In addition to a decrease in color, the fluorescence of the THC + FBBB chromophore decreased as well. The refrigerated FBBB was evaluated and did not show a difference in color between tests done on day 1 and day 7. To test the long-term stability of FBBB in the refrigerator, THC and CBD was evaluated after 45 days of being in the refrigerator. There was no decrease in color or fluorescence after 45 days, demonstrating that although the FBBB reagent is unstable at room temperature, it is remains stable at refrigerated temperatures for at least 45 days. The stability of FBBB as a preloaded salt on the PSPME substrate was also evaluated at different temperatures. Ten microliters of 0.1% FBBB were pipetted onto 3.5 mm PSPME substrates, and the solvent was allowed to evaporate. These substrates were left at room temperature , refrigerated temperatures , and freezing temperatures . They were evaluated with using a “Painted Lady” hemp extract 15 min, 1 h, 2 h, 3 h, and 4 h after FBBB was loaded onto the substrate. For the substrates left at room temperature there was a loss of orange color between 15 min and an hour. After the first hour, barely any reaction could be visualized, once again showing that FBBB is not a stable reagent at room temperature. The preloaded substrates stored at low temperatures , produced a consistent orange color from 15 min to 4 h. This shows that FBBB could be preloaded onto a PSPME substrate and kept stable at cold temperatures. However, it should be noted that the orange color produced when the FBBB is preloaded is duller and less intense than the color produced when FBBB is applied after the extract.