Despite remaining illegal at a federal level and in about one-third of states, the legal cannabis industry in the U.S. has become a nearly $11 billion business — a figure that is predicted to double by 2022. The boom of the industry, which exceeded $244 million in tax, license, and fee revenue for the state of Colorado in 2018, is also a boon for scientists, specifically analytical chemists, as testing facilities and instrument companies sprout up like, well, weeds. Cannabis complexity According to Bob Clifford, the general manager of marketing for Shimadzu Scientific Instruments, which has a North American headquarters in Columbia, Md., “Cannabis is a complex plant with more than 500 compounds, and every state and country has different testing requirements.” The cannabis plant and the methods by which it can be consumed provide several challenges for scientists. Closely related to the hops used to flavor beer, each cannabis strain or cultivar can contain a different number of various cannabinoids, which are the pharmacologically active ingredients; and terpenes, which provide so-called entourage effects. Unlike hops, however, cannabis is ingested via a variety of methods — by smoking, vaping, drinking, eating, or applying — and through a myriad of vectors: joints or electronic cigarettes, in any type of edible, or in wax and oil forms. Finally, it has several uses — for pain relief, to induce hunger and control nausea, and for recreational purposes. Because it is federally illegal, the cannabis industry in the U.S. is mired in a hodgepodge of local and regional regulations that has encouraged variability in products and in testing standards. Many states where cannabis is legal require some form of product analysis. Scientists and researchers — working alongside growers, extractors, producers, dispensaries, and law enforcement — have become key players. They have developed specialized testing labs and portable spectroscopic devices, and have leveraged analytical techniques both to assist manufacturers and to safeguard consumers. According to Clifford, cannabis and its derivative products are tested at multiple points on the production chain for cannabinoid potency and terpene content, as well as for contaminants such as pesticides and herbicides, mycotoxins and other fungal metabolites; residual solvents such as the alkanes used in extraction chemistry; and heavy metals such as arsenic, cadmium, lead, and mercury. ‘The nice thing about spectroscopy is that cost and speed are at a point where one can get useful data very quickly.’ — Dylan Wilks Orange Photonics Inc. Typically, the first step in analyzing organic plant material is chromatography in either the liquid or the gas phase, followed by an appropriate detector. For cannabinoid analysis, Clifford said use of high-performance liquid chromatography (HPLC), with either UV spectroscopy or a photodiode array detector, is the “gold standard” because of its low cost, its large linear dynamic range, and its detector stability. Pesticide analysis requires liquid or gas chromatography, which involves using more expensive triple quadrupole mass spectrometry. For heavy metal analysis, analytical chemists use inductively coupled plasma mass spectrometry because of its simultaneous, multielemental analysis. Adding to the complexity of analyzing cannabis, Clifford — who is a member of the cannabis working group of the Association of Official Analytical Chemists Stakeholder Panel on Strategic Food Analytical Methods — pointed out: “More than many other plants, cannabis absorbs from the surrounding environment. It was used as a phytoremediator of radioactive heavy metals in the USSR after the Chernobyl accident.” A commonly used fungicide, myclobutanil, provides a stark example of absorption. The wine industry uses the fungicide on grapes, often in regions where cannabis is also grown, such as northern California. Although myclobutanil is considered less harmful when swallowed and is allowed by the Environmental Protection Agency at 1 part per million on grapes, when combusted it decomposes to hydrogen cyanide, a known poison. Myclobutanil has been found on cannabis in California and has led to recalls. “We need to be very cautious in the steps we’re taking and advocate for third-party testing to ensure quality and contaminant control,” said Clifford. Shimadzu, a Japanese company, has helped set up ISO-certified, independent cannabis testing labs in the U.S. and around the world. In addition, in March 2017, the company launched a new product, the Cannabis Analyzer for Potency (Figure 1). An HPLC system with UV spectroscopic detection, the instrument capitalizes on the growth of the industry and gives growers and producers potency information on their products and plant material. Figure 1. Shimadzu’s Cannabis Analyzer for Potency uses high-performance liquid chromatography (HPLC) and UV spectroscopy to determine cannabinoid content and is designed for use by nonspecialists. Courtesy of Shimadzu Scientific Instruments. From its software to its protocols to its standards, Shimadzu’s cannabis analyzer has been designed for use by nonscientists, said Clifford. “We should be able to look at a cannabis cultivar like a box of cereal with a food label. Here’s the percentage of cannabinoids, terpenes, etc. Transparency and simplification — those are the goals.” Mars technology for marijuana “Working with a botanical product is very difficult,” said Dylan Wilks, chief technology officer of Elkins, N.H.-based Orange Photonics Inc. Medical cannabis has been legal in New Hampshire since 2013, and possession of cannabis quantities up to three-quarters of an ounce has been decriminalized since 2017. “Obviously, our clients cannot send us their samples,” he said. Wilks is a third-generation spectroscopist whose grandfather founded a half dozen instrument companies, primarily on the East Coast. In contrast to Shimadzu’s cannabis analyzer, which is a tabletop instrument, Orange Photonics has built LightLab, a similar device that takes advantage of the trend in portable spectroscopy (Figure 2). LightLab uses HPLC combined with UV spectroscopic detection at multiple fixed wavelengths and is housed in a rugged suitcase so it can be carried into the field. Figure 2. Orange Photonics Inc.’s portable cannabis analyzer uses UV spectroscopy at fixed wavelengths and can be carried into the field. Courtesy of Orange Photonics Inc. “It’s selective separation spectroscopy,” said Wilks. “We’ve combined liquid chromatography with the same spectroscopy elements used on the Curiosity Mars rover.” He said they chose to work in the UV because it helps discriminate cannabinoids that tend to have overlapping chemical signatures. While there are more than 100 known cannabinoids, most instruments, including LightLab, test for about eight, including Δ9-tetrahydrocannabinol (THC), which produces a euphoric effect, and cannabidiol (CBD), a pain reliever and antidepressant. Wilks chose fixed wavelength UV rather than broad-spectrum light sources because it is more stable when equipment is transported. Orange Photonics clients, he said, are primarily the growers themselves, as well as contract extractors, who distill the oils from the cannabis plant that are used in products such as vaping cartridges and edibles. Wilks doesn’t see their device as a replacement for chromatography combined with mass spectrometry. Instead, it aids quality control as well as research and development of new products or cultivars. “The nice thing about spectroscopy is that cost and speed are at a point where one can get useful data very quickly. I think that’s what the industry needs, and that’s what we’re hoping to fill as a niche,” he said. “Cannabinoids are quite expensive. The more you can get out, the more efficient your process is — the better. You’ll know you’re not leaving money on the table.” VUV spectroscopy “It reminds me of the pain medicine boom,” said Kevin Schug, University of Texas at Arlington professor of analytical chemistry. “Everybody was starting their own pain med lab, but at least those were federally regulated.” Schug’s lab leverages analytical techniques for several purposes, including wastewater remediation and hydraulic fracturing contaminant detection. They have recently embarked on studies of cannabinoids, and in January 2018 reported using gas chromatography with vacuum ultraviolet (VUV) spectroscopy to differentiate cannabinoids, their metabolites, and derivatives1. “The cannabis boom provided a massive opportunity for us to contribute to the scientific literature,” he said. For the “report, Schug teamed up with VUV Analytics Inc. of Cedar Park, Texas, where he’s a member of the scientific advisory board, despite the plant remaining illegal in the state (Figure 3). Figure 3. Kevin Schug’s research group at the University of Texas at Arlington uses a gas chromatography detector with vacuum ultraviolet spectroscopy to differentiate cannabinoids with similar structures. Courtesy of Allegra Leghissa/the University of Texas at Arlington. The biggest problem with mass spectrometry around compounds like cannabinoids, he said, was that they exist in the plant as a complex isomeric mixture with similar fragmentation patterns. Instead, for structural differentiation, VUV absorption down to 125 nm in the gas phase allows for half a nanometer spectral resolution. “The separation becomes less of a strict requirement to get a reliable analysis,” Schug said. “That means you can push separations faster. The VUV detector is also quite fast at 100-hertz response rate, so you can really keep up with a fast separation.” ‘We should be able to look at a cannabis cultivar like a box of cereal with a food label. Here’s the percentage of cannabinoids, terpenes, etc. Transparency and simplification — those are the goals.’ — Bob Clifford Shimadzu Scientific Instruments The VUV technique can also determine terpene content, while portable spectroscopic devices generally cannot because in most cultivars terpenes exist below the detection limits for those types of instruments. Schug has also used VUV spectroscopy to detect terpenes in hops2, and the craft beer industry seems poised to benefit from the growth of the legal cannabis market. Orange Photonics’ Wilks said their device could also be used in hops analysis with a terpene detection kit add-on. The craft beer market has continued to grow in the U.S. despite a slowdown in overall beer sales. The future of hemp Wilks points to the hemp industry as a growing market for spectroscopic devices. In many ways, the legal hemp industry seeks an outcome opposite to that of the cannabis industry — that is, to cultivate plants with lower, not higher, amounts of euphoric cannabinoids. While rich in CBD, hemp is defined by U.S. government agencies as a cannabis plant having no more than 0.3 percent THC. The most recent farm bill passed by Congress in December 2018 reclassified hemp as an agricultural crop, allowing growers to purchase federally subsidized crop insurance, among other benefits. It also made industrial hemp-derived CBD legal. Industrial hemp has gone from being grown on less than 5000 acres in the U.S. in 2015 to more than 30,000 acres in 2018. www.linkedin.com/in/farooqtheahmed Acknowledgments The author would like to thank Bob Clifford, Shimadzu Scientific Instruments; Dylan Wilks, Orange Photonics Inc.; and Kevin Schug, the University of Texas at Arlington. References 1. A. Leghissa et al. (2018). Detection of cannabinoids and cannabinoid metabolites using gas chromatography with vacuum ultraviolet spectroscopy. Sep Sci plus, Vol. 1, https://doi.org/10.1002/sscp.201700005. 2. A. Leghissa et al. (2017). Determination of cannabinoids from a surrogate hops matrix using multiple reaction monitoring gas chromatography with triple quadrupole mass spectrometry. J Sep Sci, Vol. 41, https://doi.org/10.1002/jssc.201700946.