Cannabis plants contain a wide variety of complex compounds including phytocannabinoids, terpenoids and flavonoids, found mainly in the inflorescences. Major phytocannabinoids like CBD and ∆9-THC have beneficial effects on human health. It is well known that variations in their profiles are connected to growth, light quality, humidity, the use of pesticides, harvesting and especially post-harvesting, like improper drying procedures that can facilitate fungal and microbial growth. Mass spectrometry is the tool of choice for the analysis of all these biologically active compounds along with that of heavy metals, pesticides, solvent residues, and mycotoxins to ensure the quality and safety of medicinal Cannabis products.
Introduction:
The Cannabis industry has been on a growing trend for the last few years, as many steps into the regulation and legislation of its products for medicinal and recreational use in different countries have recently changed. As examples, in April 2023 Germany announced a preliminary plan to legalize Cannabis for personal use thus allowing people to grow up to three plants at home. Mexico approved the medicinal and scientific use of Cannabis in 2017 and its recreational use in 2021. As of November 2018, physicians in the UK are legally allowed to prescribe Cannabis-based medicines (Stevens, 2018). Since the 1990s, Israel runs the longest medical Cannabis program and as of 2010, an estimated of 80,000 Israeli patients with specific medical conditions like pain associated to cancer and neuropathies, inflammatory bowel disease, epilepsy, post-traumatic stress disorder and chemotherapy-associated nausea, vomiting, fatigue, mood disorders and loss of appetite are licensed to use medicinal Cannabis (Isralowitz et al., 2021; Mirelman et al., 2019). In the USA, it is federally prohibited to consume Cannabis, although 36 states and 4 territories allow Cannabis for medical purposes although it is still illegal for physicians to prescribe it, even in states where its use is legalized (Victor et al., 2021). Nonetheless, retail sales of Cannabis products are projected to reach $51.28 billion in 2023 where most revenue will be generated in the USA (https://www.statista.com/outlook/hmo/cannabis/worldwide). This has put the Cannabis market on the spotlight as it represents a lucrative and promising business opportunity.
The history of Cannabis plants used as sources of food, fiber, and medicine dates back to 5,000 years BCE in China. Excavations in China have found perfectly preserved Cannabis in a burial dating back to 2700 years ago suggesting its use as medicinal or as a divination agent (Russo et al., 2008; Pisanti and Bifulco, 2018). Cannabis sativa L. (Cannabaceae) is originally from the Himalayan mountains and is endemic to Asia. It has three main recognized varieties C. sativa, C. indica and C. ruderalis (Small, 2015). The first two have the biggest economic value. The most common names are marihuana, hemp, hashish, cáñamo and cannabis and these include all parts of the plant, derivatives, mixtures, resins, preparations, and oils. Cannabis sativa L. produces approximately 565 secondary bioactive metabolites including phytocannabinoids, terpenoids, flavonoids and alkaloids mostly accumulated in the glandular trichomes in female inflorescences (ElSohly and Slade, 2005; Malabadi et al., 2023). The main phytocannabinoids are (-)-∆9-trans-tetrahydrocannabinol (∆9-THC) whose contents are used to determine its psychoactivity, and cannabidiol (CBD) (Mechoulam and Hanus, 2000). These two phytocannabinoids are biologically active to treat glaucoma, nausea, epilepsy, depression, neuropathic pain, multiple sclerosis, and movement disorders (Amin and Ali, 2019; Lee et al., 2018). Furthermore, they have a therapeutic potential to treat autism (Mostafavi and Gaitanis, 2020) and to alleviate Parkinson’s disease-related tremor, anxiety, pain, and quality of life (Urbi et al., 2022).
Cannabis sativa L. has three main chemovars according to the contents of the two main phytocannabinoids: ∆9-THC and CBD. The first one with a ratio of ∆9-THC/CBD >> 1 (> 0.5% ∆9-THC and < 0.5% CBD); the second is ∆9-THC/CBD ~ 1, CBD being the predominant over ∆9-THC; and the third one ∆9-THC/CBD << 1 (with low concentrations of both phytocannabinoids). The first and the second chemovar are mostly used as drugs and the third is considered as a fiber-type or hemp (ElSohly et al., 2017). These two phytocannabinoids are mostly present as carboxylic acids i.e. ∆9-trans-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) and are non-psychotropic. Both can be transformed to their neutral form either by non-enzymatic decarboxylation (Flores-Sanchez and Verpoorte, 2008), heating the plant material, long-term storage, or changes in pH (Abd-Elsalam et al., 2019).
Equally important are flavonoids present in C. sativa L. with some unique to the genus. Most flavonoids belong to the flavone-, flavonol-, aglycone- and glycoside-types, being the flavones cannflavin A, B and C the most abundant flavonoids in Cannabis leaves, seedlings, flowers, fruits, and pollen (Bautista et al., 2021; Barrett et al., 1986; Frassinetti et al., 2018; Ross et al., 2005). Cannflavins have both proven antioxidant and anti-inflammatory activities (Barrett et al., 1985; Zurier, 2003), other biological activities like neuroprotection, anticancer and antiviral have been reported. Apigenin has anxiolytic effects which makes flavonoids potentially beneficial to treat migraines, headaches, and pain (Baron, 2018).
Every Cannabis strain is unique in its phytocannabinoid, flavonoid and terpenoid profile hence they are referred as chemovars rather than strains. All these, act synergistically in the produced effects of Cannabis and this is known as the entourage effect (Silva-Sofrás and Desimone, 2023). As an example of this, a Cannabis extract containing phytocannabinoids, terpenoids and flavonoids was able to provide total relief of neuropathic pain in a rodent model for neuropathic pain when compared to pure phytocannabinoids (Comelli et al., 2008). Typically, research efforts have focused mainly on phytocannabinoids while terpenoids and flavonoids have been largely ignored. Because the chemical profile of these three groups of secondary metabolites is highly dependent not only on the chemotype but also on growth conditions, plant age, geographical location, harvesting, processing, and storing, it makes sense to characterize each group to assign a “fingerprint” to every Cannabis chemovar and thus to identify chemovars for specific medical conditions. Terpenoids are mostly profiled and quantified by gas chromatography - tandem mass spectrometry (GC-MS/MS) or gas chromatography - flame ionization detector (GC-FID) while flavonoids can be analyzed by high performance liquid chromatography-photodiode array (HPLC-PDA), if they occur in high amounts. Because both groups have isomers and isobars and some occur in very low amounts, they are good candidates for liquid chromatography - tandem mass spectrometry (LC-MS/MS).
In 1937, the USA the anti-marihuana law placed federal restrictions on Cannabis (Musto, 1972) and in the 1970s, it was classified as Schedule I substance in the USA under the Controlled Substances Act. Although in the last 20 years, a renewed interest in its use as a therapeutic agent to treat a wide range of illnesses has enabled research on its major phytocannabinoids. Nowadays, the FDA has approved several drugs containing single cannabinoids like Epidiolex containing purified CBD useful to treat rare and severe forms of epilepsy; Marinol and Syndros which contain dronabinol, a synthetic THC and Cesamet, a synthetic analogue to THC, all prescribed to treat nausea and vomiting in chemotherapy patients and loss of appetite and weight loss in patients with HIV (https://www.nccih.nih.gov/health/cannabis-marijuana-and-cannabinoids-what-you-need-to-know).
Potency is the most frequent measurement used to assess the percentage of ∆9-THC and/or CBD in plant materials. Oftentimes, other phytocannabinoids like THCA, CBDA and cannabinol (CBN) are considered in the screening panel in Cannabis products although labels only show CBD-to-∆9-THC ratios and sometimes cannabigerol (CBG). Most phytocannabinoids are structurally similar and some like CBD and ∆9-THC are isomers. The most frequently used analytical platforms are GC-MS (Nahar et al., 2020), HPLC-PDA and LC-MS/MS (Meng et al., 2018). Some shortcomings of using GC-MS are that the ratio of neutral-to-carboxylated phytocannabinoids is lost during analysis due to the high temperature in the injector and detector; long analysis times plus the fact that samples need to be derivatized. HPLC-PDA and HPLC-UV are relatively inexpensive and easy techniques when compared to LC-MS/MS although both detectors are less sensitive and less selective when it comes to co-eluting phytocannabinoids or other UV-absorbing compounds. Thus, when using a HPLC-PDA detector, an interfering peak might go unnoticed if it elutes at the same retention time as a major phytocannabinoid, thus leading to an incorrect and higher quantity (Nie et al., 2019).
LC-MS/MS is the preferred analytical tool for the separation, identification, structure elucidation and quantification of phytocannabinoids, which can quantify less abundant and isobaric compounds. It represents the best alternative for both qualitative and quantitative methods and a feasible analytical platform to analyze major and minor phytocannabinoids, their metabolites such as 11-nor-9-carboxy-THC (THCCOOH) and 11-hydroxy-THC (11-OH-THC), and glucuronide conjugates in diverse biological matrices such as breast milk (Sempio et al., 2021), blood, plasma, serum, urine, oral fluid, hair, breath and neonatal-maternal matrices (Karschner et al., 2020).
As any other plant, Cannabis plants are prone to fungal attack, bacterial infection, and diseases even in plants that are grown under controlled environments, where the most common pests found in indoor Cannabis flowers and leaves are aphids, spiders, mites, thrips, and fungi, typically Aspergillus. For this reason, growers are inclined to use pesticides or plant growth stimulators to increase their yield. Bacterial contaminations occur due to improper drying, and storage of still wet plant materials and fungal infections are the result of growing and/or storing Cannabis plants in humid conditions. Unfortunately, these infections affect plant growth and decrease product quality (Punja et al., 2019). In most countries where medicinal and recreational Cannabis use is legalized, regulatory agencies have guidelines to monitor heavy metals, mycotoxins, pesticides, microbes, and the presence of residual solvents in Cannabis oil, as quality control of Cannabis products. In the USA, the Environmental Protection Agency (EPA), will not set tolerance levels for pesticides used to grow Cannabis since the crop is federally illegal. Nevertheless, all these contaminants should be regularly monitored as they can be introduced during plant growth and processing, since consumers exposed to some of these contaminants can develop pulmonary and enteric infections, neurotoxicity, carcinogenicity, abnormal hormone production, disruption in synaptic processes, teratogenicity, poisoning among many others (Montoya et al., 2020; Dyburgh et al., 2018). Therefore, the development of analytical methods to monitor these contaminants in Cannabis plants and products is a must where testing begins at cultivation, continues during processing and storage, and ends with the final product.
One of the main challenges in analyzing Cannabis plants is the highly variable and complex amount of chemicals which poses a huge challenge to overcome matrix effects. Sample preparation, extraction and cleanup must be robust enough to ensure the removal of phytocannabinoids and other secondary metabolites so lower amounts of contaminants are able to be quantified. The most employed extraction methods to extract pesticides and mycotoxins are solid-to-liquid extraction (SLE) and quick, easy, cheap, effective, rugged, and safe (QuEChERS), the latter being recommended by the European EN 15662 method (European EN, 2008). The analytical platform should be sensitive, selective, and accurate making LC-MS/MS an ideal analytical platform for the analysis of most pesticides, due to its versatility and ability to analyze polar and semi-polar pesticides. Most pesticides can be ionized using electrospray ionization (ESI) using low resolution mass spectrometry analyzers like triple quadrupole (QQQ) or quadrupole ion trap (QTRAP), where targeted analyses are carried out as multiple reaction monitoring (MRM) experiments that allow the simultaneous analysis in a single run, of several pesticides and mycotoxins (López-Ruiz et al., 2021; Craven et al., 2019; Taylor and Birkett, 2020).
Cannabis plants can be contaminated with heavy metals i) by virtue of its bioaccumulative capacity; ii) due to cross-contamination during processing; iii) post-processing adulteration e.g. with lead to increase its weight and therefore to increase its market value (McPartland and McKernan, 2017; Busse et al., 2008); and iv) anthropogenic contaminants mainly via water, fertilizers, pesticides, and fungicides. Heavy metals are of great concern to public health since they accumulate in the body, are toxic, carcinogenic, and poisonous. Dangerous to human health as carcinogens are Cd, Hg, Pb, As, Cr and Ni (Galic et al., 2019). Inductively coupled plasma combined with mass spectrometry (ICP-MS) enables the simultaneous analysis of heavy metal analysis with high sensitivity and specificity and makes it a suitable analytical platform for trace metal profiling of Cannabis plants and products (Jones and Nelson, 2018).
Perspectives
The Cannabis industry keeps giving us new products as cosmetics, edibles, cartridges, oils, extracts and flowers. Since the 1970, there has been virtually no development of regulations for Cannabis products and now, with its legalization, comes the growing need for a reliable source of ∆9-THC and CBD to ensure the quality, safety, and integrity of commercial and medicinal products.
The development of analytical platforms is still in its early stages and there are no unified guidelines that set limits on unsafe chemicals. Regulations will have to quickly catch up with already established analytical methods to determine accurately and quantify phytocannabinoids along with monitoring the integrity of oils, extracts and flowers by routinely measuring CBN and ∆8-THC, an oxidative degradation product and an acidic isomerization artifact both of ∆9-THC, respectively.
Mass spectrometry is the preferred analytical tool due to its high selectivity and sensitivity needed to profile a complex plant matrix like Cannabis with all its structurally diverse compounds. While not all these compounds can be analyzed in one single run, all of them can be analyzed by mass spectrometry. This practice should be harmonized and unified in order to ensure that users are consuming a safe product.
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R&D Scientist, Leiden University
