Application of chromatographic methods for quality control of cannabinoid impurities in products containing Cannabidiol

Application of chromatographic methods for quality control of cannabinoid impurities in products containing Cannabidiol Dobrina Tsvetkova1, Ivanka Pencheva1, Lily Peikova1 1 Department of Pharmaceutical Chemistry; Faculty of Pharmacy, Medical University, Sofia, Bulgaria Corresponding author: Dobrina Tsvetkova ([email protected]) Received 29 August 2025♦ Accepted 4 September 2025♦ Published 14 October 2025 Citation: Tsvetkova D, Pencheva I, Peikova L (2025) Application of chromatographic methods for quality control of cannabinoid impurities in products containing Cannabidiol. Pharmacia 72: 1–13. https://doi.org/10.3897/pharmacia.72.e170419 Abstract Strict quality control of impurities in legitimate cannabidiol products and in cannabis “novel foods” is necessary due to the increased health risks from side effects associated with these impurities. The purpose of the present study is to summarize the most common impurities found in cannabidiol products, the factors that contribute to their elevated content, and the application of chromatographic methods for quality control. Light, temperature, oxidation, acidic medium, and especially their combined effects are critical in contamination with impurities. The most common impurities include delta-9-tetrahydrocannabinol, cannabinol, cannabichromene, cannabidivarin, cannabigerol, cannabiquinone, tetrahydrocannabidivarin, cannabidiolic acid, cannabigerolic acid, and tetrahydrocannabinolic acid. For the separation and determination of permissible levels of impurities in commercial cannabidiol products, HPLC and GC methods are appropriate and reliable for enhancing quality control, as they provide high sensitivity, accuracy, and precision. The most widely used techniques are HPLC-UV, HPLC-UV-PDA, HPLC-MS, HPLC-MS/MS, UHPLC-UV, UHPLC-UVPDA, UHPLC-MS, UHPLC-MS/MS, UHPSFC, GC-MS, and GC-FID. Keywords cannabidiol, GC, HPLC, impurities, quality control Introduction Cannabis species and pharmacological potential of cannabinoids Cannabis species occur in different geographical regions: Cannabis sativa Linn. grows in Europe, Cannabis indica Lam. is widespread in South Asia and Africa, and Cannabis ruderalis Janisch is specific to Central Asia (Gao et al. 2018). C. indica Lam. contains high concentrations of the psychoactive compounds tetrahydrocannabinol (THC) and tetrahydrocannabivarin (THCV), with a total cannabinoid content of about 54% (Tahir et al. 2021). From the industrial chemotype of C. sativa L., legitimate products with a high content of cannabidiol and less than 0.2% tetrahydrocannabinol are produced, including seeds, seed oil, seed tinctures, and seed extracts (Bonini et al. 2018). Medicinal cannabis has been described as effective for psychiatric, movement, and neurodegenerative diseases (Lim et al. 2017) and has been applied to improve sleep (Babson et al. 2017). Cannabinoids exert promising activity in the treatment of neurological disorders (Giacoppo et al. 2014). They are applied in the therapy of multiple sclerosis (Rudroff and Honce 2017) to decrease symptoms (Nielsen et al. 2018; Rice and Cameron 2018). Copyright Tsvetkova D et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Pharmacia 72: 1–13 DOI 10.3897/pharmacia.72.e170419 Review Article Tsvetkova D et al.: Chromatographic methods for control of impurities in Cannabidiol products2 Cannabinoids also show pharmacological potential in metabolic syndrome and neuroinflammation (Mastinu et al. 2018). Cannabinoids are used in various medical cases, such as pain (Starowicz and Finn 2017) and colitis (Couch et al. 2018). Cannabis-based drugs improve vocal blocking tics and reduce speechlessness in Gilles de la Tourette syndrome (Jakubovski and Müller-Vahl 2017). Medical cannabinoids regulate chemotherapy-induced nausea (Tafelski et al. 2016) and vomiting (Badowski 2017). The non-psychotropic cannabinoid cannabigerol (CBG) possesses antibacterial effects (Kumar 2025) and shows a neuroprotective role against Huntington’s disease (Valdeolivas et al. 2015). Cannabichromene (CBC) is important for brain function and exhibits antiviral and anti-inflammatory properties (Kumar 2025). Delta-9-tetrahydrocannabivarin (THCV) has potential benefits in the therapy of obesity and diabetes (Abioye et al. 2020). Currently, more than 100 phytocannabinoids from C. sativa L. (Cannabaceae) have been identified (Peng and Shahidi 2021). In the resin secreted from the trichomes of female plants, neutral cannabinoids and cannabinoid acids have been isolated (Hanus et al. 2016), which can be potential impurities in products containing cannabidiol. Some phytocannabinoids are non-psychotropic (Izzo et al. 2009), while others are psychotropic (Köguel et al. 2018). The more important cannabinoids and cannabinoid acids are presented in Table 1 (Hanus et al. 2016). Biosynthesis of phytocannabinoids The biosynthesis of phytocannabinoids is regulated by biosynthetic enzymes. Cannabigerolic acid is produced by the alkylation of olivetolic acid with geranyl pyrophosphate by a prenyltransferase. Cannabigerovarinic acid is formed from geranyl diphosphate and divaric acid. Through the decarboxylation of these precursors—cannabigerolic acid and cannabigerovarinic acid—the respective phytocannabinoids are formed, as presented in Table 2 (Sirikantaramas and Taura 2017). Phytocannabinoid types include: 1) Cannabigerol type and cannabidiol type: without psychoactive effect (Izzo et al. 2009). 2) Cannabicyclol type and cannabinol type: a mixture of phytocannabinoids obtained during storage in the presence of light (Hanus et al. 2016). 3) Delta-9-tetrahydrocannabinol type. Cannabidiol therapeutic effects Cannabidiol (Fig. 1) is the main pharmacologically active component of Cannabis sativa L. The reasons for developing products containing CBD as a “novel food” are related to the fact that this compound has been evaluated and found to provide benefits for different disorders (Millar et al. 2019), due to its polypharmacological effects (Castillo-Arellano et al. 2023). Cannabidiol possesses therapeutic activity in neuropsychiatric disorders (Manzoni et al. 2025), such as autism (Poleg et al. 2019) and schizophrenia (McGuire et al. 2018). CBD exhibits potential against neurodegenerative disorders, such as Alzheimer’s disease (Xiong and Lim 2021), Parkinson’s disease (de Fátima dos Santos Sampaio et al. 2024), and multiple sclerosis (de Fátima dos Santos Sampaio et al. 2024). The compound exerts beneficial effects in neurological disorders (Chayasirisobhon 2021; Singh et al. 2023), such as epilepsy (de Fátima dos Santos Sampaio et al. 2024) and neuropathic pain (Singh et al. 2023). Cannabidiol shows activity in Dravet syndrome (Miller et al. 2020) and Sturge–Weber syndrome (Kaplan et al. 2017), reducing the frequency of convulsive seizures (Szaflarski et al. 2018). Factors increasing the contamination of cannabidiol products with impurities Pure CBD and products with high levels of cannabidiol are regulated under Regulation 2015/2283 on “novel food.” Factors requiring increased quality control of cannabidiol products include (Wodak et al. 2002): 1) Use of chemotypes with high THC content for production; 2) Influence of environmental factors during incorrect storage conditions, which can initiate impurity production and the transformation of cannabidiol into toxic compounds, especially the psychoactive transdelta-9-THC (Park et al. 2022); 3) Appearance on the market of different legal and illegal products containing cannabidiol; 4) Increasing application of these products; 5) Self-medication; 6) Overdosing of cannabidiol content in products; 7) Inaccurate labeling of products with cannabidiol; 8) Risk of side effects resulting from drug–drug interactions with cannabidiol. The risk of high levels of impurities, structural analogues, or other cannabinoids (Fig. 1) in legitimate cannabidiol products is increased in cases of insufficiently strict control (McLaren et al. 2008). Cannabis contaminants limit the pharmacological use of cannabidiol due to the increased toxic effects of impurities (Huestis et al. 2019; Montoya et al. 2020). Botanically, chemotypes differ in their genetically determined CBD/THC ratio, which, according to EU Regulation 1307/2013, must be greater than 1 in cannabis products (UNODC 2007). The use of Cannabis sativa L. chemotypes with a high content of the most psychoactive trans-delta-9-THC is illegal. Its presence as an impurity in legal C. sativa L. products at high levels leads to toxic effects, and for this reason, only chemically standardized cannabis can be used (Bonini et al. 2018). Delta-9-THC acid A and delta-9-THC acid B do not exhibit psychotropic activity and are the main precursors of THC. Impurities resulting from oxidation of THC include CBN, delta-6aTHC, and delta-10a-THC. THC isomers include cis-del- Pharmacia 72: 1–13 3 ta-9-THC and delta-8-THC, the latter derived by acidic isomerization. THC intermediates include dihydrocannabinol and trihydrocannabinol. Delta-9-tetrahydrocannabivarin (D9-THCV) is another THC-type compound, mainly identified in hashish of Pakistani C. sativa L. (Hanus et al. 2016). THCA is a precursor of THC, formed by cyclization of cannabidiolic acid. More recent studies provide evidence that THCA is formed from cannabigerolic acid through oxidocyclization by the enzyme THCA synthase. Cannabigerolic acid is a precursor of CBDA, CBCA, and THCA, from which CBD, CBC, and THC are obtained by decarboxylation (Fellermeier et al. 2001). The biosynthesis of phytocannabinoids (Sirikantaramas and Taura 2017) can be influenced by various factors, such as cannabis taxonomy (Clarke and Merlin 2018), genetic factors (Hillig 2005), growth stage (Kovalchuk et al. 2020), and environmental factors (Park et al. 2022), including light (Eichhorn Bilodeau et al. 2019), temperature, water deficit, heavy metals (Park et al. 2022), and phytohormones (Burgel et al. 2020). These factors can contribute to the presence of high concentrations of important analogues in cannabidiol products, such as tetrahydrocannabinol, cannabinol, cannabichromene, cannabidivarin, cannabigerol, tetrahydrocannabidivarin, delta-8-tetrahydrocannabinol, cannabidiolic acid, cannabigerolic acid, and tetrahydrocannabinolic acid (Fig. 1), which can increase the risk of psychological side effects (Henquet et al. 2005). The development and implementation of specific analytical control procedures, including the identification and quantification of THC and other potential impurities, are necessary to prevent the distribution and use of products with psychoactive ingredient content exceeding 0.2%, the maximum permissible limit under Regulation (EC) No 327/2002. This ensures the safety of consuming authorized medicinal cannabis products containing cannabidiol and cannabis “novel foods” and limits the use of illicit products (McLaren et al. 2008). The appearance on the market of legal and illegal cannabidiol products without labeling, or with inaccurate labeling, combined with self-medication and increased product application, can lead to overdosing of CBD content. This poses serious health risks, as it can enhance side effects such as hepatic disorders, diarrhea, fatigue, and somnolence. Another health risk associated with increased cannabidiol in products is related to drug–drug interactions when used simultaneously (Huestis et al. 2019). This is connected to the influence of CBD on cytochrome P enzymes. Cannabidiol can modify enzyme activity in different ways. In some cases, it acts as an inhibitor of liver enzymes, which increases the toxic effects of other drugs due to decreased metabolism. In other cases, it can potentiate enzyme activity, decreasing the pharmacological effects of other drugs due to their increased metabolism (Huestis et al. 2019). Table 1. Cannabinoids and cannabinoid acids (Sirikantaramas and Taura 2017). Cannabinoids Cannabinoid acids Cannabidiol (CBD) Cannabidiolic acid (CBDA) Cannabidibutol (CBDB) Cannabidibutolic acid (CBDBA) Cannabidivarin (CBDV) Cannabidivarinic acid (CBDVA) Cannabidihexol (CBDH) Cannabidihexolic acid (CBDHA) Cannabidiphorol (CBDP) Cannabidiphorolic acid (CBDPA) Cannabichromene (CBC) Cannabichromenic acid (CBCA) Cannabiorcichromene (CBOC) Cannabiorcichromenic acid (CBOCA) Cannabichromevarin (CBCV) Cannabichromevarinic acid (CBCVA) Cannabicoumaronone (CBCM) Cannabicoumarononic acid (CBCMA) Cannabicyclol (CBL) Cannabicyclolic acid (CBLA) Cannabielsoin (CBE) Cannabielsoic acid (CBEA) Cannabigerol (CBG) Cannabigerolic acid (CBGA) Cannabigerovarin (CBGV) Cannabigerovarinic acid (CBGVA) Cannabinerol (CBNR) Cannabinerolic acid (CBNRA) Cannabinol (CBN) Cannabinolic acid (CBNA) Delta-8 tetrahydrocannabinol (D8-THC) Delta-8 tetrahydrocannabinolic acid (D8-THCA) Delta-9 tetrahydrocannabinol (D9-THC) Delta-9 tetrahydrocannabinolic acid (D9-THCA) Delta-9 tetrahydrocannabivarin (D9-THCV) Delta-9 tetrahydrocannabivarinic acid (D9-THCVA) Tetrahydrocannabutol (THCB) Tetrahydrocannabutolic acid (THCBA) Table 2. Phytocannabinoids in Cannabis sativa L. obtained from biosynthesis (Sirikantaramas and Taura 2017). Cannabigerolic acid Delta-9 tetrahydrocannabinolic acid Delta-9 tetrahydrocannabinol, cannabinol Cannabidiolic acid Cannabidiol Cannabichromenic acid Cannabichromene, cannabicyclol Cannabigerovarinic acid Delta-9 tetrahydrocannabivarinic acid Delta-9 tetrahydrocannabivarin Cannabidivarinic acid Cannabidivarin Cannabichromevarinic acid Cannabichromevarin Tsvetkova D et al.: Chromatographic methods for control of impurities in Cannabidiol products4 Cannabidiol impurities associated with its production CBD in crystalline solid form is obtained either by extraction from the inflorescences of C. sativa L. or by stereoselective synthesis (Citti et al. 2019). Cannabidiol from C. sativa L. is extracted with organic solvents from inflorescences after prior decarboxylation of the acidic precursor, cannabidiolic acid. Alternatively, cannabidiolic acid can be extracted and then decarboxylated by heating to obtain CBD. The extract must undergo a dewaxing step to remove waxes, after which cannabidiol is purified by chromatography or direct crystallization from the dewaxed extract using pentane or hexane (Citti et al. 2019). CBD in Epidiolex is extracted from hemp inflorescences and manufactured in accordance with good manufacturing practices. In the patent application “Use of cannabinoids in the treatment of epilepsy,” impurities in cannabidiol extracted from hemp inflorescences are described: 0.15% cannabidiolic acid, 1% cannabidivarin, 0.15% delta-9-THC, and 0.5% CBD-C4 (Guy et al. 2016). CBD-C4 is butyl-5’-methyl-2’(prop-1-en-2-yl)-1’,2’,3’,4’- tetrahydro-[1,1’-biphenyl]-2,6-diol, an analogue of CBD with a butyl side chain in place of the conventional pentyl chain of the resorcinol moiety (Citti et al. 2019). Pure CBD can also be produced by stereoselective synthesis. The synthetic route involves acidic condensation of p-mentha-2,8-dien-1-ol with olivetol. A cannabidiol isomer named “abnormal CBD” is another impurity (Baek et al. 1985). The advantage of the synthetic method is that CBD is purer and contains fewer impurities compared to cannabidiol obtained by extraction (Alvarez et al. 2023). However, the production of compounds from plant origins can introduce plant-based impurities, though it is more convenient from environmental, economic, and chemical perspectives due to the absence of toxic organic residual solvents (such as methanol, hexane, and o-, m-, and p-xylene) employed in chemical synthesis. Another advantage is that plant-derived products do not contain the toxic by-products resulting from chemical reactions (Citti et al. 2021). Considering these advantages and disadvantages of synthetic versus natural production, it can be concluded that, due to the formation of by-products in synthetic methods and the lack of harmful solvents in natural extraction, the most suitable process for industrial CBD production—both economically and environmentally—is the extraction of cannabidiol from cannabis inflorescences (Citti et al. 2021). Legal CBD products can contain various impurities, which can arise from both natural extraction and synthetic production processes. The impurities in products with cannabidiol can be summarized in the following groups: 1) Delta-9-THC: the main psychoactive impurity, derived from hemp due to contamination during extraction (Citti et al. 2021). 2) Cannabidivarin and cannabidibutol: common in natural CBD extracts (Citti et al. 2019; Brighenti et al. 2024). Figure 1. Cannabidiol analogues as potential impurities. Pharmacia 72: 1–13 5 3) Other cannabinoid impurities: cannabigerol, cannabichromene, cannabinol, cannabidiolic acid (Takashina et al. 2022), cannabidihexol and cannabidiphorol (Brighenti et al. 2024), and cannabielsoin (Schwarzenberg et al. 2022). 4) Chemical impurities: CBD-hydroxyquinone (HU33), an oxidation product formed during storage light exposure (Thomson et al. 2023). 5) Residual solvents: methanol, hexane, and o-, m-, and p-xylene (Gidal et al. 2024). Impurities such as delta-9-THC, delta-8-THC, and the (–)-trans enantiomer of CBD have been reported in both natural and synthetic cannabidiol. Impurities in hemp-derived CBD include cannabidivarin and cannabidibutol, along with minor amounts of cannabidihexol and cannabidiphorol. The impurity profile can help determine the origin of CBD using HPLC with UV detection, UHPLC-MS, and enantioselective HPLC for the (–)-trans enantiomer of CBD (Brighenti et al. 2024). Influence of storage conditions on contamination with impurities of cannabidiol products The importance of chemical standardization and strict control of cannabis extracts and other cannabis products on the market is heightened by the fact that the main impurity, delta-9-THC, and other compounds can increase in products due to environmental factors affecting plant growth or improper storage (Park et al. 2022). These impurities are linked to health risks caused by side effects. The main toxic effects of delta-9-THC are induced psychological reactions and increased pulse rate (Huestis et al. 2019). High temperature leads to degradation and oxidation of cannabidiol. CBD is formed from cannabidiolic acid through a temperature-dependent decarboxylation process. At 130 °C for 20 min, most CBDA converts to CBD. With further heating, CBD transforms into psychotropic THC isomers and oxidative products such as cannabielsoin. The degradation rate increases with temperature, particularly at 70 °C and under acidic conditions (pH 2) and oxidative stress (Seo et al. 2022). At e-cigarette temperatures (250–400 °C), CBD degrades significantly to THC and other cannabinoids (Caprioglio et al. 2020). Cannabidiol is more stable at lower temperatures. CBD stored at 4 °C shows minimal degradation over 4 weeks, whereas storage at ambient temperature (25 °C) leads to significant formation of degradation products, including cannabielsoin and CBD-hydroxyquinone (Schwarzenberg et al. 2022). Oxidation The degradation of cannabidiol under oxidative conditions involves oxidation by air oxygen and results in several degradation products. Using UHPLC with time-of-flight mass spectrometry (TOF-MS), it was demonstrated that storage of plant-based and chemically synthesized CBD for 4 weeks under stress conditions (40 °C, 75% relative humidity, dark) and ambient conditions (25 °C, 60% relative humidity, daylight) leads to an increase in the oxidative products of cannabidiol, including cannabielsoin and CBD-hydroxyquinone (HU-331), as well as other compounds such as hydroxy-CBE, hydroxy-CBD, and dihydroxy-CBD. In contrast, liquid formulations protected from light and stored at 4 °C for 4 weeks showed only very small increases in CBD oxidative products (Schwarzenberg et al. 2022). CBD-hydroxyquinone is the oxidative product and a hepatotoxic metabolite of cannabidiol (Thomson et al. 2023). Light There is a significant increase in the formation of delta-9THC under UV-B light in the range of 280–315 nm and low humidity (Park et al. 2022). CBD-hydroxyquinone is a common impurity in cannabidiol products. It has been described that CBD-hydroxyquinone can form a reactive intermediate in solution through photoisomerization. The rapid interaction of this intermediate with oxygen leads to the formation of multiple by-products. The purple color observed in CBD solutions after continuous storage under light is a result of the anions of these by-products (Thomson et al. 2023). Acidic environment The stability of CBD can be influenced by the solvent and pH. Under experimental conditions, cannabidiol degrades more in aqueous solutions than in ethanol. Acidic conditions (pH 2.0) accelerate degradation, leading to the formation of THC and other cannabinoids (Jeong et al. 2023). By UHPLC-HRMS, it was shown that cannabidiol samples stored for 3 months in the dark at room temperature yielded THC, likely due to carbon dioxide and water from the air increasing acidity. Cyclization of CBD to THC requires an acidic medium. Under an inert atmosphere, without humidity or carbon dioxide, no THC formation has been observed even at high temperatures (Citti et al. 2021). Degradation of cannabidiol and formation of THC rarely occurred at pH 5.0 after 24 h, even at 70 °C. Transformation of CBD to THC was observed at pH 3.5 and 30 °C over a short time, and the process was accelerated by lower pH (2.0) and higher temperatures. GC/MS analyses showed that the main impurities formed under acidic conditions were delta-9-THC, cannabichromene, and ethoxy-hexahydrocannabinol (HHC). Minor impurities, such as delta-8-THC, delta-10-THC, and 9-hydroxy-HHC, were also detected (Jeong et al. 2023). Increased temperature At increased temperatures, delta-9-THC and its isomers are produced through decarboxylation, hydration, isomerization, and oxidation. These transformed cannabinoids were identified by UHPLC-Q/TOF-MS (Seo et al. 2022). Increased temperature and acidic environment Major cannabinoids (CBD, CBN, and delta-9-THC) react more quickly at high temperatures and in acidic solutions. Minimum transformation of CBD, CBN, and delta-9- Tsvetkova D et al.: Chromatographic methods for control of impurities in Cannabidiol products6 THC occurs at low temperature, slightly to moderately acidic pH, and short processing times (Jaidee et al. 2022). CBD oil stored at 5 °C decomposes more slowly compared with oil at room temperature. Optimal conditions for stability and to minimize degradation include low temperature (around 5 °C) and slightly acidic to neutral pH (4–6) (Vlad et al. 2021). Analytical pharmacopoeial methods for the separation of cannabinoids CBDV and CBDB in the control of the purity of CBD products Samples obtained by extraction of hemp contain impurities in amounts below 0.5%. CBDV has been found in the range of 0.07–0.41% and CBDB in the range of 0.08–0.19%. These values vary considerably across samples, most likely because both the hemp variety and the manufacturing process influence impurity levels. CBDV has been detected in several hemp varieties in highly variable concentrations. CBDB is also present in some hemp varieties, but its concentrations have not been consistently determined due to the lack of a corresponding analytical standard. Since CBD is usually extracted from hemp by crystallization without further purification, the structural similarity of CBDV, CBDB, and CBD results in co-crystallization of all three compounds (Citti et al. 2019). Quality control of impurities is essential to guarantee the health safety of CBD products (Alvarez et al. 2023). Despite the increasing use of CBD in pharmaceutical and cosmetic products, there is no monograph in official pharmacopoeias that includes analysis of all known impurities. The only official protocol for solid or oily CBD products is a monograph in the German DAC/NRF codex, which has legal value only in Germany. This monograph describes physicochemical properties, identification methods (including thin-layer chromatography), and purity determination methods (HPLC-UV) (DAC 2015). The monograph also specifies impurities detectable in solid CBD—cannabinol, delta-9-THC, and delta8-THC—which, together with unspecified impurities, should not exceed 0.5%. However, it does not mention two important impurities found in hemp-derived CBD: cannabidivarin and CBD-C4. These can reach relatively high concentrations in final products, up to 1% for CBDV and 0.5% for CBD-C4 (DAC 2015). Although GMP procedures vary slightly between countries, they generally follow ICH guidelines. These require identification of organic impurities in active pharmaceutical ingredients when present at ≥ 0.1% and quantification when ≥ 0.15% for drug substances with a daily dose < 2 g/day. For substances with a daily dose > 2 g/day, impurities must be identified and quantified at ≥ 0.05%. Thus, both CBDV and CBD-C4 must be determined and reported in certificates of analysis of CBD products (Citti et al. 2019). Identification of CBD-C4 in cannabis samples or CBD products has been achieved only by mass spectrometric profiling, using GC-MS (Harvey 1976) or sorption ribbon extraction coupled with laser desorption ionization mass spectrometry (STELDI-MS) (Eiras et al. 2014). Cannabidibutol, the butyl analogue of CBD, is present in hemp and can also occur in acidic form as cannabidibutolic acid. Other reported impurities include tetrahydrocannabutolic acid (THCBA) and tetrahydrocannabutol (THCB) (Citti et al. 2019). Chromatographic methods for the separation of different cannabinoids for the control of the purity of CBD products For optimization of quality control, it is important to develop analytical procedures for testing CBD product purity using reliable, accurate, and sensitive methods for determining maximum permissible levels of tetrahydrocannabinol and other impurities. This ensures the safety of authorized medicinal cannabis products and “novel foods” containing cannabidiol and limits the use of illegitimate products. For separation and quantification of cannabinoid impurities in commercial products, the following High-Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC) methods have been reported (Nahar et al. 2020): 1) HPLC with UV detection (HPLC-UV) 2) HPLC with UV-photodiode array detection (HPLCUV-PDA) 3) HPLC-mass spectrometry (HPLC-MS, HPLC-MS/ MS) 4) Ultra HPLC with UV detection (UHPLC-UV) 5) Ultra HPLC with UV-photodiode array detection (UHPLC-UV-PDA) 6) Ultra HPLC-MS (UHPLC/MS, UHPLC-MS/MS) 7) Ultra HP supercritical fluid chromatography (UHPSFC) 8) GC with mass detection (GC-MS). 9) gas chromatography with flame ionization detection (GC-FID) Analytical methods for separation of cannabidiol and tetrahydrocannabinol in CBD products Analytical methods for the separation of cannabidiol and tetrahydrocannabinol in products include near-infrared spectroscopy (Deewatthanawong et al. 2023), HPLC with UV detection (Analakkattillam et al. 2022), HPLC-UVPDA (Burnier et al. 2019; Raslan-Jaramillo et al. 2024), HPLC-MS (Citti et al. 2016; Palazzoli et al. 2018; Lee et al. 2020), UHPLC-MS methods (Hwang et al. 2023), and GC-MS (Eloh et al. 2023). Near-infrared spectroscopy has been applied for the nondestructive measurement of CBD and THC (Deewatthanawong et al. 2023). An isocratic reverse-phase HPLC method has been developed for the quantification of CBD and THC in hemp oil products on a SOLAS C18 (150 mm × 4.6 mm × 5 μm) column with a flow rate of 1.5 ml/min, mobile phase acetonitrile:water = 75:25 v/v, and UV detection at λ = 214 nm Pharmacia 72: 1–13 7 (Analakkattillam et al. 2022). An isocratic HPLC method with photodiode array detection for the determination of cannabidiol and tetrahydrocannabinol in Cannabis sativa L. oil extract has been performed with a C18 column, a mobile phase of acetonitrile:water with formic acid = 80:20 v/v, and UV detection at λ = 208 nm for CBD and λ = 280 nm for THC (Raslan-Jaramillo et al. 2024). For the assay of CBD and THC, an HPLC coupled to a diode array and quadrupole time-of-flight mass spectrometry method has been evaluated (Citti et al. 2016). For the quantification of CBD and THC in dietary supplements, an HPLC-MS/MS method has been applied (Lee et al. 2020). It has been described that HPLC-MS with triple quadrupole is used for the determination of CBD and THC in rat whole blood after oral administration (Palazzoli et al. 2018). UHPLC-MS has been reported for the analysis of cannabidiol and tetrahydrocannabinol levels in hemp seeds, hemp seed oil, and hemp-based foods (Hwang et al. 2023). For the assay of CBD and THC content in C. sativa L., a GC-MS method with a column containing 5% phenylmethylpolysiloxane (30 m × 0.25 mm × 0.25 μm), an oven temperature of 260 °C, and helium carrier gas at a flow rate of 1 ml/min has been developed (Eloh et al. 2023). Methods for separation and determination of cannabidiol, tetrahydrocannabinol, and cannabinol in CBD products For the determination of CBD, THC, and CBN in CBD products, the following methods have been described: - HPLC-MS with electrospray ionization (ESI) (Citti et al. 2016; Citti et al. 2018; Hsu et al. 2021). - GC-MS in commercial hemp seeds and hemp seed oil (Ilias et al. 2005; Jang et al. 2020). A gradient HPLC method with MS detection in positive electrospray ionization (ESI) has been applied for the assay of CBD, THC, and CBN in hemp seed oil-based cosmetic products on an Xbridge BEH Shield RP18 column, using internal standards (THC-d3, CBD-d3, and CBN-d3) and a mobile phase of 10 mM ammonium formate:water:- methanol (Hsu et al. 2021). It has been reported that CBD, THC, CBN, and THCA in cannabis samples have been analyzed in CBD products by GC-MS after headspace solid-phase microextraction (Ilias et al. 2005). Methods for separation and determination of CBDV and CBDB in CBD products HPLC-UV and HPLC-MS have been developed for the identification and determination of CBDV and CBDB in CBD extracted from hemp. In the HPLC method with UV detection at λ = 228 nm, a Poroshell C18 column (30 mm × 150 mm × 2.7 μm) has been used, with a mobile phase composed of 0.1% formic acid in eluent A (water) and eluent B (acetonitrile), and a flow rate of 0.5 ml/ min (Citti et al. 2016). Methods for separation and determination of different cannabinoid impurities in CBD products Methods for the determination of cannabinoid impurities in cannabis plant extracts, CBD oil supplements, and CBD liquid products are presented in Table 3. HPLC methods with diode array detectors have been described for the determination of the following cannabinoids present in the cannabis plant: 1) CBD, CBDA, delta-9-THC, and delta-9-THCA—in the cannabis plant (Hädener et al. 2019). 2) CBD, CBN, CBDA, and delta-9-THC—in oil supplements, after liquid extraction with acetonitrile (Madej et al. 2021). 3) CBD, CBG, CBN, CBDA, CBGA, THC, and THCA— in the cannabis plant (de Backer et al. 2009). Table 3. Chromatographic methods for determination of cannabinoid impurities in CBD products. Cannabinoid impurities Mrthods CBD, THC HPLC-UV-PDA (Burnier et al. 2019) CBD, CBDA, THC, THCA HPLC-UV-PDA (Hädener et al. 2019) CBD, CBN, CBDA, THC HPLCUV (Madej et al. 2021) CBD, CBG, CBN, CBDA, CBGA, THC, THCA HPLC-UV (de Backer et al. 2009) CBD, CBC, CBG, CBL, CBN, CBDA, CBGA, 8-THC, 9-THC, THCAA, THCV HPLC-UV (Gul et al. 2015) CBD, CBC, CBG, CBN, CBDV, CBGA, CDBA, 8-THC, 9-THC, THCAA HPLC-UV (Hall et al. 2022) CBDV, CBDB HPLC-UV; HPLC-MS (Citti et al. 2016) CBG, CBGV, CBGB HPLC-UV, HPLC-MS (Tolomeo et al. 2021) CBD, CBG, CBN, CBDA, THC, THCA, THCV HPLC-MS (Merone et al. 2021) CBD, CBG, CBN, 9-THC, 11-hydroxy-THC, THCV, THCA HPLC-MS (Sobolesky et al. 2019) CBD, CBDA, THC, THCA-A HPLC-MS/MS (Meng et al. 2018) CBD, CBG, CBN, CBDA, CBGA, THCA HPLC-MS/MS (Takashina et al. 2020) CBD, CBG, CBN, CBDA, CBGA, 8-THC, 9-THC, THCA A, THCV UHPLC-UV-PDA, UHPLC-MS (Wang et al. 2017) CBD, CBC, CBG, CBN, CBDA, CBGA. 8-THC, 9-THC, THCA A UHPLC-UV (Deidda et al. 2021) CBC, CBG, CBCV, CBDB, CBDH, CBDP, CBDV, CBGV UHPLC-MS (Brighenti et al. 2024) CBD, CBC, CBG, CBN, CBDA, CBGA, 8-THC, 9-THC, THCA A UHPSFC –UV (Deidda et al. 2021) CBD, CBC, CBG, CBN, 9-THC GC-MS (Ahmed et al. 2021; Amirav et al. 2021) CBD, 9-THC, 8-THC, 9-THCA, 4-iso-9-THC, 8-iso-9-THC GC-MS (Shuda et al. 2024) CBD, CBG GC-FID (Baranauskaite et al. 2020) 4.8-epoxy-iso-THC, 8-hydroxy-iso-9-THC, cannabicitran (CBT) HPLC, GC (Radwan et al. 2023) Tsvetkova D et al.: Chromatographic methods for control of impurities in Cannabidiol products8 A gradient RP-HPLC with a UV detector and an internal standard, 4-androstene-3,17-dione, has been evaluated for the separation of cannabinoids in cannabis. After extraction from the plant with methanol:chloroform = 9:1, CBD, CBC, CBG, CBL, CBN, delta-8-THC, delta-9THC, CBDA, CBGA, THCAA, and THCV have been analyzed (Gul et al. 2015). HPLC-UV and HPLC-MS have been applied for the analysis of cannabigerovarin and cannabigerobutol (Tolomeo et al. 2021). For the simultaneous quantification of CBD, CBN, CBG, CBDA, THC, THCA, and THCV, an HPLC-MS method has been applied using a Hypersil Gold PFP (50 mm × 2.1 mm × 1.9 μm) column, with water:2 mM ammonium formate:0.2% formic acid as the mobile phase at a flow rate of 0.4 ml/min (Merone et al. 2021). An isocratic HPLC-MS/MS method with a triple quadrupole mass spectrometer for the simultaneous analysis of CBD, CBDA, THC, and THCA-A in oils has been described (Meng et al. 2018). The advantages of ultrahigh performance liquid chromatography using columns with a particle size of 2.7 μm are increased sensitivity, better separation, and decreased time for analysis. The advantages of ultrahigh performance supercritical fluid chromatography (UHPSFC) include lower amounts of organic solvents for the mobile phase and shorter times for analysis. For the separation of CBD, CBC, CBG, CBN, CBDA, CBGA, delta-8-THC, delta-9-THC, and THCA-A, the following methods have been described: 1) A gradient UHPLC-UV method on a Poroshell C18 column (150 mm × 2.1 mm × 2.7 μm) with a temperature of 30 °C, mobile phase A: water with 0.1% formic acid, and mobile phase B: acetonitrile with 0.1% formic acid, and UV detection at λ = 214 nm (Deidda et al. 2021). 2) A gradient UHPSFC method with the following conditions: analytical column Torus 1-AA (1-aminoanthracene) (100 mm × 3.0 mm × 1.7 μm) with a temperature of 40 °C, a mobile phase of methanol:water = 98:2 v/v, flow rate 1.5 ml/min, and UV detection at λ = 214 nm (Deidda et al. 2021). 3) UHPSFC with photodiode array and mass detection (Wang et al. 2017). UHPLC-MS and HPLC-UV have been described for the determination of cannabichromene, cannabichromevarin, cannabidibutol, cannabidivarin, cannabigerol, cannabigerovarin, and other minor impurities such as cannabidihexol and cannabidiphorol. An enantioselective HPLC has confirmed the (–)-trans enantiomers (Brighenti et al. 2024). For the GC separation of impurities CBC, CBD, CBG, CBN, CBDA, CBDV, CBGA, delta-8-THC, delta-9-THC, THCA, and THCV in cannabidiol products, the following types of stationary phases are used (Stefkov et al. 2022): 1) Dimethylpolysiloxane-silphenylene 2) Dimethylpolysiloxane-dimethyl-diphenyl 3) Non-polar stationary phases, such as 5%-diphenyl-dimethylpolysiloxane 4) Stationary phases with intermediate polarity, such as cyanopropyl-phenyl, dimethylpolysiloxane, and phenylmethylpolysiloxane. For the separation of CBC and CBD from other phytocannabinoids, 100% dimethylpolysiloxane columns (Stefkov et al. 2022) or two-dimensional GC with columns of different polarities, a medium-polar and a non-polar (Omar et al. 2014), are used. The detectors often applied in GC analysis of impurities in cannabidiol products are GC-FID, GC-MS with single quadrupole (GCQ), triple quadrupole (GC-QQQ), and quadrupole-time-of-flight (GC-Q-TOF) mass analyzers (Stefkov et al. 2022), as well as GC-vacuum UV (VUV) (Leghissa et al. 2018). In GC-MS analysis, electron impact ionization (EI) is preferred, while chemical ionization and atmospheric pressure ionization are used rarely (Stefkov et al. 2022). Abbreviations CBC cannabichromene CBCA cannabichromenic acid CBCM cannabicoumaronone CBCMA cannabicoumarononic acid CBCV cannabiorcichromene CBCVA cannabichromevarinic acid CBD cannabidiol CBDA cannabidiolic acid CBDB cannabidibutol CBDBA cannabidibutolic acid CBDH cannabidihexol CBDHA cannabidihexolic acid CBDP cannabidiphorol CBDPA cannabidiphorolic acid CBDV cannabidivarin CBDVA cannabidivarinic acid CBE cannabielsoin CBEA cannabielsoic acid CBG cannabigerol CBGA cannabigerolic acid CBGV cannabigerovarin CBL cannabicoumaronone CBLA cannabicyclolic acid CBN cannabinol CBNA cannabinolic acid CBNR cannabinerol CBNRA cannabinerolic acid CBOC cannabiorcichromene CBOCA cannabiorcichromenic acid D8-THC delta-8 tetrahydrocannabinol D9-THC delta-9 tetrahydrocannabinol D8-THCA delta-8 tetrahydrocannabinolic acid D9-THCA delta-9 tetrahydrocannabinolic acid D9-THCV delta-9 tetrahydrocannabivarin D9-THCVA delta-9 tetrahydrocannabivarinic acid GC gas chromatography FID flame ionization detection HPLC high-performance liquid chromatography Pharmacia 72: 1–13 9 PDA photodiode array detection MS mass spectrometr Q-TOF quadrupole-time-of-flight SFC supercritical fluid chromatography THCB tetrahydrocannabutol THCBA tetrahydrocannabutolic acid Conclusion The presence of a high content of impurities, structural analogues, or other cannabinoids in legitimate products with cannabidiol is enlarged in cases of incorrect storage conditions. The appearance on the market of different legal and illegal products containing cannabidiol, self-medication, and insufficiently strict control of these products lead to enhanced health risks for people taking them. Use for production of products of botanical chemotypes with high THC, and the influence of environmental factors, such as light, temperature, and water deficit during incorrect storage conditions, can initiate the production of impurities and the transformation of cannabidiol to its toxic impurities, especially the most psychoactive trans-delta-9-tetrahydrocannabinol. Inappropriate storage conditions can be a reason for the presence in cannabidiol products of high concentrations of impurities, such as cannabinol, cannabigerol, cannabichromene, cannabidivarin, delta-8-tetrahydrocannabinol, tetrahydrocannabidivarin, cannabidiolic acid, cannabigerolic acid, tetrahydrocannabinolic acid, and other impurities, which can increase the risk of psychological and other side effects. In order to avoid these health risks, increased and strict quality control of medical cannabis products containing cannabidiol and of “novel foods” from cannabis is needed. This aim can be realized by the application of reliable analytical procedures for testing the purity of CBD-containing products. For optimized quality control, an important goal is the development and application of analytical procedures for testing the purity of CBD-containing products. The most sensitive methods for the quantitative determination of the maximum permissible content of tetrahydrocannabinol and other impurities in cannabidiol products are HPLC-UV, HPLC-UV-PDA, HPLC-MS, HPLC-MS/ MS, UHPLC-UV, UHPLC-UV-PDA, UHPLC-MS, UHPLC-MS/MS, UHPSFC, GC-MS, and GC-FID. The use of HPLC and GC methods can be recommended as appropriate and reliable methods for increased quality control, as they provide high accuracy and precision. The application of HPLC and GC methods would contribute to ensuring the safety of authorized medical cannabis products containing cannabidiol and “novel foods” from cannabis, as well as limiting the use of illegitimate products with a high content of impurities that do not meet the requirements under European regulations. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statements The authors declared that no clinical trials were used in the present study. The authors declared that no experiments on humans or human tissues were performed for the present study. The authors declared that no informed consent was obtained from humans, donors, or donors’ representatives participating in the study. The authors declared that no experiments on animals were performed for the present study. The authors declared that no commercially available immortalized human or animal cell lines were used in the present study. Use of AI No use of AI was reported. Funding This study is financed by the Medical University – Sofia, Bulgaria. Grant 2024 Research Project for funding research projects at MU-Sofia, 8302/27.11.2023, Contract No. 144/29.05.2024. Author contributions All authors have contributed equally. Author ORCIDs Dobrina Tsvetkova https://orcid.org/0000-0002-4284-8101 Ivanka Pencheva https://orcid.org/0000-0002-8974-1694 Lily Peikova https://orcid.org/0000-0002-8030-3479 Data availability All of the data that support the findings of this study are available in the main text. References Abioye A, Ayodele O, Marinkovic A, Patidar R, Akinwekomi A, Sanyaolu A (2020) Δ9-Tetrahydrocannabivarin (THCV): a commentary on potential therapeutic benefit for the management of obesity and diabetes. Journal of Cannabis Research 2(1): 6. https://doi.org/10.1186/ s42238-020-0016-7 Ahmed AQ, Noshad D, Li PC (2021) Quantification of cannabinoids in cultivars of Cannabis sp. by gas chromatography–mass spectrometry. Chromatographia 84(8): 711–717. https://doi.org/10.1007/s10337021-04060-9 Alvarez JC, Pelissier AL, Mura P, Goullé JP (2023) Cannabidiol (CBD): Analytical and toxicological aspects. Therapie 78(6): 639–645. https://doi.org/10.1016/j.therap.2023.02.006 Amirav A, Neumark B, Margolin Eren KJ, Fialkov AB, Tal N (2021) Cannabis and its cannabinoids analysis by gas chromatography–mass