In this work, two different UHPLC-UV methods were applied for cannabinoid analysis based on national pharmacopeial methods (OMC, USP). They differed in the chromatographic conditions, particularly column properties and mobile phases (described in section 2. Material and Methods). Use of these methods resulted in different cannabinoid separations, and therefore possible quantification of analytes. The main disadvantage of using the OMC chromatographic conditions (Method I) was the coelution of CBG and CBGA. Thus, their concentrations in the sample could only be expressed as the sum. However, this method succeeded in separating CBNA and ∆⁸-THC, which was not possible by the USP method (Method II). CBNA monitoring was necessary for the ointment extracts due to the possible degradation of ∆⁹-THCA during storage. Therefore, Method I was preferred for the extract analysis. On the other hand, CBG and CBGA resolution was essential for detailed chemical characterization of cannabis chemotype inflorescences, which was achievable by Method II. This dual approach allowed selection of the best method according to the sample properties and the required compound quantification.

Analysis of decarboxylation was not the primary objective of this paper. However, it became a stimulus for discussion due to many inconsistencies between previous studies. Baratta et al. (2019) tested the time for cannabinoid extraction (30, 60, 120 min) into olive oil and the influence of two decarboxylation processes of the raw material in comparison with no preheating. For warm extraction, they used a boiling water bath. Based on their results, an extended time of extraction was deemed not necessary (120 min) and they recommended an optimal extraction time of 60 min under the given conditions. In the present study, a 30 min extraction time under an infrared lamp was found to be sufficient for olive oil and Vaseline, with no need for lengthy preparation. Moreover, in Baratta et al.’s study, after 120 min heating of raw (non-decarboxylated) cannabis in the water bath, the acidic forms still prevailed over the decarboxylated ones, similarly to the current study. Comparing two different decarboxylation conditions, higher levels of decarboxylated forms were achieved when conditions of 140°C for 30 min were applied compared to a lower temperature for a longer period (115°C for 40 min) (Baratta et al., 2019). Romano and Hazekamp (2013) heated chemotype I cannabis at 145°C for 30 min, which resulted in complete decarboxylation of ∆⁹-THCA. However, it should be noted that such a high temperature was probably beyond the evaporation point of ∆⁹-THC (Wang et al., 2016). In the present work, preheating the raw material at 121°C for 30 min followed common practice in Czech pharmacies. In addition, Baratta et al. (2019) recommended homogenization and spreading of the material in a thin layer for uniform heating (maximum 5 mm, preferably 1–2 mm), as well as the Italian monograph (maximum thickness of the cannabis layer 1 cm) (Società Italiana Farmacisti Preparatori, 2016). In the present work, trimmed inflorescences were heated without pre-homogenization. These findings indicate that temperature and pre-homogenization of the plant material, with emphasis on layer thickness, are key aspects for effective decarboxylation.

In light of the above observations, the decarboxylation process in cannabis flowers should be optimized and studied to achieve higher yields of the decarboxylated forms. The process of decarboxylation optimization could be problematic in the case of chemotype II samples since they contain both major cannabinoids. ∆⁹-THCA decarboxylates more rapidly than CBDA, potentially increasing levels of CBN, which is restricted in medical cannabis products. Previous kinetic studies reported unexplained losses of reactants or products during heating in the case of CBDA decarboxylation (Wang et al., 2016; Citti et al., 2018; Moreno et al., 2020). Despite this, acidic cannabinoids may also be desirable compounds in dermatological applications due to cannabinoid synergistic interactions. Indeed, the entourage effect has been extensively discussed and studied (Russo, 2011; Koltai and Namdar, 2020),. For example, cannabinoid-cannabinoid synergy resulted in an increased plasma concentration of CBDA when the extract was administered orally in comparison with single molecule administration (Anderson et al., 2021).

To the best of our knowledge, our study is the first to report the extractive capabilities of a wide range of POBs for phytocannabinoids. Casiraghi et al. (2020) studied the influence of vehicle-related aspects on the skin permeation process. However, although they tested different formulations (liquids, semisolids), they only investigated pure CBD and not extracts or other cannabinoids. Majority of the studies have aimed to the development of innovative methods for the preparation of cannabis galenic oil formulations (Società Italiana Farmacisti Preparatori, 2016; Casiraghi et al., 2018; Baratta et al., 2019; Ternelli et al., 2020; Baratta et al., 2021) and their quality assessment (Bettiol et al., 2019; Dei Cas et al., 2020), or cannabinoid extraction using lipid-based vehicles (Aguirre et al., 2023). Nevertheless, Aguirre et al.’s work did not focus on stability testing of the prepared formulations. In the present study, the three most commonly used medical cannabis chemotypes were investigated. All tested POBs showed, to some extent, extractive capabilities for cannabinoids. The highest extraction efficiencies (EE, %) were observed for olivae oleum and Synderman^®, whereas the lowest were for cream bases. However, the lower content of cannabinoids in other POBs extracts may have been due to differences in the filtration process, i.e., wetting of the cotton gauze and glassware by the POBs.

The optimal time for quantitative cannabinoid extraction induced by heat was shown to be 30 min since a decrease in cannabinoid content (specifically ∆⁹-THCA) in olivae oleum and vaselinum album was observed at longer time (up to 120 min). Furthermore, re-extraction of the plant material used in the first 30 min extraction was examined. As a result, the ∆⁹-THC levels were below the LLOQ (<0.025%). Thus, adding more ointment base did not increase the cannabinoid extractability into olivae oleum or vaselinum album. Levels of CBN (degradation product of THC), which are limited by implementing decree (Ministry of Health and Ministry of Agriculture, 2015), were below the method LLOQ in all extracts, consistent with the findings of a previous study of olive oil extracts (Citti et al., 2016) and Baratta et al. (2019), where CBN was present in minimal amounts in the oils.

The Italian monograph stipulates a 1:10 ratio (1 g of cannabis/10 mL of olive oil) for the preparation of cannabis oil extracts in pharmacies (Società Italiana Farmacisti Preparatori, 2016), which corresponds to preparation of a 10% extract (w/v). By adhering to this ratio, the extraction rate of total CBD (sum of CBDA and CBD) was about 78% in olive oil (Citti et al., 2016). In the present work, 5% extracts (w/w) with approximately 70% of the extractive yield of ∆⁹-THC and CBD in olive oil extracts were prepared since this concentration is generally formulated in Czech pharmacy practice. Similarly, Citti et al. (2016) concluded that the stability of cannabinoids in solution (olive oil and 96% ethanol) was dependent on storage temperature (8°C versus 25°C). Furthermore, they observed higher decomposition of ∆⁹-THC compared to other cannabinoids, matching our obtained data.

In the present study, cannabinoid extraction from homogenized plant material into alcohol solvents (96% ethanol and isopropanol) at laboratory temperature was also investigated. A procedure that could be routinely performed in a pharmacy with basic equipment was compared with ultra-sonic extraction, which generally results in higher yields. However, the ∆⁹-THC recoveries were comparable for both preparation methods and extraction solvents. The extraction efficiency for cannabinoids was almost 100% for both tested alcohols. The main advantage of our method is that there is no high loss of biologically active molecules during the extraction and filtration process. Furthermore, cannabis tinctures could be easily incorporated into suitable alcohol-tolerant ointment bases and titrated to the required cannabinoid concentration. In this case, an isopropanol tincture would be preferable as it is less drying to the skin compared to ethanol. On the other hand, isopropanol is only applied externally, whereas ethanol tinctures and extracts could also be used orally or via inhaler. Moreover, propylene glycol, which can also be used for oral drug administration, showed relatively high ∆⁹-THC extraction efficiency (70%) (Table 3). However, Citti et al. (2016) observed rapid decomposition of cannabinoids in 96% ethanolic extract during storage. Therefore, the tinctures should be mixed with an appropriate POB immediately after preparation.

During preparation and storage, changes in the structure of some POBs were observed, especially for AmiFarm^®, Pentravan^® and AquaNeoFarm^® unguentum. Therefore, these ointment bases are not recommended for the preparation of extracts by the protocol proposed here. Besides this experiment, in our laboratory, the incorporation of cannabis extracts (supercritical fluid CO₂ extract (SFE), evaporated alcohol extract) into Ambiderman (AmiFarm^®) has been tested. The result was a homogeneous cream with no observed disintegration or change in structure even during storage for months at low temperatures (refrigerator). However, the stability of cannabinoids in the ointment was not monitored. This cream base contains a water phase consisting of carbomer gel (polyacrylic acid) which could affect the stability of active ingredients. In the current study, a rapid decrease in ∆⁹-THC and acidic cannabinoid forms was observed in AmiFarm^® extracts stored at laboratory temperature or in a refrigerator. Since the Ambiderman cream contained the highest amount of water of all the tested POBs (70% of water), a high water content could have a negative impact on THC stability via hydrolysis. Moreover, the stability of galenic preparations of Ambiderman depends on the concentration of incorporated compounds and resulting pH. Conversely, CBD was stable in the cream extracts at both temperatures during the monitoring time. These observations suggest that cannabinoids are variably stable in ointment bases depending on their physical and chemical properties.

The Pentravan^® cream was expected to be a promising ointment base since it penetrates through skin layers due to its chemical composition and could be used for transdermal administration of cannabinoids into the bloodstream, which is desirable for patients who cannot inhale or take cannabis orally. However, its structure disintegrated during the heated extraction and storage. Furthermore, the cream formulation was not easy to filter through cotton gauze to remove the plant particles. In future work, incorporation of a cannabis extract (SFE, ethanolic, oil) into the cream could be tested with monitoring of its stability.

The law novelization and recent extension of the Czech implementing decree (Ministry of Health and Ministry of Agriculture, 2015; Ministry of Health and Ministry of Agriculture, 2022) have allowed prescription of medical cannabis extracts for treatment, which could potentially be used for the preparation of galenic formulations. This could facilitate the preparation of ointments because with a known concentration of substances in the extract, it would be easier to dilute the cannabinoid content and standardize the final product. Besides dermatological applications, some algesiologists have expressed interest in the transdermal administration of medical cannabis for pain therapy. Nevertheless, extracts are not a commonly available drug form in Czech medical practice. Moreover, physicians and patients have experienced a shortage of medical cannabis flowers in the Czech market in the past. A similar situation might also be expected for extracts in the future. Therefore, it is essential to have a protocol for the preparation of cannabis-based galenic formulations from raw plant material (inflorescence) that could be easily employed by pharmacists. In general, the usual preparation procedures in pharmacies are unnecessarily time-exhaustive. The preparation procedure proposed in this work is simple and less time-consuming and could be applied routinely even in the absence of medical cannabis extracts.

This research paper is primarily focused on the phytocannabinoid extraction and stability in cannabis-based galenic formulations for topical application. We foresee an opportunity for future related studies that should be dedicated to terpenoid evaluation as these compounds are also the main active constituents of cannabis with pharmacological effects including antimicrobial or anti-inflammatory activities (Hanuš and Hod, 2020). Moreover, the presented results on the stability of the active compounds may be beneficial for the pharmaceutical practice, since medical cannabis is prescribed with a shelf life of 1 month with possible prolongation depending on the physical and chemical properties of the prepared galenic formulations (Czech Pharmacopoeia, 2017-Appendix, 2022). However, the assays described in Pharmacopoeia should be performed to assess the quality of the prepared formulations, including microbiological safety.