2.1. Cannabinoids Content
Current ambiguous all-purpose regulations allow huge variations in the quality and safety of the CBD-based preparations available on the market and clear labelling regarding the exact concentration of CBD is not yet mandatory. Our results demonstrate that CBD concentrations were not always in accordance with producer information (Table 1
). As a matter of fact, nine out of 14 tested samples presented concentrations that differed notably from the declared amount, while the remaining five preserved CBD levels within optimal limits (the variation was less than 10%). Our analysis also revealed that two preparations (particularly oils 8 and 10) exhibited higher levels of CBD than those specified by producers, while in another two (samples Oil_3 and Oil_14) the CBD content was far inferior to the stated values. In one sample, the theoretical CBD concentration was not indicated on the label and therefore values obtained could not be compared to the producer’s statement. Taken together, the results highlighted the extreme variability of the commercialised CBD oil preparations, justifying the need for stricter regulations/controls. Precise information regarding the composition of each lot that is available on the market is crucial for consumers who have to be able to properly adapt the recommended dose to the available/purchased preparation [9
]. These results are in agreement with those obtained from a preliminary study toward the labeling accuracy of cannabidiol extracts preparations from products available on the US market. In the tested products, 26% contained less CBD than labeled, which could negate any potential clinical response [30
]. The over labeling of CBD products in the study was similar in magnitude to levels that triggered warning letters to 14 businesses in 2015–2016 from the US Food and Drug Administration suggesting that there is a continued need for federal and state regulatory agencies to take steps to ensure label accuracy of these consumer products.
Although CBD is a principal constituent of the examined cannabis oil extracts, the original plant is only capable of producing its acid form, cannabidiolic acid (CBDA). Decarboxylation of CBDA catalysed by thermal exposure during extraction conditions leads to the conversion of CBDA to the CBD as the corresponding decarboxylated (neutral) counterpart. Therefore, the determination of CBDA is important in order to evaluate the CBDA decarboxylation rate and effectiveness of the reaction during the extraction process [31
]. Interestingly, looking through the web sites of the CBD oil producers enrolled in this survey, it can be found that some of them published an analytical report in which only the total CBD content as the sum of CBD + (0.877 CBDA) is reported. This is quite problematic as the biological effects of the neutral and acidic forms are remarkably different [5
]. Generally, expressing the CBD content as a sum of the acidic and neutral forms is conditioned by the analytical method applied. Concretely, it occurs when gas chromatography (GC), one of the most commonly used analytical platforms for cannabinoid analysis, is used [31
]. It involves the heating of the sample at high temperature in the injector prior to the chromatographic separation that leads inevitably to the decarboxylation of the cannabinoid acids. Therefore, the analytical result is the sum of the acid and neutral forms. The GC method is still officially employed by the authorities for the determination of cannabinoids, but obviously is unsuitable. A few research groups continue to suggest that an accurate cannabinoid profile should be evaluated by determining the acid and neutral forms separately [12
]. Results obtain in this study confirms this necessity. Employing the LC-HRMS technology, we were able to distinguish the acidic form from neutral CBD, and to examine the wide concentration range. As can be seen in Table 1
, in the majority of the samples the CBDA concentration was found to be negligible compared to the amount of CBD (for example, samples Oil_4 and Oil_15). On the contrary, there were a few samples with a significant amount of CBDA. A striking example is Oil_3, in which the CBDA content exceeded CBD, and only the sum of both forms justified the CBD percentage declared by the producer. Furthermore, it is evident that the label concentration of CBD in Oil_12 is reached only when the sum of both forms is considered, bearing in mind the significant amount of CBDA.
Nevertheless, all producers underline that their manufacturing methods yield the so called full spectrum extract, which means that hemp extracts contain different phyto-cannabinoids, including THC, CBN, CBG, THCA, CBGA and others, depending from cannabis strain and extraction method. In order to achieve full-spectrum in a hemp extract, the profile of bioactive compounds that a plant flower contains must be transferred into the extract itself without compromising any aspect of the profile.
In comparison with previous works available on cannabis oil [9
] we employed a HRMS method that provided more complete information regarding the cannabinoids profile and amount in the oil composition. Actually, besides CBD as a principal cannabinoid, we were able to detect and to quantify the six most significant cannabinoids, including the essential ones (THC, THCA and CBDA) along with quantification of CBN, CBG and CBGA. The obtained results clearly show that 12 out of 14 samples contained THC which is attention-grabbing because of its potential intoxicating activity. The THC content showed the considerable variability in the analysed samples, but was mainly at the levels describable as low (0.2%) [17
]. Only one among all THC-positive sample (Oil_6) contained a considerable amount of THC (0.35%), which is matter of concern because the manufacturer declared the product to be THC-free. This result highlights the importance of also specifying the amount of THC or any another intoxicating cannabinoid present in commercialised CBD oils.
CBN was quantifiable in the vast majority of samples (except Oil_14). Its detection is of great importance as it is not considered to be a natural cannabinoid but rather an artefact formed by THC oxidation during plant aging, by use of an inadequate extraction procedure or inappropriate storage conditions [32
]. Therefore, its determination may assist in the evaluation of the quality of CBD oils with regards to the raw plant material used, extraction method applied and storage. For example, in the sample Oil_13 the quantity of the CBN was more than twice the amount of THC. Considering CBN as a degradation product of THC, it would be better to think through the sum of THC+CBN as a relevant parameter for the evaluation of initial THC concentration in the oil extract. In addition, CBN, though much less psychoactive than THC, express sedative effects [33
] which is why its content should be indicated on the label along with THC. It is well known that THC derives from the decarboxylation of tetrahydrocannabinolic acid (THCA) [20
], and this is the reason why the amount of THCA was quantified in this study. Our results did not reveal any significant presence of either THCA (Table 1
) or cannabigerolic acid (CBGA) which is the precursor of the all other cannabinoid acids. CBGA gives by decarboxylation cannabigerol (CBG) that either was completely absent or present in minor quantities. The quantification of CBGA and CBG did not turn out to be imperative, but their presence could serve as a confirmation that the oil sample contains a natural, full spectrum cannabis extract. Furthermore, employing the retrospective analysis, several other minor “untargeted” compounds were detected by means of the Orbitrap (Thermo Fisher Scientific, San Jose, CA, USA) ®
analyser. Among others (data not shown) it is important to highlight the persistent occurrence of CBDV in all analysed samples. Figure 1
shows the fragmentation pattern of CBDV and CBD. Bearing in mind that this compound has expressed significant physiological activity [33
] and that accompanies the CBD as its analogue, it should be included in any quality evaluation of full spectrum CBD oil preparations. Besides, we noticed that when the hemp seed oil was used as matrix, the signal of CBDV augments notably, which means that maybe one portion of CBDV derives from hemp oil, not from flower extract [34
Bedrolite oil extract (Oil_1) obtained by a recently published procedure [12
], is a defined galenic formulation that has been used for distinct therapeutic purposes. It was included in this study as a “reference material” from a well-defined starting material (cannabis plant variety) and made using a standardized/authorized preparation procedure. There are at least two reasons to use the cannabinoid profile of Bedrolite oil extract as a reference point in the evaluation of CBD-rich hemp oils. Firstly, it can be considered as a full-spectrum extract
that preserves the natural ratios of cannabinoids, any impurities that can compromise the experiments should be absent. Secondly, many consumers tend to replace galenic oil preparations (such Bedrolite oil extract) with CBD-rich hemp oil extract, due to the fact that a medical prescription is required for the former. Our study revealed that Bedrolite oil extract contains 0.8% of CBD. This is in agreement with theoretical percentage (0.9%) that should be found in the Bedrolite oil extract: the inflorescence contains 9% of CBD and the dilution ratio during the extraction is 1:10. However, as regards the cannabinoids profile (Table 1
), it is evident that the quantities of CBDA, THC, THCA and CBGA are inferior compared with CBD-rich hemp oil extracts. These data are of great importance as they highlight the reduced concentration of all cannabinoids in Bedrolite oil extract compared to CBD hemp oil extract.
The reasons for all the abovementioned variations between examined samples are numerous and multiple. The final composition of CBD-rich hemp oil extracts depends on the chemotype and quality of the industrial hemp used, but it is also conditioned by the extraction method applied. Unfortunately, not all producers indicate the extraction method used. Only four declared the use of supercritical CO2
fluid extraction, which is shown to be the method of choice in that the low temperature and inert atmosphere results in higher CBD yields [18
]. However, the main drawback of this technology is its high cost, and it is reasonable to assume that solvent extraction is also used for the inexpensive industrial processing. However, it is questionable if this is a correct choice for a product for human consumption because residual solvents (typically hexane, ethanol, isopropyl alcohol, toluene, benzene, xylene and acetone) may contaminate the final product [32
]. Without having complete information on the methods of CBD oils preparation, we investigated the occurrence of the most frequently used extraction solvents as solvent residues. Our analysis revealed the sporadic incidence of acetone (Table 2
, ketones section) that is more probably present as a lipid oxidation product rather than as a true residual solvent. Nevertheless, the presence of some volatile compounds that might be considered as problematic impurities from solvents residues was detected (Table 2
, miscellaneous section). Namely, the samples Oil_4 and Oil_6 showed the presence of 1,3-dimethylbenzene while in the sample Oil_3, 1,2,4-trimethylbenzene was detected. Those aromatic compounds were not present in galenic preparation (Oil_1).
2.2. Volatile Fingerprint: Terpene Profile and Secondary Lipid’s Oxidation Products
Terpenes and cannabinoids share biosynthetic pathways and, in fact, cannabinoids are terpenophenolic compounds. In Cannabis
plants, terpenes are secreted and stored together with cannabinoids in glandular trichomes. Considering this fact also in relation to recent evidence of the synergic action of terpenes and cannabinoids (“entourage effect”), a comprehensive survey of terpenes is fundamental for the evaluation of cannabis oil preparations as dietary supplement with therapeutic applications. Complete data concerning the terpenes profile are summarized and reported in Table 2
. Overall, up to 110 volatile compounds composed the volatile fingerprint, including 48 terpenes that are further divided into classes as presented in Figure 2
. The sample Oil_6 contained an extremely high amount of terpenes compared with all other samples. α-Pinene, β-myrcene and limonene are the most concentrated terpenes in this preparation, which points toward the extremely efficient extraction method applied. Samples 5, 12 and 14 contained a distinct number of various terpenes, although in far lower concentration compared to sample Oil_6. Apparently, these formulations were obtained by an extraction process able to preserve naturally occurring terpenes profile from initial Cannabis sativa
plants, as their terpene profile is in accordance with those already published in literature [12
]. Similarly, Bedrolite oil extract (Oil_1) contains various terpene structures, reflecting the initial plant profiling. A particular profile is observed for the sample Oil_4 that showed a different terpene fingerprint compared to the other oils as it predominantly contains monoterpene subclass molecules. It has previously been demonstrated how the preparation method used for the production of cannabis extracts is able to affect the presence of different terpenes [18
], and this is most probably reason of such a specific terpene profile found in Oil_4.
β-Myrcene and limonene accompanied by β-ocimene and trans
-caryophyllene were found in all samples, but in a much reduced amounts compared to sample 6, and their concentration differed greatly from sample to sample. α-Pinene and β-pinene were identified in the majority of samples, excluding samples Oil_3 and Oil_13. This remains unclear considering that the two pinenes are quite balanced within the different Cannabis
varieties representing around the 10% of the terpenes group and not exceeding 15–20% [20
]. The occurrence of α-terpinolene in all samples (except Oil_2 and Oil_13), might be important as this compound was suggested as a genetic marker for distinguishing two important gene pools for breeding low-THC varieties [35
]. Sample Oil_14 was particularly rich in trans
-caryophyllene followed by α-humulene. The dominance of those two sesquiterpenes over the other terpenes detected in this preparation may indicate the geographic provenience of the starting Cannabis sativa
material, and as a matter of fact, the producer specified the mountain region where the plant was cultivated.
It is also important to notice that sample Oil_15 was almost completely deprived of a terpene fraction aside from the traces of the main three mentioned above. This might indicate an inefficient/inadequate processing of the starting materials or even artificial addition of CBD to the oil matrix, as some essential cannabinoids were also missing (Table 1
). In addition, extremely low terpene content was established for samples 11 and 13, while the remaining samples had total terpene contents in the range between 174 and 1367 μg/g, with pronounced variation in composition from sample to sample. Nevertheless, qualitative and quantitative differences observed in the chemical profiles of terpene fractions are conditioned by many factors such as: hemp variety, cultivation and environmental conditions harvest time and post-harvest conditions, storage and drying of raw plants, extraction procedure applied, matrix used and finally storage of the oil formulation.
Besides hydrocarbon terpenes, oxygenated terpenoids such as linalool and α-terpineol, were found in some preparations (Table 2
, Figure 2
) with a notably high concentration again in sample Oil_6. Those compounds correspond to secondary photooxidation products of the initial terpenes. In the presence of light and singlet oxygen, terpenes are also known to undergo photooxidation leading to the formation of allylic hydroperoxides [35
In addition to the terpene compound profiles that accounted for more than 90% of the detected volatile constituents of the oils, it was possible to note the presence of other organic compounds commonly found in natural extracts such as esters, alcohols, aldehydes and ketones (Table 2
). Only a few low chain esters could actually be identified, with ethyl acetate dominating in sample Oil_3. Its presence is most likely to be due to the preparation method and could also be considered indicative of potential adulteration.
On the another hand, the detection of aldehydes and ketones suggests the initiation of lipid peroxidation of polyunsaturated fatty acids (PUFA) in the oils used as a matrix, as demonstrated in our previous work concerning the observed trends of these compounds during storage of macerated Cannabis
-derived oils [12
]. It is well documented that peroxidation of PUFA leads to the formation of a well-defined series of aldehydes and ketones such as nonenal, hexanal and pentanal, 2-heptenal, especially during storage. The rate of formation of lipid oxidation products depends strictly on several factors, among which the most important are the preparation method temperature, fatty acid composition of the oil in which Cannabis
extract was dissolved and the storage conditions (storage temperature) as recently demonstrated in a study [12
]. These parameters are crucial to define the ultimate characteristics of the final products as evidenced also by the color of the samples (Figure 3
). Other volatile decomposition compounds frequently encountered include 2-hexenal, 2-octenal, 2,4-nonadienal, 4,5-dihydroxydecenal [37
], some of which also appeared to be present in some of our samples.
For the CBD oils analyzed in this study, tree different oil typologies were used: medium chain triglycerides (MCT oil, one sample), olive oil (six samples) and hemp seed oil (eight samples). It is worth emphasizing that sample Oil_4 was almost completely deprived of lipid oxidation products (Table 2
, Figure 4
). This preparation was the only one prepared in MCT oil, which means that this kind of matrix is less susceptible to oxidative degradation than the olive or hemp seed oils declared as matrices for other preparations enrolled herein
As far as olive oil is concerned, is often used by producers as it has a strong nutritional potential, being rich in the polyunsaturated fatty acids. Moreover, FU oil (pharmaceutical grade olive oil) is used for the preparation of CBD galenic formulations [9
] as it was performed for Bedrolite oil extract.
Regarding the hemp seed oil used as a carrier for dissolving the CBD extract (hence the term “CBD hemp oil”) some clarifications regarding this kind of preparation are indispensable because misconceptions that may confuse the final users of this preparations still exist. “CBD oil” expression is typically limited to extracts in oil of flowering buds and not stalks, fibers, or seeds of each Cannabis sativa
L. variety. Hemp seeds do not contain any cannabinoids, but their contact with the resin secreted by the epidermal glands located on flowers, and leaves and/or a bad selection of the bracts of the perigonium can cause the appearance of some cannabinoids in hemp oil [34
]. Therefore, any cannabinoids detected actually represent hemp seed oil contaminants. Their concentration is influenced by the hemp variety and by the seed cleaning process. Although the cannabinoid concentration in hemp seed oils is usually extremely low, it must be determined before oil commercialization [39
]. Nevertheless, the seeds of industrial hemp plants have important uses in human nutrition [40
] and this is reason why its oil is used as an adequate, naturally resembling matrix for CBD-enriched products. Hemp seed oils represent good sources of protein, and are rich in omega-3 and omega-6 fatty acids with an ideal n3/n6 PUFA nutrition ratio according to WHO guidelines [42
]. It should be considered that hemp oil is rich in unsaturated fatty acids which are the components susceptible to oxidation phenomena during storage. Although hemp seed oil was shown to be more predisposed to peroxidation than olive oil [44
], this study did not identify any significant differences between these two matrixes as far as on-going peroxidation was concerned. Nevertheless, the critical point is to assess stability during the storage period, that is not reported or available for any of the CBD preparations analysed here. This represents a fundamental issue since the formation of lipid oxidation products is related with the decreasing concentration of cannabinoids and terpenes, as well [12
]. Therefore, the investigation of trends of compounds characterizing the formulations is essential to define the management conditions. Moreover, an adequate storage temperature would be useful to define the correct expiry date of the products as they are commercialized in EU as dietary supplements.