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 [12,31]. 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 [9,10,11,31] 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 [33,34] 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 [18,19]. 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).