3. Results and discussion

each oil in 10 mL of isopropanol. After adding 1 μg/mL of IS, 10 μL of each sample were

Romano and Hazekamp [32]

Amount inflorescence/FU oil volume 1 g:10 mL 1 g:10 mL 1 g:10 mL

Preparation time (min) 150 120 90

(98C 120 min)

Filtration Yes/filter paper Yes/filter paper Yes/filter paper

Chromatography was accomplished on an HPLC system (Thermo Fisher Scientific, San Jose, CA, USA) that was made up of a Surveyor MS quaternary pump with a degasser, a Surveyor AS autosampler with a column oven and a Rheodyne valve with a 20 μL loop. Analytical separation was carried out using a reverse-phase HPLC column 150 2 mm i.d., 4 μm, Synergi Hydro RP, with a 4 3 mm i.d. C18 guard column (Phenomenex, Torrance, CA, USA). The mobile phase contained a binary combination of 0.1% aqueous formic acid and acetonitrile. The gradient was initiated with 60% eluent 0.1% aqueous formic acid with a linear decrease up to 95% in 10 min. This condition was maintained for 4 min. The mobile phase was returned to initial conditions at 14 min, followed by a 6-min re-equilibration period. The flow rate was 0.3 mL/min. The column and sample temperatures were 30 and 5C, respectively. The mass spectrometer Thermo Q-Exactive Plus (Thermo Scientific, San Jose, CA, USA) was equipped with a heated electrospray ionisation (HESI) source. Capillary temperature and vaporiser temperature were set at 330 and 280C, respectively, while the electrospray voltage was adjusted at 3.50 kV (operating in both positive and negative mode). Sheath and auxiliary gas were 35 and 15 arbitrary units, with S lens RF level of 60. The mass spectrometer was controlled by Xcalibur 3.0 software (Thermo Fisher Scientific, San Jose, CA, USA). The exact mass of the compounds was calculated using Qualbrowser in Xcalibur 3.0 software. The FS-dd-MS<sup>2</sup> (full scan data-dependent acquisition) in both positive and negative mode was used for both screening and quantification purposes. Resolving power of FS adjusted on 140,000 FWHM at

m/z 200, with scan range of m/z 215-500. Automatic gain control (AGC) was set at 3e<sup>6</sup>

injection time of 100 ms. Fragmentation of precursors was optimised as two-stepped normalised collision energy (NCE) (25 and 40 eV). Detection was based on calculated exact mass of the protonated/deprotonated molecular ions, at least one corresponding fragment and on retention time of target compounds [12]. Extracted ion chromatograms (EICs) were

negative mode at 35,000 FWHM (m/z 200). The AGC target was set to 2e5

injection time of 200 ms. A targeted MS/MS (dd-MS<sup>2</sup>

, with an

, with the maximum

) analysis operated in both positive and

Pacifici et al. [33] Calvi et al. [30]

Yes/145C, 30 min static oven

Ultrasound (35 KHz 30 min)

(1) (2) (3)

static oven

Heating in water bath (98C 60 min)

No Yes/145C, 30 min

diluted in 890 μL of initial mobile phase from which 2 μL was injected.

Preparation's step Preparation method

Extraction process Heating in water bath

Table 1. Preparation procedures details for Bediol® macerated oils.

Decarboxylation step (conversion acid form in

62 Recent Advances in Cannabinoid Research

neutral form of cannabinoids)

#### 3.1. Quality analysis of Cannabis inflorescences

#### 3.1.1. ASE Cannabis sample preparations from Bediol® medical chemotype

The choice of the appropriate analytical approach for cannabinoid profiling in cannabis inflorescences is extremely important, considering the need for a comprehensive chemical characterisation of cannabis and derived products [34]. For these reasons, analytical techniques based on high resolution mass spectrometer (HRMS-Orbitrap), due to their excellent resolution, precision and sensitivity [35], nowadays represent the gold standard techniques for the investigation of the highly complex cannabis composition. Proper purification and extraction methodology must also be implemented and is considered crucial in order to achieve an in-depth screening of the cannabinoids in Cannabis sativa L. inflorescence [32, 33].

The traditional solvent extraction methods often used for the extraction of different bioactive compounds from plants carry certain drawbacks [30]. Often, they are time consuming, laborious, have low selectivity or low extraction yields and usually large amounts of toxic solvents are required. Emphasis has currently shifted toward the use of sub- and supercritical fluids and generally-recognised-as-safe (GRAS) solvents as also detailed elsewhere [34]. Recent advances using accelerated solvent extraction (ASE) systems, as described in several publications [35, 36] include procedures for selective removal of interferences during sample extraction, thus combining extraction and purification into a single step. ASE is considered one of the most promising extraction process because, unlike standard extraction methods, it utilises high temperature and pressure to improve the extraction of the analyte from the solid sample. These conditions enhance the diffusion of the extraction solvent throughout the sample matrix which result in the more complete dissolution and recovery of the investigated compounds. The sample to be extracted is placed in a sealed metal cell that is then allocated automatically in a heated oven chamber and filled with the extraction solvent. The extraction cell is then pressurised, allowing for an increase in the boiling point of the extraction solvent, and for the solubilisation of the analytes at a temperature higher than would be possible at atmospheric pressure. Hereafter, the sample is extracted and collected by the automated filling and voiding of the cell through repeated static cycles. Compared to other solid sample extraction techniques, ASE requires less time, consumes less solvent during extraction and, with the added benefit of automation, has proven effective for several food solid samples.

Evaluation of the performance of ASE for the extraction of natural compounds like curcuminoids, saponins, flavonolignans, terpenes, taxanes, xanthone, flavonoids and artemisinin has already been conducted, as well as the application of ASE for the characterisation of phenolic compounds from fine Alpine plant roots [37]. The advantage of applying pressure is due to the fact that it is able to force the extracting solvent into the matrix and therefore may improve extraction efficiency dramatically. To the best of our knowledge, the present study reports an ASE-based method applied to the extraction of cannabinoids from cannabis row material (inflorescences) for the first time.

Bediol® chemotype was chosen for the optimisation of the ASE working parameters as it encompasses a combination of balanced amounts of THC and CBD, two cannabinoids responsible for most of the clinical effects that medical cannabis can express. In addition, it has been repeatedly suggested that the effect of isolated THC or of any other single cannabinoid is not equivalent to that of whole cannabis preparations, since some of the bioactivity observed could be related also to the presence of acidic cannabinoids. In this context, the use of an analytical method allowing the qualitative and quantitative exhaustive extraction of neutral cannabinoids and its native, acidic forms (THCA and CBDA) from cannabis plant is fundamental to characterise different cannabis varieties, a particularly relevant point when considering medical varieties. That is why the extraction efficacy of ASE was evaluated also for THCA and CBDA.

However, the optimization of effective extraction from cannabis plant is a strategic and very important issue in cannabinoid determination, as it determines the accuracy of the whole analytical method. Therefore, several extraction solvents for ASE extraction of cannabinoids from Bediol® chemotype were evaluated herein.

The best combination in terms of relative area (area analyte/IS) was obtained using methanol as extraction solvent at room temperature and 2 extraction cycles of 5 min each, with a resulting total extraction time of 15 min (Figure 1). These results are in line with a recent study that investigated the use of different extraction methods (dynamic maceration, ultrasound, microwave and supercritical fluid extraction) for the analysis of cannabinoids from fibre-type cannabis varieties [38]. Recoveries calculated by comparing the concentrations of the extracted compounds with those from the MMC calibration curves at two different fortification levels showed an average recovery of 93 and 5.7% as coefficient of variation. Based on obtained MMC calibration curves used for the purpose of validation of ASE procedures the percentage of THC, THCA, CBD and CBDA in Bediol® inflorescence by means of LC-Q-Exactive-Orbitrap-MS analysis was calculated as being: 0.88, 5.7, 0.96 and 7.4%, respectively.

#### 3.1.2. HS-SPME and GC-MS for terpenes fingerprint from Bediol® medical chemotype

In comparison with cannabinoid derivatives, the volatile constituents of Cannabis sativa L. have received much less attention. At present, scarce emphasis has been given toward the exhaustive characterisation of the terpenes profile obtained from Cannabis chemotype standardised and certified for medical use [18, 27]. In relation to recent evidence concerning the synergic role of terpenes and cannabinoids (entourage effect) [21], the comprehensive evaluation of terpene compounds especially characterising medical strains is nowadays crucial to correctly managing Cannabis as a complete therapeutic tool. In addition, several medical applications of Cannabis flos involve the vaporisation of inflorescence by using medical vaping equipment to heat the herb thus releasing both cannabinoids and terpenes into the vapour phase. The need to understand the real terpene profile emitted by medical varieties in order to select the most appropriate varieties for therapeutic use is particularly evident. In the present study, an HS-SPME method was adopted for the preconcentration of the volatile compounds with particular focus on terpenes fraction (mono-di-tri terpenes and sesquiterpenes). HS-SPME is considered a gold analytical technique for the analysis of volatile compounds in general (ref), but scarce data are available about the application of HS-SPME in the analysis of terpenes and in general of the volatile profile from medical cannabis varieties. Nevertheless, a study published recently demonstrates the convenience of HS-SPME in the characterisation of hashish terpene profile [35]. In particular, by the means of HS-SPME, authors were able to isolate and identify a potential volatile marker that might serve as a substance by which the resin and plant material

Figure 1. Impact of extraction solvents, temperature and number of extraction cycles on extractability of cannabinoids by

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using accelerated solvent extraction (ASE) from Bediol® chemotype.

Quality Traits of Medical *Cannabis sativa* L. Inflorescences and Derived Products Based on Comprehensive… http://dx.doi.org/10.5772/intechopen.79539 65

from fine Alpine plant roots [37]. The advantage of applying pressure is due to the fact that it is able to force the extracting solvent into the matrix and therefore may improve extraction efficiency dramatically. To the best of our knowledge, the present study reports an ASE-based method applied to the extraction of cannabinoids from cannabis row material (inflorescences)

Bediol® chemotype was chosen for the optimisation of the ASE working parameters as it encompasses a combination of balanced amounts of THC and CBD, two cannabinoids responsible for most of the clinical effects that medical cannabis can express. In addition, it has been repeatedly suggested that the effect of isolated THC or of any other single cannabinoid is not equivalent to that of whole cannabis preparations, since some of the bioactivity observed could be related also to the presence of acidic cannabinoids. In this context, the use of an analytical method allowing the qualitative and quantitative exhaustive extraction of neutral cannabinoids and its native, acidic forms (THCA and CBDA) from cannabis plant is fundamental to characterise different cannabis varieties, a particularly relevant point when considering medical varieties. That is why the extraction efficacy of ASE was evaluated also for THCA and

However, the optimization of effective extraction from cannabis plant is a strategic and very important issue in cannabinoid determination, as it determines the accuracy of the whole analytical method. Therefore, several extraction solvents for ASE extraction of cannabinoids

The best combination in terms of relative area (area analyte/IS) was obtained using methanol as extraction solvent at room temperature and 2 extraction cycles of 5 min each, with a resulting total extraction time of 15 min (Figure 1). These results are in line with a recent study that investigated the use of different extraction methods (dynamic maceration, ultrasound, microwave and supercritical fluid extraction) for the analysis of cannabinoids from fibre-type cannabis varieties [38]. Recoveries calculated by comparing the concentrations of the extracted compounds with those from the MMC calibration curves at two different fortification levels showed an average recovery of 93 and 5.7% as coefficient of variation. Based on obtained MMC calibration curves used for the purpose of validation of ASE procedures the percentage of THC, THCA, CBD and CBDA in Bediol® inflorescence by means of LC-Q-Exactive-Orbitrap-MS analysis was

for the first time.

64 Recent Advances in Cannabinoid Research

CBDA.

from Bediol® chemotype were evaluated herein.

calculated as being: 0.88, 5.7, 0.96 and 7.4%, respectively.

3.1.2. HS-SPME and GC-MS for terpenes fingerprint from Bediol® medical chemotype

In comparison with cannabinoid derivatives, the volatile constituents of Cannabis sativa L. have received much less attention. At present, scarce emphasis has been given toward the exhaustive characterisation of the terpenes profile obtained from Cannabis chemotype standardised and certified for medical use [18, 27]. In relation to recent evidence concerning the synergic role of terpenes and cannabinoids (entourage effect) [21], the comprehensive evaluation of terpene compounds especially characterising medical strains is nowadays crucial to correctly managing Cannabis as a complete therapeutic tool. In addition, several medical applications of Cannabis flos involve the vaporisation of inflorescence by using medical vaping equipment to heat the herb thus releasing both cannabinoids and terpenes into the vapour phase. The need

Figure 1. Impact of extraction solvents, temperature and number of extraction cycles on extractability of cannabinoids by using accelerated solvent extraction (ASE) from Bediol® chemotype.

to understand the real terpene profile emitted by medical varieties in order to select the most appropriate varieties for therapeutic use is particularly evident. In the present study, an HS-SPME method was adopted for the preconcentration of the volatile compounds with particular focus on terpenes fraction (mono-di-tri terpenes and sesquiterpenes). HS-SPME is considered a gold analytical technique for the analysis of volatile compounds in general (ref), but scarce data are available about the application of HS-SPME in the analysis of terpenes and in general of the volatile profile from medical cannabis varieties. Nevertheless, a study published recently demonstrates the convenience of HS-SPME in the characterisation of hashish terpene profile [35]. In particular, by the means of HS-SPME, authors were able to isolate and identify a potential volatile marker that might serve as a substance by which the resin and plant material could be discriminated. Volatiles in some Bedrocan® varieties have been previously investigated for their terpene content by GC-FID [29], a technique that provides only a partial volatile profile and is severely limited, as it does not furnish the identification of unknown volatiles, as is feasible with GC-MS facilities accompanied by adequate, up-dated mass spectrum libraries [31, 40].

is in contrast to previously published data for Bediol® inflorescence [29]. This finding is remarkable because the Bediol® chemotype is obtained by hybridising the Bedrocan variety (high THC content) with CBD-predominant varieties. Although the mechanisms underlying the regulation of terpene synthesis in cannabis plants remain to be elucidated, it is possible that

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Besides the chemical composition of the terpene fraction of Bediol® inflorescence that is comprehensively documented herein, the sesquiterpene fraction was also investigated in detail (Figure 3). This flos was particularly rich in trans-caryophyllene which is typical for most of Cannabis sativa L. varieties [19, 41, 42], but the significant amount of selina-3,7(11)-dione might be more specific to the Bediol® chemotype. In addition, by the means of mass spectrometry it was possible to identify a compound with a sesquiterpene structure which does not correspond to any known substance from this class. Considering its abundance, a profound examination of this "new", unknown compound is mandatory, as it could be used as a specific

Also, this chemotype was principally rich in esters, volatile compounds responsible for, and associated with, "fruity" flavour notes (Figure 4). The most abundant ester found is butanoic acid-hexyl ester, which is recognised by its sweet, apple, and apple peel flavour [43]. Its domination in the ester profile of Bediol® candidates this compound as the principal natural

In line with the approval by the Italian Ministry of Health of a decree that regulates the cultivation, processing, and therapeutic uses of Cannabis [16], there has been increasing request for the medicinal oil extracts obtained from the dried flowers [43]. A standardised protocol for oily preparations is therefore also required, but until now has not been formulated. In this

Figure 3. Representative sesquiterpenes fraction extracted from Bediol® chemotype by means of HS-SPME and identified

3.2. Quality analysis of Bediol® oil formulations: cannabinoids and VOC profile

selective, individual breeding could influence terpene proportion profiles [22].

flavouring substance for this Cannabis sativa L. chemotype.

Bediol® marker.

using the GC-MS (μg/g).

Furthermore, the terpenes were extracted using ethanol as an extraction solvent [29] and then quantified by using a calibration curve constructed by using generic internal standard. This approach is usually limitative as the polarity of the solvent could dramatically influence the terpene profile obtained and lead to the underestimation of the complex mixture of secondary metabolites emitted by plants as a result [40]. Methods involving headspace sampling appear to be the most opportune option to investigate cannabis volatile profile to obtain a representative profile of their volatile constituents avoiding interference potentially brought by predominant cannabinoids in the resulting chromatogram [41].

It is worth mentioning that the terpenes family includes a great variety of compounds (monodi-tri and sesquiterpenes) with pronounced chemical differences which consequentially aggravate the dissimilarities in terms of potential clinical effects. It was possible to identify more than 40 monoterpenes in Bediol® medical chemotype by using the optimised HS-SPME and GC-MS. The most representative are presented in Figure 2. As a general consideration, βmyrcene was the predominant terpene in Bediol® chemotype as was reported previously [22, 29, 41]. Moreover, this is an extremely important finding as this monoterpene demonstrates a prominent narcotic-like effect that is seemingly responsible for the 'couch lock' phenomenon frequently associated with modern cannabis phenomenology [24]. Furthermore, five other monoterpenes, namely α-terpinolene, β-ocimene, β-phellandrene α-and β-pinene are the major monoterpenes in Bediol® chemotype, as was revealed for other Cannabis sativa L. varieties [42]. Interestingly, our analysis revealed the presence of limonene (930 μg/g), which

Figure 2. Representative terpenes fraction extracted from Bediol® chemotype by means of HS-SPME and identified using the GC-MS (μg/g).

is in contrast to previously published data for Bediol® inflorescence [29]. This finding is remarkable because the Bediol® chemotype is obtained by hybridising the Bedrocan variety (high THC content) with CBD-predominant varieties. Although the mechanisms underlying the regulation of terpene synthesis in cannabis plants remain to be elucidated, it is possible that selective, individual breeding could influence terpene proportion profiles [22].

could be discriminated. Volatiles in some Bedrocan® varieties have been previously investigated for their terpene content by GC-FID [29], a technique that provides only a partial volatile profile and is severely limited, as it does not furnish the identification of unknown volatiles, as is feasible with GC-MS facilities accompanied by adequate, up-dated mass spectrum libraries

Furthermore, the terpenes were extracted using ethanol as an extraction solvent [29] and then quantified by using a calibration curve constructed by using generic internal standard. This approach is usually limitative as the polarity of the solvent could dramatically influence the terpene profile obtained and lead to the underestimation of the complex mixture of secondary metabolites emitted by plants as a result [40]. Methods involving headspace sampling appear to be the most opportune option to investigate cannabis volatile profile to obtain a representative profile of their volatile constituents avoiding interference potentially brought by predom-

It is worth mentioning that the terpenes family includes a great variety of compounds (monodi-tri and sesquiterpenes) with pronounced chemical differences which consequentially aggravate the dissimilarities in terms of potential clinical effects. It was possible to identify more than 40 monoterpenes in Bediol® medical chemotype by using the optimised HS-SPME and GC-MS. The most representative are presented in Figure 2. As a general consideration, βmyrcene was the predominant terpene in Bediol® chemotype as was reported previously [22, 29, 41]. Moreover, this is an extremely important finding as this monoterpene demonstrates a prominent narcotic-like effect that is seemingly responsible for the 'couch lock' phenomenon frequently associated with modern cannabis phenomenology [24]. Furthermore, five other monoterpenes, namely α-terpinolene, β-ocimene, β-phellandrene α-and β-pinene are the major monoterpenes in Bediol® chemotype, as was revealed for other Cannabis sativa L. varieties [42]. Interestingly, our analysis revealed the presence of limonene (930 μg/g), which

Figure 2. Representative terpenes fraction extracted from Bediol® chemotype by means of HS-SPME and identified using

inant cannabinoids in the resulting chromatogram [41].

[31, 40].

66 Recent Advances in Cannabinoid Research

the GC-MS (μg/g).

Besides the chemical composition of the terpene fraction of Bediol® inflorescence that is comprehensively documented herein, the sesquiterpene fraction was also investigated in detail (Figure 3). This flos was particularly rich in trans-caryophyllene which is typical for most of Cannabis sativa L. varieties [19, 41, 42], but the significant amount of selina-3,7(11)-dione might be more specific to the Bediol® chemotype. In addition, by the means of mass spectrometry it was possible to identify a compound with a sesquiterpene structure which does not correspond to any known substance from this class. Considering its abundance, a profound examination of this "new", unknown compound is mandatory, as it could be used as a specific Bediol® marker.

Also, this chemotype was principally rich in esters, volatile compounds responsible for, and associated with, "fruity" flavour notes (Figure 4). The most abundant ester found is butanoic acid-hexyl ester, which is recognised by its sweet, apple, and apple peel flavour [43]. Its domination in the ester profile of Bediol® candidates this compound as the principal natural flavouring substance for this Cannabis sativa L. chemotype.

#### 3.2. Quality analysis of Bediol® oil formulations: cannabinoids and VOC profile

In line with the approval by the Italian Ministry of Health of a decree that regulates the cultivation, processing, and therapeutic uses of Cannabis [16], there has been increasing request for the medicinal oil extracts obtained from the dried flowers [43]. A standardised protocol for oily preparations is therefore also required, but until now has not been formulated. In this

Figure 3. Representative sesquiterpenes fraction extracted from Bediol® chemotype by means of HS-SPME and identified using the GC-MS (μg/g).

CBN, CBG and CBGA [30]. Applying this method, we were able to determine the cannabinoid profile in Bediol® chemotype oils prepared by three different methods, as described in the

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Method 3 (realised by applying a preheating/ultrasounds assisted extraction), showed the highest extraction yields of the neutral cannabinoids CBD and THC. In contrast, method 1 provided the maximal concentrations of THCA, CBDA and CBGA, as a preheating step was not involved. At present, it is important to emphasise that, in the field of the therapeutic uses of cannabinoids related to pharmacological and clinical effects, THC and CBD in their neutral forms are of primary interest, even if there is growing attention toward the acidic forms

Furthermore, apart from the targeted compounds revealed, several other untargeted cannabinoids were detected, as well. HRMS analysis has proven to be very useful also in the retrospective evaluation of untargeted isomeric cannabinoids. The structural interpretation of untargeted compounds was accomplished from the mass spectra collected in the FS and corresponding dd-MS<sup>2</sup> scan mode, and relied on the information found in the literature [30, 45, 46, 47, 49] and mass spectrum libraries [48]. In this respect, Q-Exactive-Orbitrap-MS analyser is often used in order to obtain structural information of the compounds detected as it provides accurate mass identification for both the precursor and the product ions. Among untargeted molecules, we verified the presence of THCV and CBDV that expressed the same fragmentation behaviour as their C5 equivalents but differed in fragments that contained the C3 side chain [30]. The presence and further quantification of those two compounds seems to be essential as it was revealed that in three models of seizure, cannabis-derived "botanical drug substances" rich in CBDV and CBD exerted significant anticonvulsant effects that were not mediated by the CB1 receptor and were of comparable efficacy with purified CBDV [50]. On the other hand, it is well-known that THCV (also as THC) binds to CB1 and CB2 receptors and acts as a cannabimimetic agonist [50, 51]. Therefore, the pharmacological potency of CBDV and THCV is substantial and, regardless of their relatively small amounts in oil preparations, they may contribute to the physiological efficiency of the overall cannabinoids profile

Moreover, in the Bediol® oil extract samples in full scan negative acquisition mode at least four different cannabinoids with the same molecular ions (m/z 343.1915) but different retention times were noted (Figure 5). Their appearance and intensity varies according to the preparation method used. The fragmentation pattern of peaks at retention time (RT) 9.91 and 12.24 min correspond to tetrahydrocannabinolic acid—C4 (THCA-C4) and cannabidiolic acid—C4 (CBDA-C4). Those two

Preparation method THC CBD CBN CBG THC-A CBD-A CBG-A 1 [32] 370 23 2010 56 10 0.5 7 0.8 8300 507 14,120 1002 260 23 2 [33] 4520 102 5503 89 56 7 125 21 1808 201 1208 750 114 15 3 [30] 5214 87 7304 108 47 4 102 12 487 42 29 0.75 18 6

Table 2. Quantitative analysis of main cannabinoids from Bediol®'s macerated oil preparations obtained by three

[18], at least as far as Bediol® oil preparation is concerned.

different preparation procedures (μg/g, mean SD, n = 3).

materials and methods section.

(Table 2) [3].

Figure 4. Esters fraction extracted from Bediol® chemotype by means of HS-SPME and identified using the GC-MS (μg/g).

context, cannabis extraction was performed using olive oil and a standardised medicinal cannabis "flos" (according to pharmaceutical standards) [31, 34, 39, 44, 45].

HPLC-MS/MS based analysis has recently been employed for the analysis of cannabinoids in plant materials, extracts and biological matrices [8, 29, 45]. This detection technique has proven to be particularly trustworthy, as there is no risk of native cannabinoids decomposition (decarboxylation of cannabinoid acids during the analysis), which may compromise the accurate assessment of the overall cannabinoids profile. Currently, the most widely used analysers for cannabinoids quantification are the triple quadrupole instruments, which possess excellent sensitivity and selectivity [31, 46]. However, they do not allow structural identification of "non-target" compounds.

In this respect, high-resolution accurate mass (HRMS) analyser such as Q-Exactive-Orbitrap-MS, offers the possibility to operate generating an "in-depth" qualitative analysis of thousands of compounds in complex biological, environmental or food matrixes providing insights beyond what is currently achievable with classic mass spectrometry instrumentation. Orbitrap mass spectrometer technology is rapidly developing also for cannabinoids profiling in different matrices, because it uniquely provides accurate molecular masses and specific fragmentation patterns for detected species. Moreover, HRMS acquisition mode accumulates all sample data, enabling identification of "unpredicted" compounds with cannabinolic structure and retrospective data analysis without the need to re-run samples.

As an example, a simultaneous identification of 24 synthetic and natural cannabinoids for a wide variety of samples such as herbal cannabis plant material by means of Orbitrap was reported [3]. Moreover, our research group has also recently published results concerning HPLC-Q-Exactive-Orbitrap-MS method for the determination of the seven most important cannabinoids, including four essential cannabinoids (THC, CBD, THCA and CBDA) accompanied with quantification of CBN, CBG and CBGA [30]. Applying this method, we were able to determine the cannabinoid profile in Bediol® chemotype oils prepared by three different methods, as described in the materials and methods section.

Method 3 (realised by applying a preheating/ultrasounds assisted extraction), showed the highest extraction yields of the neutral cannabinoids CBD and THC. In contrast, method 1 provided the maximal concentrations of THCA, CBDA and CBGA, as a preheating step was not involved. At present, it is important to emphasise that, in the field of the therapeutic uses of cannabinoids related to pharmacological and clinical effects, THC and CBD in their neutral forms are of primary interest, even if there is growing attention toward the acidic forms (Table 2) [3].

Furthermore, apart from the targeted compounds revealed, several other untargeted cannabinoids were detected, as well. HRMS analysis has proven to be very useful also in the retrospective evaluation of untargeted isomeric cannabinoids. The structural interpretation of untargeted compounds was accomplished from the mass spectra collected in the FS and corresponding dd-MS<sup>2</sup> scan mode, and relied on the information found in the literature [30, 45, 46, 47, 49] and mass spectrum libraries [48]. In this respect, Q-Exactive-Orbitrap-MS analyser is often used in order to obtain structural information of the compounds detected as it provides accurate mass identification for both the precursor and the product ions. Among untargeted molecules, we verified the presence of THCV and CBDV that expressed the same fragmentation behaviour as their C5 equivalents but differed in fragments that contained the C3 side chain [30]. The presence and further quantification of those two compounds seems to be essential as it was revealed that in three models of seizure, cannabis-derived "botanical drug substances" rich in CBDV and CBD exerted significant anticonvulsant effects that were not mediated by the CB1 receptor and were of comparable efficacy with purified CBDV [50]. On the other hand, it is well-known that THCV (also as THC) binds to CB1 and CB2 receptors and acts as a cannabimimetic agonist [50, 51]. Therefore, the pharmacological potency of CBDV and THCV is substantial and, regardless of their relatively small amounts in oil preparations, they may contribute to the physiological efficiency of the overall cannabinoids profile [18], at least as far as Bediol® oil preparation is concerned.

context, cannabis extraction was performed using olive oil and a standardised medicinal canna-

Figure 4. Esters fraction extracted from Bediol® chemotype by means of HS-SPME and identified using the GC-MS (μg/g).

HPLC-MS/MS based analysis has recently been employed for the analysis of cannabinoids in plant materials, extracts and biological matrices [8, 29, 45]. This detection technique has proven to be particularly trustworthy, as there is no risk of native cannabinoids decomposition (decarboxylation of cannabinoid acids during the analysis), which may compromise the accurate assessment of the overall cannabinoids profile. Currently, the most widely used analysers for cannabinoids quantification are the triple quadrupole instruments, which possess excellent sensitivity and selectivity [31, 46]. However, they do not allow structural identification of

In this respect, high-resolution accurate mass (HRMS) analyser such as Q-Exactive-Orbitrap-MS, offers the possibility to operate generating an "in-depth" qualitative analysis of thousands of compounds in complex biological, environmental or food matrixes providing insights beyond what is currently achievable with classic mass spectrometry instrumentation. Orbitrap mass spectrometer technology is rapidly developing also for cannabinoids profiling in different matrices, because it uniquely provides accurate molecular masses and specific fragmentation patterns for detected species. Moreover, HRMS acquisition mode accumulates all sample data, enabling identification of "unpredicted" compounds with cannabinolic structure and

As an example, a simultaneous identification of 24 synthetic and natural cannabinoids for a wide variety of samples such as herbal cannabis plant material by means of Orbitrap was reported [3]. Moreover, our research group has also recently published results concerning HPLC-Q-Exactive-Orbitrap-MS method for the determination of the seven most important cannabinoids, including four essential cannabinoids (THC, CBD, THCA and CBDA) accompanied with quantification of

bis "flos" (according to pharmaceutical standards) [31, 34, 39, 44, 45].

retrospective data analysis without the need to re-run samples.

"non-target" compounds.

68 Recent Advances in Cannabinoid Research

Moreover, in the Bediol® oil extract samples in full scan negative acquisition mode at least four different cannabinoids with the same molecular ions (m/z 343.1915) but different retention times were noted (Figure 5). Their appearance and intensity varies according to the preparation method used. The fragmentation pattern of peaks at retention time (RT) 9.91 and 12.24 min correspond to tetrahydrocannabinolic acid—C4 (THCA-C4) and cannabidiolic acid—C4 (CBDA-C4). Those two


Table 2. Quantitative analysis of main cannabinoids from Bediol®'s macerated oil preparations obtained by three different preparation procedures (μg/g, mean SD, n = 3).

Figure 5. Extracted ion chromatograms from retrospective data analysis which point toward the presence of CBDA-C4; THCA-C4 and CBCA-C4.

amount of terpenes, followed by methods 3 and 2 (Table 3). This was predictable, as method 1 did not include preheating for decarboxylation, thus the terpene fraction was preserved with evident domination of β-Myrcene. Although preheating the plant material released more of the known active neutral cannabinoids, it simultaneously led to the loss of components such as

Figure 6. Extracted ion chromatograms from retrospective data analysis which point toward the presence of CBDVA;

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As regards lipid oxidation products, the opposite trend was shown among the three preparation procedures. In particular, method 3, realised without any heating step, showed minor concentrations of lipid oxidation products. The macerated oil obtained using the method by Romano-Hazekamp (method 1) contained the highest levels of oxidation products, compared with the other two procedures, as expected. This can be related to preparation conditions in which the oil is heated at 98C for 120 min. The data concerning the formation of lipid oxidation products in cannabis medical oil preparations are extremely limited [30]. The occurrence of aldehydes in the sample obtained by method 1 indicates the initiation of lipid peroxidation of polyunsaturated fatty acids (PUFA) from oils used as a matrix [52, 53]. 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 [54]. The formation rate of lipid oxidation products depends closely on several factors among which the most important are: method preparation temperature, fatty acid composition of oil in which cannabis extract is dissolved and storage conditions [55]. These parameters are crucial to define the ultimate characteristics of the final products to be used for medical treatment. Finally, the presence of 2-furancarboxaldehyde in the oil sample obtained by method 1 confirmed that preheating initiates the series of reactions that leads to the formation of potentially toxic

terpenes by degradation or evaporation.

THCVA and CBCVA.

compounds.

acids are respectively homologues of main acids (THCA and CBDA) from which they differ just in the butyl side chain (instead of pentyl). In addition, the presence of the peak 10.31 and its fragmentation profile indicate the presence of cannabichromenic acid C4 (CBCA-C4). In a completely analogous way, the extracted ion chromatograms for m/z 329.17580 confirm the occurrence of THCVA and CBDVA, the acidic precursors of the above-mentioned THCV and CBDV, just for the oil samples from methods 1 and 2 (Figure 6). Additionally, the oil extract obtained by extraction method 3 revealed the presence of cannabichromevarinic acid (CBCVA). This compound, like its neutral counterpart cannabichromevarin CBCV, is not supported by adequate research work to fully understand its eventual distinctive pharmacological and physiological behaviour. However, the fact that extraction method 3 (preheating/ultrasounds) transfers this compound from the inflorescence to the medicinal oil has to be taken into consideration, especially when the signals of THCVA and CBDVA were practically absent in extract 3. This is most likely due to different kinetics of extraction performed by ultrasound that preserves the benzopiranic structure of CBCVA.

All in all, our retrospective analysis of Bediol® medical oil provides clear evidence of the need to develop a standardised procedure for extraction, especially in terms of time and extraction method, since they unambiguously affect the chemical composition of the final product, thus influencing the pharmacological effect of the medicinal preparation that is eventually dispensed to patients.

As far as VOCs profile is concerned, all three preparation methods extracted substantial amounts of terpenes, resembling the profile obtained for the Bediol® inflorescence. Comparing the three different preparation methods, it can be observed that method 1 extracted the highest Quality Traits of Medical *Cannabis sativa* L. Inflorescences and Derived Products Based on Comprehensive… http://dx.doi.org/10.5772/intechopen.79539 71

Figure 6. Extracted ion chromatograms from retrospective data analysis which point toward the presence of CBDVA; THCVA and CBCVA.

amount of terpenes, followed by methods 3 and 2 (Table 3). This was predictable, as method 1 did not include preheating for decarboxylation, thus the terpene fraction was preserved with evident domination of β-Myrcene. Although preheating the plant material released more of the known active neutral cannabinoids, it simultaneously led to the loss of components such as terpenes by degradation or evaporation.

acids are respectively homologues of main acids (THCA and CBDA) from which they differ just in the butyl side chain (instead of pentyl). In addition, the presence of the peak 10.31 and its fragmentation profile indicate the presence of cannabichromenic acid C4 (CBCA-C4). In a completely analogous way, the extracted ion chromatograms for m/z 329.17580 confirm the occurrence of THCVA and CBDVA, the acidic precursors of the above-mentioned THCV and CBDV, just for the oil samples from methods 1 and 2 (Figure 6). Additionally, the oil extract obtained by extraction method 3 revealed the presence of cannabichromevarinic acid (CBCVA). This compound, like its neutral counterpart cannabichromevarin CBCV, is not supported by adequate research work to fully understand its eventual distinctive pharmacological and physiological behaviour. However, the fact that extraction method 3 (preheating/ultrasounds) transfers this compound from the inflorescence to the medicinal oil has to be taken into consideration, especially when the signals of THCVA and CBDVA were practically absent in extract 3. This is most likely due to different kinetics of extraction performed by ultrasound that preserves the

Figure 5. Extracted ion chromatograms from retrospective data analysis which point toward the presence of CBDA-C4;

All in all, our retrospective analysis of Bediol® medical oil provides clear evidence of the need to develop a standardised procedure for extraction, especially in terms of time and extraction method, since they unambiguously affect the chemical composition of the final product, thus influencing the pharmacological effect of the medicinal preparation that is eventually dis-

As far as VOCs profile is concerned, all three preparation methods extracted substantial amounts of terpenes, resembling the profile obtained for the Bediol® inflorescence. Comparing the three different preparation methods, it can be observed that method 1 extracted the highest

benzopiranic structure of CBCVA.

pensed to patients.

THCA-C4 and CBCA-C4.

70 Recent Advances in Cannabinoid Research

As regards lipid oxidation products, the opposite trend was shown among the three preparation procedures. In particular, method 3, realised without any heating step, showed minor concentrations of lipid oxidation products. The macerated oil obtained using the method by Romano-Hazekamp (method 1) contained the highest levels of oxidation products, compared with the other two procedures, as expected. This can be related to preparation conditions in which the oil is heated at 98C for 120 min. The data concerning the formation of lipid oxidation products in cannabis medical oil preparations are extremely limited [30]. The occurrence of aldehydes in the sample obtained by method 1 indicates the initiation of lipid peroxidation of polyunsaturated fatty acids (PUFA) from oils used as a matrix [52, 53]. 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 [54]. The formation rate of lipid oxidation products depends closely on several factors among which the most important are: method preparation temperature, fatty acid composition of oil in which cannabis extract is dissolved and storage conditions [55]. These parameters are crucial to define the ultimate characteristics of the final products to be used for medical treatment. Finally, the presence of 2-furancarboxaldehyde in the oil sample obtained by method 1 confirmed that preheating initiates the series of reactions that leads to the formation of potentially toxic compounds.


Compound class/name Preparation method

Sesquiterpenes

Furans

1 [32] 2 [33] 3 [30]

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Quality Traits of Medical *Cannabis sativa* L. Inflorescences and Derived Products Based on Comprehensive…

Eucalyptol 5.2 0.58 3.14 0.76 4.84 0.46 β-Phellandrene 52.00 7.57 27.25 4.37 35.83 1.57 Cis-ocimene 2.70 0.20 1.47 0.24 0.72 0.11 γ-Terpinene 13.87 1.13 14 2.36 8.50 0.48 β-Ocimene 107.22 6 49.0 6.7 64.88 1.15 p-Cymene 11.86 1.11 6.7 0.63 4.7 0.49 α-Terpinolene 253.3 20.9 157.78 19.46 197.14 1.08 1,3,8-p-Menthatriene 0.63 0.01 0.37 0.03 0.27 0.04 p-Cymenyl 6.3 0.18 6.84 1.46 8.07 0.33 Isomenthone n.d. 0.16 0.02 0.57 0.08 4,8-Epoxy-p-menth-1-ene 12.11 0.12 4.57 1.01 2.80 0.27 β-Linalool 0.89 0.05 0.83 0.19 0.66 0.05 p-Menth-2-en-1-ol 0.42 0.05 n.d. n.d. 4-Terpineol 2.60 0.01 2.65 0.78 2.61 0.18 Verbenol 2.41 0.13 1.56 0.63 2.21 0.08 1,8-Menthadien-4-ol 7.00 0.32 5.34 1.55 6.15 0.25 α-Terpineol 4.66 0.15 3.63 1.15 3.45 0.20 Borneol 1.07 0.16 0.89 0.26 0.77 0.02 p-Menth-1-en-3-ol 0.85 0.03 0.39 0.06 0.25 0.03 Trans-3-caren-2-ol 1.00 0.05 0.64 0.11 0.52 0.04 Cuminol 4.60 0.36 3.42 0.66 4.29 0.23

α-Santalene 0.94 0.16 0.61 0.08 0.57 0.06 α-Bergamotene 4.66 1.03 3.17 0.63 4.28 0.83 α-Guaiene 8.94 2.17 6.97 1.14 7.05 1.93 Trans-caryophyllene 27.64 4.78 20.60 3.11 21.07 3.13 α-Humulene 10.62 2.35 7.11 1.39 8.00 1.73 δ-Guaiene 7.50 2.11 5.84 0.94 5.90 1.41 β-Selinene 1.15 0.26 0.83 0.11 0.90 0.29 α-Selinene 1.78 0.07 1.07 0.11 1.90 0.45 α-Farnesene 0.63 0.20 0.42 0.06 0.54 0.16 Selina-3,7(11)-diene 7.40 2.30 5.60 0.78 6.65 1.93 Nerolidol 0.37 0.08 0.35 0.11 0.46 0.18

2-Furancarboxaldehyde 0.32 0.05 n.d. n.d.

Compound class/name Preparation method

72 Recent Advances in Cannabinoid Research

Alcohols

Aldehydes

Ketones

Esters

Mono/di/triterpenes

1 [32] 2 [33] 3 [30]

1-Hexanol 31.10 2.8 15 1.3 13.15 2.12 3-Hexen-1-ol 1.10 0.14 0.56 0.12 0.7 0.1 2-Ethyl-1-hexanol 0.22 0.03 n.d. n.d. 3,3,6-Trimethyl-1,5-heptadien-4-ol 13.1 0.5 7.3 1.93 5.3 0.45 α-Toluenol 0.16 0.03 0.10 0.02 0.08 0.02

2-Methyl-butanal 0.42 0.05 n.d. n.d. 3-Methyl-butanal 0.26 0.03 n.d. n.d. Hexanal 1.51 0.13 n.d. n.d. Heptanal 1.06 0.29 n.d. n.d. 2-Hexenal 1.90 0.22 n.d. n.d. Octanal 0.54 0.09 0.36 0.01 0.04 0.02

6-Methyl-5 hepten-2 one 1.8 0.15 0.98 0.14 0.28 0.08 3-Methyl-3-cyclohexen-1-one 3.01 0.67 0.58 0.14 0.19 0.05

α-Pinene 109 1.4 12.37 2.54 29.0 0.39 α-Thujene 5.41 0.45 2.12 0.34 2.71 0.11 Camphene 2.27 0.15 0.67 0.09 0.30 0.01 β-Pinene 55.04 7.0 14.57 1.54 17.20 0.67

Sabinene 1.82 0.14 0.2 0.07 n.d. δ-3-Carene 18.4 1.93 6.62 0.90 7.44 0.13 α-Phellandrene 19.00 2.21 10.67 1.93 5.57 0.51 β-Myrcene 1074.2 30 227.77 35.1 458.0 2.74 α-Terpinene 13.90 1.27 10.20 1045 16.56 1.14 Limonene 32.4 4.13 14.39 1.75 18.17 1.38

Acetic acid-methyl ester 0.41 0.09 n.d. n.d. 3-Hexen-1-ol-acetate 0.51 0.02 0.22 0.03 0.18 0.01 Propanoic acid-hexyl ester 1.84 0.01 0.99 0.17 0.90 0.1 Propanoic acid-2-methyl-hexyl ester 2.47 0.01 1.55 0.25 1.70 0.09 Butanoic acid-hexyl ester 21.01 0.21 10.80 2.72 16 0.82 Hexanoic acid-hexyl ester 1.78 0.54 1.23 0.28 1.43 0.22 Benzoic acid-2-amino-methyl ester 0.55 0.04 0.53 0.16 0.53 0.04



drugs and/or psychotropic substances for scientific purposes. The present paper is partially funded and realised within the project ITALIAN MOUNTAIN LAB, Ricerca e Innovazione per l'ambiente ed. i Territori di Montagna—Progetto FISR Fondo integrativo speciale per la ricerca.

Quality Traits of Medical *Cannabis sativa* L. Inflorescences and Derived Products Based on Comprehensive…

1 Department of Agricultural and Environmental Sciences—Production, Landscape,

2 CRC-Ge.S.Di.Mont.—Centre for Applied Studies in the Sustainable Management and Protection of the Mountain Environment, CRC-Ge.S.Di.Mont—Università degli Studi di

3 Department of Health, Animal Science and Food Safety, Università degli Studi di Milano,

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\*, Luca Giupponi1,2, Valeria Leoni1,2 and

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, Radmila Pavlovic1,2, Sara Panseri3

\*Address all correspondence to: sara.panseri@unimi.it

Board of Family Medicine. 2011;24:452-462

Epilepsy & Behavior. 2017;70:288-291

2008;7:615-639

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Agroenergy, Università degli Studi di Milano, Milan, Italy

Author details

Annamaria Giorgi1,2

Milano, Brescia, Italy

Milan, Italy

References

Lorenzo Calvi<sup>1</sup>

Table 3. Volatile compounds extracted and identified by HS-SPME-GC/MS in Bediol® oil obtained from different preparation methods.
