2. Materials and methods

#### 2.1. Chemical and reagents

cannabis-based medicines in Poland can be sold if they are made in pharmacies with the use of

The current status of cannabis highlights that, since it causes "psychoactive activity," its use in medicine should follow the legal provisions of member states, including "control of the use of narcotics and psychotropic substances" [17]. European countries have an obligation to control cannabis according to the three UN Conventions on Drug Control that require them to restrict

At an EU level there are no harmonised laws on the recreational and medical use of cannabis

As an example, medical cannabis in Italy represents a multifaceted reality [16, 18]. At present varieties Bedrocan, Bediol, Bedica and Bedrolite produced by company Bedrocan from Netherlands [19] and the new strain FM2 produced by the Military Pharmaceutical Chemical Works of Florence, Italy (authorised in November 2015 with a Ministerial Decree) can be prescribed to treat a wide range of pathological conditions [16]. In relation to this, Italian galenic pharmacies are authorised to prepare precise cannabis doses for vaping, herbal teas, resins, micronised capsules and oils [20]. The latter, prepared by using European Pharmacopoeia olive oil (FU) as extraction solvent has received great attention due to the easiness with which dosage can be modulated or titrated during the treatment period. Also, oil formulations are high-steamed

As regards Cannabis sativa composition, beyond and besides cannabinoids, a substantial amount of the approximately 500 compounds (terpenes, flavonoids, stilbenoids, fatty acids, alkaloids, carbohydrates, and phenols) are described [21]. Terpenes represent the volatile component of the plant and have been proven to have a synergic action with cannabinoids [19]. Cannabis plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence [22]. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties [23]. Differences between the pharmaceutical properties of different cannabis strains have been attributed to interactions (or an 'entourage effect') between cannabinoids and terpenes [24]. Terpenes themselves exhibit a wide array of pharmacological properties, including interaction with the mammalian endocannabinoid system: sesquiterpene β-caryophyllene interacts with mammalian cannabinoid receptors [25, 26]. Some terpenes like β-myrcene, limonene and linalool

drug supplies and use it exclusively for medical and scientific purposes.

because of the extended bioavailability of the active compounds contained.

display anxiolytic, antibacterial, anti-inflammatory, and sedative effects, too [27].

come with two distinct approaches.

The chemical complexity of cannabis makes its pharmaceutical standardisation challenging and must include well-defined methodologies that would characterise the plant chemotype and the herbal drug as well as extraction procedures. As a matter of fact, it was found that the concentrations of target cannabinoids obtained for the same plant chemotype originating from different suppliers varied by more than 25% [28]. This lack of standardisation could be over-

The first is a botanical issue and points toward strict control of varieties and strains during cultivation in order to assure the highest homogeneity in the final plants, especially if the

and the member states themselves decide whether to legalise them.

an imported substance.

58 Recent Advances in Cannabinoid Research

All HPLC or analytical grade chemicals were from Sigma (Sigma-Aldrich, St. Louis, MO, USA). Formic acid 98–100% was from Fluka (Sigma-Aldrich, St. Louis, MO, USA). Ultrapure water was obtained through a Milli-Q system (Millipore, Merck KGaA, Darmstadt, Germany). For headspace (HS) analysis, the SPME coating fibre (DVB/CAR/PDMS, 50/30 μm) was from Supelco (Bellefonte, PA, USA). Acetonitrile, 2-propanol, formic acid LC-MS grade were purchased from Carlo Erba (Milan, Italy). CBD, THC, CBN, CBG, CBNA, THCA, CBGA were purchased from Sigma Aldrich (Round Rock, Texas). High intensity planetary mill Retsch (model MM 400, Retsch, GmbH, Retsch-Allee, Haan) was used to obtain representative aliquots of cannabis flos samples powder.

#### 2.2. Cannabis plant material and superfine grinding (SFG) sample preparation

Bediol® medical Cannabis chemotype that contains 6.5% THC and 8% CBD as standardised and certified by the company Bedrocan was used for all analyses. It was selected as representative because it represents the most common medical variety actually prescribed alone or in combination for several pathologies. Superfine cannabis inflorescence powder was prepared using mechanical grinding-activation in an energy intensive vibrational mill. Different samples (1.0 g each) were ground in a high intensity planetary mill. The mill was vibrating at a frequency of 25 Hz for 1 min, using two 50 mL jars with 20 mm stainless steel balls. Prior to use, jars were precooled with liquid nitrogen. The speed differences between balls and jar result in the interaction of frictional and impact forces, releasing high dynamic energies. The interplay of all these forces results in the very effective energy input of planetary ball mills. Mechano-chemical technology has been developed and successfully adopted in different fields (synthesis of superfine powder, surface modification, drug and pharmaceutical applications) and could represent a novel research tool.

according to previous published research, in order to prevent possible matrix alterations ensuring the most efficient adsorption of volatile compounds onto the SPME fibre [15, 16]. To keep the temperature constant during analysis, the vials were maintained in a cooling block (CTC Analytics, Zwingen, Switzerland). At the end of the sample equilibration time (30 min), a conditioned (60 min at 280C) SPME fibre was exposed to the headspace of the sample for 120 min using a CombiPAL system injector autosampler (CTC Analytics, Zwingen, Switzerland). All analytical parameters had already been validated in our previous research [32].

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Analyses were performed with a Trace GC Ultra coupled to a Trace DSQII quadrupole mass spectrometer (MS) (Thermo-Fisher Scientific, Waltham, MA, USA) equipped with an Rtx-Wax column (30 m 0.25 mm i.d. 0.25 μm film thickness) (Restek, Bellefonte, PA, USA). The oven temperature program was: from 35C, held for 8 min, to 60C at 4C/min, then from 60 to 160C at 6C/min and finally from 160 to 200 at 20C/min. Helium was the carrier gas, at a flow rate of 1 mL/min. Carry over and peaks originating from the fibres were regularly assessed by running blank samples. After each analysis fibres were immediately thermally desorbed in the GC injector for 5 min at 250C to prevent contamination. The MS was operated in electron impact (EI) ionisation mode at 70 eV. An alkane mixture (C8-C22, Sigma R 8769, Saint Louis, MO, USA) was run under the same chromatographic conditions as the samples to calculate the Kovats retention indices (RI) of the detected compounds. The mass spectra were obtained by using a mass selective detector, a multiplier voltage of 1456 V, and by collecting the data at a rate of 1 scan/s over the m/z range of 35–350. Compounds were identified by comparing the retention times of the chromatographic peaks with those of authentic compounds analysed under the same conditions when available, by comparing the Kovats retention indices with the literature data and through the National Institute of Standards and Technology (NIST) MS spectral database. The quantitative evaluation was performed using the internal standard procedure and the results were finally expressed as μg/g or mg/g IS equivalents of each volatile

Three different methods for oil preparation were performed and evaluated. The preparation conditions were selected on the basis of previously published methods [31]. Briefly, common issues for all three methods were the amount of Bediol® inflorescence used (1 g) and the European Pharmacopoeia (FU) olive oil volume (10 mL) that served as extraction matrix. The crucial differences concerning the preheating temperature of the inflorescence to perform the decarboxylation step and extraction process are highlighted in Table 1. After extraction and cooling down (methods 1 and 2) the oils were filtrated and subsequently prepared for LC-Q-Exactive-Orbitrap-

The cannabinoid profile in plants and the corresponding oil were assessed applying the method recently published with particular emphasis on method development [31]. In order to perform HPLC-Q-Exactive-Orbitrap®-MS analysis, samples extracted with ASE were prepared as indicated in Section 2.4, while oil samples were prepared by dissolving 100 mg of

compound. All analyses were done in triplicate.

2.6. Cannabinoids LC-Q-Exactive-Orbitrap-MS analysis

2.5. Cannabis macerated oil preparations

MS analysis.

#### 2.3. Accelerated Solvent Extraction (ASE) for cannabinoid analysis

All extractions to define the cannabinoid profile of Bediol® medical chemotype were executed using an ASE 350 (Thermo-Fisher Scientific, Waltham, MA, USA). 34-mL stain steel cells were used for the extraction. 100 mg of Cannabis flos powder obtained by using SFG was weighed and then homogenised with an equal weight of diatomaceous earth and transferred into the cell. Then, 100 μL of extraction solution containing the IS (diazepam 1 mg mL<sup>1</sup> ) was added. Different extraction solvents were tested and were: methanol, methanol:CH3Cl (9:1), hexane, acetonitrile and ethanol. Diatomaceous earths were added in order to fill the remaining empty part of the cell. Room temperature of 25C, pressure (1500 psi), number of static cycles (2 cycles, 5 min each), purging time (60 s with nitrogen) and rinse volume (90%) were used for the study. Organic extracts were finally collected in 66 mL vials and treated with sodium sulphate to remove any possible humidity. Afterwards, the extract was collected and dried under vacuum in a centrifugal evaporator. The residue was dissolved in 1 mL of acetonitrile and after proper dilution, 2 μL were submitted to analysis by HPLC-Q-Exactive-Orbitrap-MS. Validation was performed according to the European Union SANTE/2015 guidelines usually adopted to test ASE performance especially for trace residue analysis [31].

The method was completely optimised investigating the typologies of extraction solvents, number of extraction cycles and extraction temperature to define the optimum analytical conditions as well. To realise the matrix-matched calibration curves (MMCs) blank samples (100 mg officinal plant previously analysed for the absences of cannabinoids) were used and spiked with appropriate standard solution of THC, THC-A, CBD, CBD-A and CBN covering the concentration range from 0.1 to 10 μg g<sup>1</sup> . Recoveries were calculated by comparing the concentrations of the extracted compounds with those from the MMC calibration curves at two different fortification levels (1.0 and 10 μg g<sup>1</sup> ).

#### 2.4. HS-SPME and GC-MS analysis for terpenes investigation

One gram of oil or 100 mg of inflorescence previously grinded were weighed and put into 20 mL glass vials along with 100 μL of the IS (4-nonylphenol, 2000 μg/mL in 2-propanol). Each vial was fitted with a cap equipped with a silicon/PTFE septum (Supelco, Bellefonte, PA, USA). A temperature of 37C was selected as both the extraction and equilibration temperature according to previous published research, in order to prevent possible matrix alterations ensuring the most efficient adsorption of volatile compounds onto the SPME fibre [15, 16]. To keep the temperature constant during analysis, the vials were maintained in a cooling block (CTC Analytics, Zwingen, Switzerland). At the end of the sample equilibration time (30 min), a conditioned (60 min at 280C) SPME fibre was exposed to the headspace of the sample for 120 min using a CombiPAL system injector autosampler (CTC Analytics, Zwingen, Switzerland). All analytical parameters had already been validated in our previous research [32].

Analyses were performed with a Trace GC Ultra coupled to a Trace DSQII quadrupole mass spectrometer (MS) (Thermo-Fisher Scientific, Waltham, MA, USA) equipped with an Rtx-Wax column (30 m 0.25 mm i.d. 0.25 μm film thickness) (Restek, Bellefonte, PA, USA). The oven temperature program was: from 35C, held for 8 min, to 60C at 4C/min, then from 60 to 160C at 6C/min and finally from 160 to 200 at 20C/min. Helium was the carrier gas, at a flow rate of 1 mL/min. Carry over and peaks originating from the fibres were regularly assessed by running blank samples. After each analysis fibres were immediately thermally desorbed in the GC injector for 5 min at 250C to prevent contamination. The MS was operated in electron impact (EI) ionisation mode at 70 eV. An alkane mixture (C8-C22, Sigma R 8769, Saint Louis, MO, USA) was run under the same chromatographic conditions as the samples to calculate the Kovats retention indices (RI) of the detected compounds. The mass spectra were obtained by using a mass selective detector, a multiplier voltage of 1456 V, and by collecting the data at a rate of 1 scan/s over the m/z range of 35–350. Compounds were identified by comparing the retention times of the chromatographic peaks with those of authentic compounds analysed under the same conditions when available, by comparing the Kovats retention indices with the literature data and through the National Institute of Standards and Technology (NIST) MS spectral database. The quantitative evaluation was performed using the internal standard procedure and the results were finally expressed as μg/g or mg/g IS equivalents of each volatile compound. All analyses were done in triplicate.

#### 2.5. Cannabis macerated oil preparations

because it represents the most common medical variety actually prescribed alone or in combination for several pathologies. Superfine cannabis inflorescence powder was prepared using mechanical grinding-activation in an energy intensive vibrational mill. Different samples (1.0 g each) were ground in a high intensity planetary mill. The mill was vibrating at a frequency of 25 Hz for 1 min, using two 50 mL jars with 20 mm stainless steel balls. Prior to use, jars were precooled with liquid nitrogen. The speed differences between balls and jar result in the interaction of frictional and impact forces, releasing high dynamic energies. The interplay of all these forces results in the very effective energy input of planetary ball mills. Mechano-chemical technology has been developed and successfully adopted in different fields (synthesis of superfine powder, surface modification, drug and pharmaceutical applications) and could represent a novel

All extractions to define the cannabinoid profile of Bediol® medical chemotype were executed using an ASE 350 (Thermo-Fisher Scientific, Waltham, MA, USA). 34-mL stain steel cells were used for the extraction. 100 mg of Cannabis flos powder obtained by using SFG was weighed and then homogenised with an equal weight of diatomaceous earth and transferred into the

Different extraction solvents were tested and were: methanol, methanol:CH3Cl (9:1), hexane, acetonitrile and ethanol. Diatomaceous earths were added in order to fill the remaining empty part of the cell. Room temperature of 25C, pressure (1500 psi), number of static cycles (2 cycles, 5 min each), purging time (60 s with nitrogen) and rinse volume (90%) were used for the study. Organic extracts were finally collected in 66 mL vials and treated with sodium sulphate to remove any possible humidity. Afterwards, the extract was collected and dried under vacuum in a centrifugal evaporator. The residue was dissolved in 1 mL of acetonitrile and after proper dilution, 2 μL were submitted to analysis by HPLC-Q-Exactive-Orbitrap-MS. Validation was performed according to the European Union SANTE/2015 guidelines usually adopted to test

The method was completely optimised investigating the typologies of extraction solvents, number of extraction cycles and extraction temperature to define the optimum analytical conditions as well. To realise the matrix-matched calibration curves (MMCs) blank samples (100 mg officinal plant previously analysed for the absences of cannabinoids) were used and spiked with appropriate standard solution of THC, THC-A, CBD, CBD-A and CBN covering

concentrations of the extracted compounds with those from the MMC calibration curves at two

).

One gram of oil or 100 mg of inflorescence previously grinded were weighed and put into 20 mL glass vials along with 100 μL of the IS (4-nonylphenol, 2000 μg/mL in 2-propanol). Each vial was fitted with a cap equipped with a silicon/PTFE septum (Supelco, Bellefonte, PA, USA). A temperature of 37C was selected as both the extraction and equilibration temperature

. Recoveries were calculated by comparing the

) was added.

cell. Then, 100 μL of extraction solution containing the IS (diazepam 1 mg mL<sup>1</sup>

2.3. Accelerated Solvent Extraction (ASE) for cannabinoid analysis

ASE performance especially for trace residue analysis [31].

2.4. HS-SPME and GC-MS analysis for terpenes investigation

the concentration range from 0.1 to 10 μg g<sup>1</sup>

different fortification levels (1.0 and 10 μg g<sup>1</sup>

research tool.

60 Recent Advances in Cannabinoid Research

Three different methods for oil preparation were performed and evaluated. The preparation conditions were selected on the basis of previously published methods [31]. Briefly, common issues for all three methods were the amount of Bediol® inflorescence used (1 g) and the European Pharmacopoeia (FU) olive oil volume (10 mL) that served as extraction matrix. The crucial differences concerning the preheating temperature of the inflorescence to perform the decarboxylation step and extraction process are highlighted in Table 1. After extraction and cooling down (methods 1 and 2) the oils were filtrated and subsequently prepared for LC-Q-Exactive-Orbitrap-MS analysis.

## 2.6. Cannabinoids LC-Q-Exactive-Orbitrap-MS analysis

The cannabinoid profile in plants and the corresponding oil were assessed applying the method recently published with particular emphasis on method development [31]. In order to perform HPLC-Q-Exactive-Orbitrap®-MS analysis, samples extracted with ASE were prepared as indicated in Section 2.4, while oil samples were prepared by dissolving 100 mg of


obtained with an accuracy of 2 ppm m/z from total ion chromatogram (TIC) engaging the m/z corresponding to the molecular ions [M+H]+ 315,23145 for CBD and THC, 311,20020 for CBN. 317,24716 for CBG and 311,2024 for CBN. In ESI– the molecular ions [MH] considered were

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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

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

357,2164 for CBDA and THCA, while CBGA was detected by 359,22269.

3.1.1. ASE Cannabis sample preparations from Bediol® medical chemotype

screening of the cannabinoids in Cannabis sativa L. inflorescence [32, 33].

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

3. Results and discussion

3.1. Quality analysis of Cannabis inflorescences

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

each oil in 10 mL of isopropanol. After adding 1 μg/mL of IS, 10 μL of each sample were diluted in 890 μL of initial mobile phase from which 2 μL was injected.

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> , with an injection time of 200 ms. A targeted MS/MS (dd-MS<sup>2</sup> ) analysis operated in both positive and negative mode at 35,000 FWHM (m/z 200). The AGC target was set to 2e5 , with the maximum 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 obtained with an accuracy of 2 ppm m/z from total ion chromatogram (TIC) engaging the m/z corresponding to the molecular ions [M+H]+ 315,23145 for CBD and THC, 311,20020 for CBN. 317,24716 for CBG and 311,2024 for CBN. In ESI– the molecular ions [MH] considered were 357,2164 for CBDA and THCA, while CBGA was detected by 359,22269.
