molecules
Article New Methods for the Comprehensive Analysis of Bioactive Compounds in Cannabis sativa L. (hemp)
Federica Pellati 1,* , Virginia Brighenti 1, Johanna Sperlea 1,2, Lucia Marchetti 1, Davide Bertelli 1 and Stefania Benvenuti 1
1 Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 103, 41125 Modena, Italy; [email protected] (V.B.); [email protected] (J.S.); [email protected] (L.M.); [email protected] (D.B.); [email protected] (S.B.) 2 Faculty of Agricultural Sciences, Nutritional Sciences, and Environmental Management, Justus-Liebig University of Giessen, Goethestrasse 58, 35390 Giessen, Germany * Correspondence: [email protected]; Tel.: +39-059-2058565
Received: 21 September 2018; Accepted: 12 October 2018; Published: 14 October 2018
Abstract: Cannabis sativa L. is a dioecious plant belonging to the Cannabaceae family. The main phytochemicals that are found in this plant are represented by cannabinoids, flavones, and terpenes. Some biological activities of cannabinoids are known to be enhanced by the presence of terpenes and flavonoids in the extracts, due to a synergistic action. In the light of all the above, the present study was aimed at the multi-component analysis of the bioactive compounds present in fibre-type C. sativa (hemp) inflorescences of different varieties by means of innovative HPLC and GC methods. In particular, the profiling of non-psychoactive cannabinoids was carried out by means of HPLC-UV/DAD, ESI-MS, and MS2. The content of prenylated flavones in hemp extracts, including cannflavins A and B, was also evaluated by HPLC. The study on Cannabis volatile compounds was performed by developing a new method based on headspace solid-phase microextraction (HS-SPME) coupled with GC-MS and GC-FID. Cannabidiolic acid (CBDA) and cannabidiol (CBD) were found to be the most abundant cannabinoids in the hemp samples analysed, while β-myrcene and β-caryophyllene were the major terpenes. As regards flavonoids, cannflavin A was observed to be the main compound in almost all the samples. The methods developed in this work are suitable for the comprehensive chemical analysis of both hemp plant material and related pharmaceutical or nutraceutical products in order to ensure their quality, efficacy, and safety.
Keywords: Cannabis sativa L.; hemp; cannabinoids; flavonoids; terpenes; HPLC; GC; MS.
1. Introduction In recent years, Cannabis sativa L. has been the focus of the attention of the scientific community all over the world, definitely becoming one of the most studied plants [1]. It is one of the two representatives of the Cannabaceae family and nowadays it is widely distributed all over the world. C. sativa has been cultivated for a long time for medicinal, food, and abuse purposes, together with its use as a source of textile fibre [1,2]. The taxonomy of the plant has always represented a critical issue, due to the huge variability within the same genus [1–3]. Recently, a monotypic classification has been preferred, in which one species (C. sativa) is recognised and it is divided into different chemotypes on the basis of the specific cannabinoid profile [3]. Nonetheless, in the pharmaceutical field, only two phenotypes are considered, on the basis of the content in the psychoactive principle ∆9-tetrahydrocannabinol (∆9-THC) [4]. In detail, the first one is drug-type Cannabis, which is rich in psychoactive ∆9-THC, and is used for medicinal or recreational purposes [4]; the second one is fibre-type Cannabis (commonly
Molecules 2018, 23, 2639; doi:10.3390/molecules23102639 www.mdpi.com/journal/molecules Molecules 2018, 23, x 2639 FOR PEER REVIEW 22 of of 21 known as hemp or industrial hemp), used for both textile or food purposes, which has content of Δ9- THCknown below as hemp the legal or industriallimit of 0.2 hemp),%–0.3% used [2,5] and for bothis rich textile in non or-psychoactive food purposes, cannabinoids. which has Currently, content of 69∆9 -THChemp belowvarieties the have legal been limit approved of 0.2–0.3% for [2 ,commercial5] and is rich use in non-psychoactiveby the European Community cannabinoids. [6]. Currently, 69 hempThe varieties pharmaceutical have been interest approved in this for plant commercial has been use mainly by the European addressed Community to drug-type [6 ].Cannabis, thanksThe to pharmaceuticalits new therapeutic interest application in this plants [7], has while been hemp mainly is addressedat the moment to drug-type under-employedCannabis, thanksin this ambit.to its new However, therapeutic the number applications of studies [7], whilefocused hemp on the is at characterisation the moment under-employed of fibre-type hemp in this varieties ambit. andHowever, on the the evaluation number of studiesthe biological focused potential on the characterisation of non-psychoactive of fibre-type compounds hemp has varieties increased and onlately the [2,3,8evaluation–16]. of the biological potential of non-psychoactive compounds has increased lately [2,3,8–16]. The chemist chemistryry of C. sativa is known to be very complex; indeed, several chemical classes have have been identified identified in in the the plant, plant, encompassing encompassing terpenes, terpenes, carbohydrates, carbohydrates, fatty acidsfatty andacids their and esters, their amides, esters, amides,amines, amines, phytosteros, phytosteros, phenolic phenolic compounds, compounds, and cannabinoids and cannabinoids [17]. From [17]. aFro chemicalm a chemical point point of view, of view,the latter the are latter meroterpenoids are meroterpenoids and they and are they specific are ofspecific this plant of this [2, 17 plant]. Usually, [2,17]. the Usually, most abundant the most abundantcannabinoids cannabinoids present in present fibre-type in fibre plants-type are plants cannabinoic are cannabinoic acids, such acids, as cannabidiolic such as cannabidiolic acid (CBDA) acid (CBDA)and cannabigerolic and cannabigerolic acid (CBGA), acid followed (CBGA), by their followed decarboxylated by their forms, decarboxylated namely cannabidiol forms, namely (CBD) cannabidioland cannabigerol (CBD) (CBG) and (Figure cannabigerol1)[ 9]. In (CBG) plant (Figure tissues, cannabinoids1) [9]. In plant are tissue biosynthesizeds, cannabinoids in the acid are biosynthesizedform; environmental in the factors, acid form; such environmental as heat and light, factors, generally such induceas heat a and spontaneous light, generally decarboxylation induce a spontaneousprocess to form decarboxylation decarboxylated process cannabinoids to form [decarboxylated2,4,9,16,18,19]. cannabinoids [2,4,9,16,18,19].
OH O OH
OH
HO HO
Cannabidiolic acid Cannabidiol (CBDA) (CBD)
OH O OH
OH
HO HO
Cannabigerolic acid Cannabigerol (CBGA) (CBG) FigureFigure 1. 1. ChemicalChemical structures structures of of main main non non-psychoactive-psychoactive cannabinoids in hemp inflorescences. inflorescences.
Among nonnon-psychoactive-psychoactive cannabinoids, cannabinoids, CBD CBD represents represents the the most most promising promising one one from from the ppharmaceuticalharmaceutical point of view, due to its high high anti anti-oxidant-oxidant and and anti anti-inflammatory-inflammatory activity, activity, in addition to its anticonvulsant, anticonvulsant, anxiolytic, neuroprotective,neuroprotective, and antibiotic properties [[2,4,202,4,20–23].]. CBDA has antimicrobial and and anti anti-nausea-nausea properties properties [2,20,22], [2,20,22], wh whileile CBG has anti anti-inflammatory,-inflammatory, antimicrobial and analgesic activities [2,20,22,24]. [2,20,22,24]. CConcerningoncerning other other phenolic compounds, compounds, several several flavonoids flavonoids have have been been identified identified in in C.C. sativa sativa,, belonging mainly mainly to to flavones flavones and and flavonols flavonols [25]. [25 In]. particular, In particular, cannflavin cannflavin A and A B andrepresent B represent hemp- specifichemp-specific methylated methylated isoprenoid isoprenoid flavones flavones(Figure 2) (Figure [25]. Several2)[ 25 biological]. Several effects biological have been effects ascribed have tobeen hemp ascribed flavonoids, to hemp including flavonoids, anti including-inflammatory, anti-inflammatory, anti-microbial, anti-microbial, neuroprotective neuroprotective,, and anti- proliferativeand anti-proliferative activities activities [17]. In particular [17]. In, particular, cannflavin cannflavin A and B A are and known B are to known possess to an possess anti- inflammatoryan anti-inflammatory action [17] action and microsomal [17] and microsomal prostaglandin prostaglandin E2 synthase (mPGES E2 synthase-1) and (mPGES-1) 5-lipoxygenase and (55-lipoxygenase-LO) have been (5-LO) identified have been as identified their molecularas their molecular targets targets [26]. [ In26 ]. addition In addition to to flavonoids, flavonoids, dihydrostildihydrostilbenoidsbenoids repr representesent another class of polyphenolic substances isolated from hemp, of which canniprene is is the the main main representative representative (Figure (Figure 2)2)[ [25].25]. As As to tocanniprene, canniprene, it has it has been been demonstrated demonstrated to exertto exert an an anti anti-inflammatory-inflammatory activity activity as as well, well, by by inhibiting inhibiting the the production production of of prpro-inflammatoryo-inflammatory eicosanoids [8]. [8].
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O O OH OH OH O HO O HO O HO O O OH
OH O OH O OH O OH
Cannfllaviin A Cannfllaviin B Canniiprene (CFL-A) (CFL-B) FigureFigure 2. ChemicalChemical structures structures of main flavonoids flavonoids and related compounds in hemp inflorescences.inflorescences.
As regards the other compounds present in hemp, terpenes are responsible for the characteristic aroma of the plant. The The main main v volatilesolatiles detected detected in in the the aerial aerial parts parts of of the the plant plant include include both both mono mono-- and sesquit sesquiterpeneserpenes [[14,15,17,2714,15,17,27–29],], withwith β--myrcenemyrcene and β--caryophyllenecaryophyllene as the most most representative representative compounds (Figure (Figure 3),3), respectiv respectively.ely. As As regards regards monoterpenes, monoterpenes, β-myrceneβ-myrcene is known is known to poss toess possess anti- inflammatory,anti-inflammatory, analgesic analgesic,, and andanxiolytic anxiolytic propertie propertiess [17]. [As17]. for As sesquiterpenes, for sesquiterpenes, β-caryophylleneβ-caryophyllene was wasfound found to be to an be antian anti-inflammatory-inflammatory agent agent and and to to exert exert a a gastric gastric cytoprotector cytoprotector activity; activity; it it has has been demonstrated also also to to be be able able to to bind bind to to the the canna cannabinoidbinoid receptors receptors type type 2 2(CB (CB2) 2and,) and, in inthis this context, context, it canit can be be considered considered as as a phytocannab a phytocannabinoidinoid [17]. [17 ].
H H
-Myrcene -Caryophyllene Figure 3. Chemical structures of main terpenes in hemp inflorescences inflorescences..
Some biological activities of cannabinoids are are known to to be enhanced by the presenc presencee of other secondary metabolites in in C. sativa extracts, such such as as in in case casess of of sleeping disorders disorders and and anxiety anxiety [30]. [30]. This effect hashas beenbeen attributedattributed toto aa strictstrict interactioninteraction between between cannabinoids cannabinoids and and terpenes, terpenes, resulting resulting in in a asynergistic synergistic action action [30 [30].]. As As an an example, example, terpenes terpenes areare ableable toto increaseincrease blood-brainblood-brain barrier permeability and they they can can also also interact interact with with neurotransmitter neurotransmitter receptors, receptors, thus contributing thus contributing to cannabinoid-mediated to cannabinoid- mediatedanalgesic and analgesic psychotic and effects psychotic [17]. Finally, effects also [17]. flavonoids Finally, may also modulate flavonoids the pharmacokineticsmay modulate the of pharmacosome cannabinoids,kinetics of bysome means cannabinoids, of the inhibition by means of hepatic of the P450inhibition enzymes of hepatic [17]. P450 enzymes [17]. With this in view,view, the the development development and application application of advanced analytical methods for a comprehensive analysisanalysis ofofC. C. sativasativaextracts extracts plays plays a a pivotal pivotal role role in in order order to to have have a reliablea reliable evaluation evaluation of oftheir their chemical chemical composition composition both both for a higherfor a higher reproducibility reproducibility of biological of biological assays and assays also to and guarantee also to guaranteethe efficacy the and efficacy safety and in their safety pharmaceutical in their pharmaceutical use. use. In the light of allall thethe above,above, thethe presentpresent studystudy waswas aimedaimed at thethe firstfirst multi-componentmulti-component analysis of the bioactive compounds present in hemp female inflorescencesinflorescences of different varieties, including cannabinoids, hemp hemp-specific-specific phenolics,phenolics, and volatile compounds. Innovative HPLC and GC methods were developed and and applied applied in in this this study study to to the the chemical chemical classes classes of of compounds compounds cited cited above. above. In particular,In particular, the theprofiling profiling of non of- non-psychoactivepsychoactive cannabinoids cannabinoids in hemp in hemp extracts extracts was carried was carried out by means out by 2 ofmeans HPLC of HPLCwith UV/DAD, with UV/DAD, ESI-MS ESI-MS,, and ESI and-MS ESI-MS2 detection.detection. A new A RP new-HPLC RP-HPLC-UV/DAD,-UV/DAD, ESI-MS ESI-MS,, and 2 ESIand-MS ESI-MS2 method,method, together together with with a selective a selective extraction extraction protocol, protocol, was wasdeveloped developed as well as well and and applied applied for thefor thedetermination determination of hemp of hemp phenolics phenolics (including (including cannflavin cannflavin A, cannflavin A, cannflavin B, and B, canniprene). and canniprene). The Thestudy study on C. on sativaC. sativa volatilevolatile compounds compounds was was performed performed by by developing developing a a ne neww method method based based on headspace solid solid-phase-phase microextraction (HS-SPME)(HS-SPME) coupled with GC-MSGC-MS and GC GC-FID.-FID. The analytical methods developed inin thisthis studystudy representrepresent reliable reliable and and useful useful tools tools for for a completea complete characterization characterization of ofhemp hemp plant plant material material and and extracts extracts to beto be used used in thein the pharmaceutical pharmaceutical and and nutraceutical nutraceutical fields. fields.
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2. Results and Discussion Molecules 2018, 23, 2639 4 of 21 2.1. Extraction of Non-Psychoactive Cannabinoids and Flavonoids from Hemp Inflorescences The2. development Results and Discussion and application of a suitable extraction procedure covers a crucial role to achieve 2.1. a reliable Extraction characterization of Non-Psychoactive of Cannabinoids a plant material. and Flavonoids For from what Hemp concerns Inflorescences cannabinoids, a simple dynamic maceration (DM) at room temperature was applied by using ethanol (EtOH) as the The development and application of a suitable extraction procedure covers a crucial role to extractionachieve solvent, a reliable which characterization has been demonstrated of a plant material. to be For the what best concerns solven cannabinoids,t for the extraction a simple of these bioactivedynamic compounds maceration [9]. (DM)EtOH at roomis indeed temperature a good was solvent applied for by using the extraction ethanol (EtOH) of asmany the extraction phenolics from plant materialsolvent, [31 which]. As has a matter been demonstrated of fact, cannflavins to be the bestA and solvent B were for thedetected extraction in hemp of these ethanolic bioactive extracts as well, compounds but, conversely [9]. EtOH to is cannabinoids, indeed a good solvent their content for the extraction in the inflorescences of many phenolics was from extremely plant low [8,10,25];material in addition, [31]. As the a matter concomitant of fact, cannflavins presence A and of Bcannabinoids were detected in in hemp the same ethanolic extract extracts made as them well, but, conversely to cannabinoids, their content in the inflorescences was extremely low [8,10,25]; difficult into addition,be quantified. the concomitant For this presence reason, of two cannabinoids different in protocols the same extractwere madeoptimised them difficultand app tolied be in this work forquantified. the extraction For this of reason, non-psychoactive two different protocols cannabinoids were optimised and flavonoids and applied from in this hemp. work for the Startingextraction with of the non-psychoactive extraction conditions cannabinoids previously and flavonoids optimised from hemp. for cannabinoids, the method was modified as Startingfollows. with First, the extractionthe solvent conditions was properly previously chosen optimised in forord cannabinoids,er to have a the good method yield was of hemp flavonoidsmodified from asthe follows. plant First,material; the solvent to do wasthis properly EtOH, chosenacetone in and order ethyl to have acetate a good ( yieldEtOAc of) hemp were tested. flavonoids from the plant material; to do this EtOH, acetone and ethyl acetate (EtOAc) were tested. The extracts were also concentrated 25:1 before HPLC analysis, in order to increase the intensity of The extracts were also concentrated 25:1 before HPLC analysis, in order to increase the intensity of the the UV/DADUV/DAD signal. signal. Of Ofthe the solvents solvents tested, tested, acetoneacetone resulted resulted in beingin be theing best the onebest and one it wasand thereforeit was therefore selected selectedfor use forin usethe insubsequent the subsequent steps steps (Figure (Figure 44).).
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0 10 12 14 16 18 20 22 24 min Figure 4.Figure HPLC 4.-HPLC-UV/DADUV/DAD chromatograms chromatograms of of a ahemp hemp extract (sample (sample C6) C6) in acetone in acetone (blue line),(blue EtOAc line), EtOAc (red line)(red and line) EtOH and EtOH(green (green line), line), recorded recorded at at 342 342 nm.nm. For For peak peak numbering, numbering, see Table see1 Table. 1. In order to reduce any possible interference from cannabinoids in the concentrated extract, In ordera sample to pre-treatmentreduce any possible was considered interference necessary. from Indeed, cannabinoids the amount ofin cannabinoidsthe concentrated in hemp extract, a sample inflorescences pre-treatment is much was higher considered than that necessary. of flavonoids; Indeed, thus, thethe peaks amount related of to thesecannabinoids compounds in hemp inflorescencesin the extracts is much may higher interfere than with that the of quantification flavonoids; of thus, other the constituents peaks related at 210 nm. to these Conversely compounds to in other phenolics, cannabinoids can dissolve also in non-polar solvents (i.g. n-hexane, chloroform, the extracts may interfere with the quantification of other constituents at 210 nm. Conversely to other dichloromethane (DCM)) [9]; hence, a sample pre-treatment by means of DM repeated three times phenolics,with cannabinoids a non-polar solvent, can including dissolven-hexane also and in its non combination-polar solv withents DCM, (i.g. ethyl ethern-hexane, and toluene, chloroform, dichlorometwas testedhane to(DCM be suitable)) [9]; to hence, remove a cannabinoids. sample pre After-treatment this treatment, by means the residue of DM was repeated extracted three three times with a nontimes-polar with solvent, acetone by including means of DM. n-hexane As shown and in its Figure combination5, n-hexane with alone DCM resulted, ethyl in being ether the mostand toluene, was testedsuitable to be solvent suitable for the to sampleremove pre-maceration cannabinoids. in comparison After this with treatment, other non-polar the residue solvents was tested. extracted Indeed, its combination with DCM or ethyl ether or toluene, led to a decrease in the final amount of three times with acetone by means of DM. As shown in Figure 5, n-hexane alone resulted in being flavonoids extracted from hemp. the most suitable solvent for the sample pre-maceration in comparison with other non-polar solvents tested. Indeed, its combination with DCM or ethyl ether or toluene, led to a decrease in the final amount of flavonoids extracted from hemp.
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10 12 14 16 18 20 22 24 min 18 19 20 21 22 23 24 min 0 0 (a) (b) 10 12 14 16 18 20 22 24 min 18 19 20 21 22 23 24 min (a) (b) Figure 5. (a) Effect of different pre-maceration procedures on flavonoid extraction from hemp Figure(sample 5. (C6).(aa)) Effect Effect Chromatograms of of different different pre-maceration obtained pre-maceration at 342 procedures nm procedures with onpre flavonoid on-maceration flavonoid extraction with extraction n- fromhexane from hemp (3x) hem (sample (bluep (sampleC6).line), Chromatograms with C6). n -Chromatogramshexane (2x) obtained + dichloromet obtained at 342 nm hatane 342 with (DCM nm pre-maceration with) (1x) pre (red-maceration line), with withn-hexane withn-hexane n- (3x)hexane (2 (bluex) + (3x)ethyl line), (blue ether with line),n-(1x)hexane with(green(2x) n- line),hexane + dichloromethane with (2x) n -+hexane dichloromet (2 (DCM)x) + htolueneane (1x) (DCM (red(1x)) line),(pink(1x) (red withline line),).n- Forhexane with peak n (2x)numbering,-hexane + ethyl (2x) ethersee + ethyl Table (1x) ether 1. (green (b ) (1x)line),Effect (green with of line),n- prehexane-maceration with (2x)n-hexane + toluene with (2 x)n -+ (1x)hexane toluene (pink on(1x) line). cannabinoid (pink For line peak). Forremoval numbering, peak numbering, from see hemp Table see 1 (sample .(Tableb) Effect1. ( C6).b) of Effectpre-macerationChromatograms of pre-maceration with obtainedn-hexane with at 210onn-hexane cannabinoid nm with on pre cannabinoid removal-maceration from removal with hemp n - (samplehexanefrom hemp (3x)C6). (blue Chromatograms (sample line) C6). and Chromatogramsobtainedwithout atpre 210-maceration nm obtained with pre-maceration(red at 210line). nm For withpeak with prenumbering,n-hexane-maceration (3x) see (bluewithTable line)n1.-hexane and without (3x) (blue pre-maceration line) and without(red line). pre For-maceration peak numbering, (red line). see For Table peak1. numbering, see Table 1.
Finally, in order to facilitate the the removal removal of of cannabinoids, cannabinoids, a a decarboxylation decarboxylation of of the the plant plant material, material, priorFinally, to to the the in pre-maceration order pre-maceration to facilitate with withthen-hexane, removal n-hexane, was of cannabinoids, also was taken also into taken a accountdecarboxylation into in account order toof in convertthe order plant cannabinoic tomaterial, convert priorcannabinoicacids to into the their acids pre neutral-maceration into their counterparts, neutral with countern-hexane, whichparts, are was morewhich also soluble are taken more in into thissoluble accountsolvent. in this In in solvent. particular, order In to particular, theconvert plant ◦ ◦ cannabinoicthematerial plantwas materialacids heated into was their at 120 heated neutralCfor at counter 1 120 hor °Cparts, at for 80 which 1C h for or are 2 at h, more 80respectively. °Csoluble for 2in h, Afterthis respectively. solvent. this decarboxylation In particular, After this thedecarboxylationstep, plant the pre-maceration material step, was the heated process pre- maceration at with 120n -hexane °C processfor 1 and h with the or at extraction n 80-hexane °C for with and 2 acetone h,the respectively. extraction previously with After optimised aceto thisne decarboxylationpreviouslywere applied. optimised As step, shown the were in pre Figure applied.-maceration6 , the As peak shown process areas in with related Figure n-hexane to 6, flavonoidsthe and peak the areas decreased extraction related after with to the flavonoids aceto heatingne previouslydecreasedprocess and optimisedafter the the removal heating were of cannabinoidsprocessapplied. and As the shown was removal not in significantly Figure of cannabinoids 6, the increased; peak was areas therefore,not significantly related it was to flavonoids notincreased; further decreasedtherefore,pursued. after Init was the the lightnot heating further of all process the pursued. above, and aIn the two-step the removal light extraction,of of all cannabinoids the above, based a on wastwo an -notstep initial significantly extraction, pre-maceration basedincreased; on of thean therefore,initialplant material pre it- macerationwas with not furthern-hexane of the pursued. and plant on material In the the subsequent light with of nall-hexane extraction the above, and with a on two acetone, the-step subsequent extraction, was selected extraction based as theon with bestan initialacetone,protocol pre was- formaceration hemp selected flavonoids. ofas the best plant protocol material for with hemp n- hexaneflavonoids. and on the subsequent extraction with acetone, was selected as the best protocol for hemp flavonoids. mAU mAU
120 mAU 7 mAU 120 2500 100 7 1 4 2500 2000 100 1 4 80
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500 0 20 0 10 12 14 16 18 20 22 24 min 0 18 19 20 21 22 23 24 min 0 10 12 14 16 (a) 18 20 22 24 min 18 19 20 21 (b) 22 23 24 min (a) (b) Figure 6. (a) Effect of different thermal treatments on flavonoid extraction from hemp (sample C6).
FigureChromatograms 6. (a) Effect obtained of different at 342 th nmermal without treatments thermal on treatment flavonoid (blue extraction line), withfrom thermal hemp (sample treatment C6). at FigureChromatograms80 ◦C 6. for (a 2) hEffect (red obtained line),of different with at thermal342 th ermalnm without treatment treatments thermal at 120on flavonoidtreatment◦C for 1 h (green(blueextraction line), line). from with For hempthermal peaknumbering, (sample treatment C6). see at Chromatograms80Table °C 1for.( 2b )h Effect(red obtained line), of different with at 342 thermal thermalnm without treatment treatments thermal at 120 on treatment °C cannabinoid for 1 h (blue (green removalline), line). with For from thermal pe hempak numb treatment (sampleering, C6).atsee 80TableChromatograms °C for 1 .2 ( bh) (red Effect line), obtained of differentwith thermal at 210 thermal nm treatment without treatment at thermal 120s on °C cannabinoid for treatment 1 h (green (blue removal line). line), For from with pe akhemp thermal numb (sampleering, treatment seeC6). TableChromatogramsat 80 1◦. C(b for) Effect 2 h (red obtainedof different line), withat 210thermal thermal nm without treatment treatment thermals on at cannabinoid 120treatment◦C for 1(blue hremoval (green line), line). fromwith Forhempthermal peak (sample treatment numbering, C6). at Chromatograms80see °C Table for 12. h (red obtained line), with at 210 thermal nm without treatment thermal at 120 treatment °C for 1 h (blue (green line), line). with For thermal peak numbering, treatment atsee 80Table °C for 1. 2 h (red line), with thermal treatment at 120 °C for 1 h (green line). For peak numbering, see TableThe 1. optimized extraction techniques, based on dynamic maceration at room temperature, did not causeThe the optimized degradation extraction of the compounds techniques, of based interest on or dynamic the formation maceration of artefacts. at room temperature, did not Thecause optimized the degradation extraction of thetechniques, compounds based of intereston dynamic or the maceration formation ofat artefacts.room temperature, did not cause the degradation of the compounds of interest or the formation of artefacts. Molecules 2018, 23, x; doi: FOR PEER REVIEW www.mdpi.com/journal/molecules Molecules 2018, 23, x; doi: FOR PEER REVIEW www.mdpi.com/journal/molecules Molecules 2018, 23, 2639 6 of 21 Molecules 2018, 23, x FOR PEER REVIEW 6 of 21
2.2.2.2. HPLC Metho Methodsds for the Analysis of Non-PsychoactiveNon-Psychoactive Cannabinoids and Flavonoids in HempHemp ExtractsExtracts The aim of the present study was to provide a comprehensive analysis of the main bioactive compounds in fibre-typefibre-type C.C. sativa sativa inflorescences.inflorescences. In In this perspective, the developmentdevelopment of HPLC methods for thethe determinationdetermination ofof non-psychoactivenon-psychoactive cannabinoids cannabinoids and and hemp-specific hemp-specific phenolics phenolics was was a criticala critical aspect aspect of the of research. the research. RP-HPLC RP-HPLC on C18 oncolumns, C18 columns, with acidic with methanol acidic (MeOH)methanol or ( acetonitrileMeOH) or (ACN)acetonitrile used (ACN as the) used strong as solventthe strong eluent solvent and eluent acidic and H2O acidic as the H weak2O as the one, weak is frequently one, is frequently used for theused analysis for the of analysis cannabinoids of cannabinoids in C. sativa in C.extracts sativa [extracts3,9,13,16 [3,9,13,16].]. Indeed, Indeed, the use the of an use acidic of an mobile acidic phasemobile improves phase improves the separation the separation performance, performance, providing providing better peak better shape peak and shape enhanced and resolution, enhanced alongresolution, with along the enhancement with the enhancement of their ionization of their ionization in HPLC-ESI-MS in HPLC and-ESI- MSMS2 andexperiments MS2 experiments [3,9,13]. For[3,9,13]. acidification, For acidification, HCOOH HCOOH is usually is usually employed employed [3,9,13 [3,9,13].]. For our For purpose, our purpose, an Ascentis an Ascentis Express Express C18 (150C18 (150× 3.0 × 3.0 mm mm I.D., I.D., 2.7 2.7µm, µm, Supelco) Supelco) as as the the chromatographic chromatographic column column and and aa gradientgradient mobilemobile phase composed of of H H2O2O and and ACN, ACN, both bot containingh containing 0.1% 0.1% of HCOOH of HCOOH were selected, were selected, on the basis on the of abasis previous of a workprevious [9]. work [9]. The gradient gradient elution elution with with the thesame same mobile mobile phase phase was then was properly then properly modified modifiedfor hemp flavonoids, for hemp sinceflavonoids, peaks since corresponding peaks corresponding to cannflavins to cann Aflavins and B wereA and observed B were observed to elute to with elute the with solvent the solvent front. Underfront. Under the final the conditions, final conditions, a good a resolutiongood resolution of peaks of peaks belonging belonging to cannflavins to cannflavins A and A B and and B other and hempother hemp phenolics phenolics was achieved.was achieved. Representative Representative chromatograms chromatograms obtained obtained by by using using the the twotwo HPLC methodsmethods are shown in Figure7 7..
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40 200
20 100 2 0 3 0 0 5 10 15 20 25 30 min 0 5 10 15 20 25 min (a) (b)
Figure 7. (a) Representative HPLC-UV/DAD chromatogram of a hemp extract (sample C6) at 342 nm obtainedFigure 7. by(a) usingRepresentative the analytical HPLC method-UV/DAD optimised chromatogram for flavonoids of a hemp and relatedextract (sample compounds. C6) at For 342 peak nm numbering,obtained by seeusing Table the1 .(analyticalb) Representative method optimised HPLC-UV/DAD for flavonoids chromatogram and related of a compounds. hemp extract For (sample peak C6)numbering, at 210 nm see obtained Table 1 by. (b using) Representative the analytical HPLC method-UV/DAD optimised chromatogram for cannabinoids. of a hemp For peak extract numbering, (sample seeC6) Table at 2101. nm obtained by using the analytical method optimised for cannabinoids. For peak numbering, see Table 1. 2.3. Identification of Non-Psychoactive Cannabinoids and Flavonoids in Hemp Extracts 2.3. IdentificationThe identification of Non-Psychoactive of secondary Cannabinoids metabolites and in Flavonoids hemp extracts in Hemp was Extracts carried out by combining the informationThe identification obtained of secondary from UV/DAD metabolites and in MS hemp detectors. extracts was For carried a complete out by characterisation, combining the theinformation experimental obtained data were from compared UV/DAD with and the MS literature detectors. [3,9 ,12 For,32 a] and complete with commercially characterisation, available the referenceexperimental standards. data were compared with the literature [3,9,12,32] and with commercially available referenceThe UVstandards. spectra were used to preliminarily assign the chromatographic peaks to a chemical class. As aThe matter UV ofspectra fact, cannabinoicwere used to acids, preliminarily such as assign CBDA the and chromatographic CBGA, can be easily peaks distinguished to a chemical class. from theirAs a decarboxylatedmatter of fact, cannabinoic counterparts acids, (CBD such and as CBG, CBDA respectively) and CBGA, by c theiran be UV/Vis easily spectra:distinguished CBDA from and CBGAtheir de arecarboxylated indeed characterised counterparts by (CBD three absorptionand CBG, respectively) maxima (λmax by), their one at UV/Vis 220–223 spectra: nm, a secondCBDA and one at 266–270 nm and a third one around 305 nm; CBD and CBG show a first λ at 210–215 nm and an CBGA are indeed characterised by three absorption maxima (λmax), one at 220max–223 nm, a second one additional one at 270 nm [9,13]. Canniprene showed a UV/Vis spectrum comparable to that of neutral at 266–270 nm and a third one around 305 nm; CBD and CBG show a first λmax at 210–215 nm and an cannabinoids,additional one with at 270 a λnmmax [9,13].at 204 Canniprene nm and a minor showed one a at UV/Vis 280 nm. spectrum Also cannflavins comparable can to be that recognised of neutral on the basis of their UV/Vis spectra, since they possess a representative chromophore: indeed, the spectra cannabinoids, with a λmax at 204 nm and a minor one at 280 nm. Also cannflavins can be recognised on the basis of their UV/Vis spectra, since they possess a representative chromophore: indeed, the spectra of cannflavins A and B showed a first λmax at 213–215 nm, a second one at around 273–274 nm and, finally, one at 342 nm.
Molecules 2018, 23, x; doi: FOR PEER REVIEW www.mdpi.com/journal/molecules Molecules 2018, 23, 2639 7 of 21
of cannflavins A and B showed a first λmax at 213–215 nm, a second one at around 273–274 nm and, finally, one at 342 nm. As regards MS and MS2, the experiments were carried out both in the positive and in the negative ion mode. The MS and MS2 data are shown in Table1. In particular, cannabinoic acids showed a higher intensity of the signals in the negative ion-mode, as opposed to decarboxylated compounds and canniprene, which ionised better in the positive ion mode. Conversely to cannabinoids, flavonoids (cannflavin A and B) were only detectable when positively ionised. As regards the MS data acquired in the positive ion mode, both CBDA and CBGA generated sodium adduct ions [M + Na]+ [3], in addition to their pseudo-molecular ions [M + H]+. CBGA was easily distinguished from CBDA, thanks to its different molecular weight. As to their MS2 spectra carried out in the positive ion mode, both CBDA and CBGA showed base peaks related to the loss of water (−18 u) at m/z 341 and 343, respectively. CBDA showed an additional product ion at m/z 285, 2 attributable to the loss of a C4H8 group [9,33], which was not observed in the MS spectrum of CBGA. Both CBG and CBD showed product ions at m/z 233 and 207 in the MS2 experiments performed in the positive ion mode, although their relative intensity was different. The product ion at m/z 259 in the 2 MS spectrum of CBD was due to the loss of a C4H8 group [9,33], probably from the side alkyl chain, which was absent in the MS2 spectrum of CBG. As to canniprene, the MS spectrum carried out in the positive ion mode showed the pseudo-molecular ion [M+H]+ at m/z 343 and a base peak at m/z 287, due to the loss of a C4H8 group of the side prenyl group. In the MS spectra of cannflavins A and B only the pseudo-molecular ion [M + H]+ was observed. The MS2 spectra of these compounds showed only one intense fragment at m/z 313, generated after the loss of the side prenyl moiety. As for the MS data obtained in the negative ion mode, both CBDA and CBGA generated product ions corresponding to the loss of water (−18 u) and of CO2 (−44 u), together with their pseudo-molecular ions [M − H]−. Concerning the MS2 spectra acquired in the negative ion-mode, CBG generated a product ion at m/z 247, due to the loss of a C5H8 group; additional fragments were observed at m/z 297, 271, and 204. The fragmentation of CBD in the negative ion-mode provided almost exclusively one product ion at m/z 245, attributable to the loss of a C5H8 group, following a retro Diels-Alder reaction, which is a common fragmentation pathway in the field of natural substances when an ESI ion source is employed [9,34]. Canniprene generated its pseudo-molecular ion [M − H]− at m/z 341 in the negative ion mode; its MS2 spectrum showed a major product ion at m/z 326, attributable to the loss of a methyl group. It should be specified that the two peaks that precede each cannflavin belong to different flavonoids, which have not been identified. Even though the UV/Vis spectrum was observed to resemble those of flavones, the molecular mass and the fragmentation pattern was found to be different in the MS and MS2 experiments.
2.4. Validation Data of the HPLC Methods Over the concentration range tested, the two HPLC-UV/DAD methods showed good linearity (r2 > 0.999) for the reference standards chosen in this study (Table S1). The limit of detection (LOD) values for chrysoeriol and canniprene were 0.4 and 0.1 µg/mL, respectively, while for cannabinoids it ranged from 0.4 and 0.8 µg/mL. The limit of quantification (LOQ) values obtained for cannflavins and canniprene were 1.3 and 0.3 µg/mL, respectively, while for cannabinoids it was in the range 1.3–2.5 µg/mL (Table S1). The low intra- and inter-day % relative standard deviation (%RSD) for retention times (≤2.4%) and peak areas (≤3.2%) relative to the target compounds (Table S2) and their low intra- and inter-day SD values for content (≤21.3 µg/g) (Table S3) indicate the high precision of both the chromatographic system and the extraction procedure. By taking into account all the information described above, it can be concluded that this method is a reliable tool for the identification and quantification of flavonoids in fibre-type hemp, conforming to the ICH guidelines. Molecules 2018, 23, 2639 8 of 21
Table 1. Retention times, UV, MS, and MS2 data of the main non-psychoactive cannabinoids, flavonoids and related compounds in hemp extracts.
a 2 a b 2 b Peak Number Compound tR (min) UV λmax (nm) MS (m/z) MS (m/z) MS (m/z) MS (m/z) 1 CBDA 13.8 225,269,307 359, 341 * 219 (100), 261 (55), 285 (33), 232 (22) 357 339 (100), 313 (9) 2 CBGA 15.7 223,268,305 361, 343 * 219 (100), 261 (49), 233 (19) 359 341 (100), 359 (6) 3 CBG 17.1 207,231sh,273 317 207 (100), 233 (40), 280 (10) 315 271 (100), 204 (80), 247 (51), 297 (49) 4 CBD 17.9 209,232sh,276 315 259 (100), 233 (26), 221 (24), 207 (24) 313 245 (100), 210 (6) 5 CFL-B 14.5 215,273,342 370 313 (100) - - 6 Canniprene 16.9 204,280 343, 287 * 255 (100), 227 (98), 269 (11) 341 326 (100), 269 (84), 283 (43), 257 (12) 7 CFL-A 21.1 214,274,342 437 313 (100) - - Experimental conditions as described in Sections 3.4 and 3.5. a Positive ion mode. b Negative ion mode. * Precursor ion. Molecules 2018, 23, 2639 9 of 21
2.5. Quantitative Analysis of Non-Psychoactive Cannabinoids in Hemp by HPLC-UV/DAD Quantitative data related to the content of non-psychoactive cannabinoids in hemp inflorescences determined by means of the HPLC-UV/DAD method are shown in Table2. Since all the samples analysed are fibre-type hemp varieties, it is not surprising that the most abundant cannabinoids were represented by CBDA and CBD [9]. In particular, samples C1, C3, and C6 showed the typical cannabinoid profiles of the fresh plant material, with CBDA as the main compound, followed by CBD. On the other hand, samples C2, C4, and C5 showed the opposite behaviour: as a matter of fact, CBD represented the most abundant cannabinoid in these samples, followed by CBDA. This profile may be attributed either to an aged plant material or to a non-optimal drying process applied to the inflorescences or to unsuitable storage conditions that may have led to a partial conversion of the parent cannabinoic acid into its neutral counterpart, via a decarboxylation process, which naturally occurs with time under the action of heat and light [9]. The same trend was observed also for the other two major non-psychoactive cannabinoids, namely CBGA and CBG, though their content was about 10 times lower in comparison to CBDA and CBD and, in some cases, not quantifiable. Nevertheless, the amount of non-psychoactive cannabinoids determined in the analysed samples is in agreement with what is described in the literature for the content of cannabinoids in hemp female inflorescences [9].
Table 2. Amount (mg/g) of the main non-psychoactive cannabinoids in hemp inflorescences a.
Compound C1 C2 C3 C4 C5 C6 CBDA 22.6 ± 2.5 4.1 ± 1.7 36.4 ± 1.0 4.1 ± 0.1 3.7 ± 0.2 17.6 ± 0.4 CBGA 0.4 b 0.2 ± 0.1 0.6 b 0.2 b 2.6. Quantitative Analysis of Flavonoids and Related Compounds in Hemp by HPLC-UV/DAD The content of the two flavonoids cannflavin A and B and the stilbenoid canniprene in the hemp samples analysed in this work is shown in Table3. Canniprene was not detected in any of the samples analysed, even though its presence has been previously reported in the literature for fibre-type hemp varieties, comprising C2 and C3 [8]. However, Allegrone et al. [8] observed that a huge variation in the amount of canniprene in hemp inflorescences may occur; this can explain why this compound was not detected in any of the samples analysed in this study [8]. As regards flavonoids, cannflavin A was observed to be the most abundant one in all the samples, with the only exception of sample C3, which had a high content of both cannflavins A and B. These quantification data are in good agreement with the existing literature [8,10,25]. Table 3. Amount (µg/g) of the main flavonoids and related compounds in hemp inflorescences a. Compound C1 C2 C3 C4 C5 C6 CFL-A 163.4 ± 2.4 109.4 ± 5.7 318.9 ± 5.8 59.9 ± 3.4 89.5 ± 6.7 140.6 ± 12.5 CFL-B 100.9 ± 1.7 21.0 ± 2.6 412.2 ± 4.0 25.3 ± 0.2 61.4 ± 5.7 75.9 ± 6.6 Canniprene 2.7. Extraction of Volatile Compounds from Hemp by HS-SPME The terpene composition of hemp inflorescences has been found to enhance the biological activity of hemp-based products [30]; in this perspective, the development of a fast and reliable procedure for the extraction of the volatile compounds from the plant material is highly recommended for its complete characterisation. Molecules 2018, 23, 2639 10 of 21 Molecules 2018, 23, x FOR PEER REVIEW 10 of 21 HS-SPMEHS-SPME is is being being increasingly increasingly selected selected as as the the technique technique of choiceof choice in thein the ambit ambit ofthe of the extraction extraction of volatileof volatile compounds compounds in the in field the field of natural of natural product product research research [14,15 ,[14,15,27,28].27,28]. In this In study, this study, a new HS-SPMEa new HS- procedureSPME procedure was optimised was optimised in order in to order achieve to achieve a reliable a reliable characterisation characterisation of the volatiles of the volatiles from hemp from inflorescences.hemp inflorescences. To do this, To do sample this, sample C3 was C used3 was for used the optimisationfor the optimisation of the HS-SPME of the HS conditions,-SPME conditions, as the essentialas the essential oil from oil the from same the variety same wasvariety also was available. also available. Firstly,Firstly, the the performance performance of of two two different different SPME SPME fibres fibres was was assessed, assessed, including including both both a a polydimethylsiloxanepolydimethylsiloxane (PDMS) (PDMS and) a divinylbenzene-carboxen-polydimethylsiloxaneand a divinylbenzene-carboxen-polydimethylsiloxane (DVB/CAR/ PDMS)(DVB/CAR/PDMS fibre. The representative) fibre. The representa GC-FID chromatogramstive GC-FID chromatograms of the HS profile of theof hemp HS profile inflorescences of hemp obtainedinflorescences by using obtained the PDMS by using and the the PDMS DVB/CAR/PDMS and the DVB/CAR/PDMS fibres are shown fibres inare Figure shown8. in In Figure order 8. toIn compare order to thecompare fibre performance,the fibre performance, the same the temperature, same temperature, equilibrium equilibrium time, and time extraction, and extraction time weretimeemployed. were employed. The extraction The extraction efficiency efficiency of volatile of volatile compounds compounds was foundwas found to be to higher be higher when when the DVB/CAR/PDMSthe DVB/CAR/PDMS fibre fibre was was used; used; this this fibre fibre was was therefore therefore selected selected for thisfor this work. work. pA pA 1600 1400 800 1200 600 1000 800 400 600 400 200 200 0 0 0 5 10 15 20 25 30 35 40 45 min 0 5 10 15 20 25 30 35 40 45 min (a) (b) FigureFigure 8. 8.Effect Effect of of fibre fibre type type on on the the HS-SPME-GC-FID HS-SPME-GC-FID profiles profiles of of hemp hemp inflorescences inflorescences (sample (sample C3): C3): (a()a PDMS) PDMS fibre; fibre; (b ()b DVB/CAR/PDMS) DVB/CAR/PDMS fibre.fibre. ForFor the the outcome outcome of of the the overall overall extraction extraction procedure, procedure, it it is is also also important important to to properly properly set set the the extractionextraction temperature, temperature, together together with with the the equilibrium equilibrium time time and and the the extraction extraction time. time. On On the the basis basis of of ◦ previousprevious data data available available in in the the literature literature [ 14[14,15,27,28],,15,27,28], temperatures temperatures of of 40, 40, 60, 60 and, and 80 80 C,°C, equilibrium equilibrium timestimes of of 20 20 and and 30 30 min min and and extraction extraction times times of of 10 10 and and 20 20 min min were were evaluated evaluated in in this this study. study. As As concerns concerns thethe effect effect of of temperature, temperature, a a lower lower temperature temperature was was found found to to be be much much better, better, since since an an increase increase in in the the extractionextraction temperature temperature led led to to a a loss loss in in monoterpenes monoterpenes with with respect respect to to the the sesquiterpenes. sesquiterpenes. In In general, general, ◦ thethe chromatographic chromatographic profile profile of of hemp hemp volatile volatile compounds compounds at at 40 40 C°C was was characterised characterised by by higher higher peak peak areasareas (Figure (Figure9); 9); therefore, therefore, it it was was finally finally selected selected as as the the optimal optimal extraction extraction temperature. temperature. Being Being the the samplesample submitted submitted to toa relatively a relatively low lowtemperature, temperature, longer longer equilibrium equilibrium and extraction and extraction times are times usually are requiredusually forrequired a satisfactory for a satisfactory recovery ofrecovery volatile of compounds; volatile compounds; indeed, higher indeed, peak higher areas peak were areas obtained were fromobtained hemp from when hemp an equilibriumwhen an equilibrium time and time an extraction and an extraction time of 30time and of 2030 and min, 20 respectively, min, respectively, were appliedwere ap (Figureplied (Figure 10). 10). InIn Figure Figure 11 , 11, a representative a representative GC-FID GC chromatogram-FID chromatogram obtained obtained with the final with HS-SPME the final conditions HS-SPME isconditions shown, reflecting is shown, the reflecting profile of the the profile essential of the oil distilledessential from oil distilled the same from plant the material. same plant material. Molecules 2018, 23, x; doi: FOR PEER REVIEW www.mdpi.com/journal/molecules Molecules 2018, 23, x FOR PEER REVIEW 11 of 21 Molecules 2018, 23, 2639 11 of 21 Molecules 2018pA, 23, x FOR PEER REVIEW 11 of 21 Molecules 20181600 , 23, x FOR PEER REVIEW 11 of 21 1400 pA 16001200 pA 1000 1400 1600 800 1200 1400 600 1000 1200 400 800 1000 200 600 800 0 400 600 0 5 10 15 20 25 30 35 40 45 min 200 400 0 200 0 5 10 15 20 (a)25 30 35 40 45 min 0 pA 0 5 10 15 20 25 30 35 40 45 min 1750 (a) 1500pA (a) 1750 1250 pA 150010001750 12507501500 10005001250 7502501000 500 0 750 0 5 10 15 20 25 30 35 40 45 min 250 500 0 250 0 5 10 15 20 (b)25 30 35 40 45 min 0 pA 0 5 10 15 20 25 30 35 40 45 min 1600 (b) 1400 pA (b) 16001200 pA 1000 1400 1600 800 1200 1400 600 1000 1200 400 800 1000 200 600 800 0 400 600 0 5 10 15 20 25 30 35 40 45 min 200 400 0 200 (c) 0 5 10 15 20 25 30 35 40 45 min 0 0 5 10 15 20 (c) 25 30 35 40 45 min Figure 9. Effect of temperature on the HS-SPME-(GCc) -FID profiles of hemp inflorescences (sample C3) FigureFigurewith the 9.9. EffectDVB/CAR/PDMS of temperature fibre: on ( thea) 40 HS-SPME-GC-FIDHS °C;-SPME (b) 60-GC °C;- FID(c) 80 profilesprofiles °C. of hemp inflorescencesinflorescences (sample C3) Figure 9. Effect of temperature on the HS-SPME-GC-FID profiles of hemp inflorescences (sample C3) withwith thethe DVB/CAR/PDMSDVB/CAR/PDMS fibre: fibre: (a (a) )40 40 °C;◦C; (b (b) )60 60 °C;◦C; (c ()c )80 80 °C.◦C. pA with the DVB/CAR/PDMS fibre: (a) 40 °C; (b) 60 °C;pA (c) 80 °C. 1600 1600 pA pA 1400 1400 1600 pA 1600 pA 1200 1600 1200 1600 1400 1400 1000 1400 1000 1400 1200 1200 800 1200 800 1200 1000 1000 600 1000 600 1000 800 800 400 800 400 800 600 600 200 600 200 600 400 400 0 400 0 400 200 200 0 5 10 15 20 25 30 35 40 45 min 0 200 5 10 15 20 25 30 35 40 45 min 200 0 0 (a) (b) 0 0 5 10 15 20 25 30 35 40 45 min 0 0 5 10 15 20 25 30 35 40 45 min 0 5 10 15 20 (a) 25 30 35 40 45 min 0 5 10 15 (20 b) 25 30 35 40 45 min Figure 10. Effect of the (equilibriuma) time and extraction time on the HS-SPME(b)- GC-FID profiles of FigureFigurehemp 10. inflorescences10. EffectEffect of of the the equilibrium(sample equilibrium C3) time withtime and theand extraction DVB/CAR/PDMS extraction time time on the on fibre HS-SPME-GC-FIDthe atHS 40-SPME °C: - (GCa) profilesequilibrium-FID profiles of hemp and of Figure 10. Effect of the equilibrium time and extraction time on◦ the HS-SPME-GC-FID profiles of inflorescenceshempextraction inflorescences time (sample of 20 and (sample C3) 10 with min, C3) the respectively; with DVB/CAR/PDMS the DVB/CAR/PDMS (b) equilibrium fibre at 40and fibreC: extraction at (a ) 40 equilibrium °C: time (a) equilibriumof and30 and extraction 20 min, and hemp inflorescences (sample C3) with the DVB/CAR/PDMS fibre at 40 °C: (a) equilibrium and timeextractionrespectively. of 20 andtime 10 of min, 20 and respectively; 10 min, respectively; (b) equilibrium (b) equilibrium and extraction and time extraction of 30 and time 20 min,of 30 respectively.and 20 min, extraction time of 20 and 10 min, respectively; (b) equilibrium and extraction time of 30 and 20 min, respectively. pA respectively. pA 1600 pA pA 1400 800 1600 pA pA 1200 1600 1400 800 600 1000 1400 800 1200 800 1200 600 1000 400 600 600 1000 800 400 800 400 600 200 400 200 600 400 200 0 400 200 0 200 0 200 5 10 15 20 25 30 35 40 45 min 0 5 10 15 20 25 30 35 40 45 min 0 (a) 0 (b) 0 0 5 10 15 20 25 30 35 40 45 min 0 0 5 10 15 20 25 30 35 40 45 min 0 5 10 15 (20 a) 25 30 35 40 45 min 0 5 10 15 20(b) 25 30 35 40 45 min FigureFigure 11. 11. (a(a)) GC-FID GC-FID( chromatograma chromatog) ram of of hemp hemp inflorescencesinflorescences obtainedobtained by by applying applying(b) the the HS-SPME HS-SPME procedureFigureprocedure 11. optimized (optimizeda) GC-FID (sample (sample chromatog C3); C3) (ram; b(b)) GC-FID GC of hemp-FID chromatogram chromatogram inflorescences of of obtained the the essential essential by applyingoil oil of of hemp hemp the sample sample HS-SPME C3. C3. Figure 11. (a) GC-FID chromatogram of hemp inflorescences obtained by applying the HS-SPME procedure optimized (sample C3); (b) GC-FID chromatogram of the essential oil of hemp sample C3. procedure optimized (sample C3); (b) GC-FID chromatogram of the essential oil of hemp sample C3. Molecules 2018, 23, x; doi: FOR PEER REVIEW www.mdpi.com/journal/molecules Molecules 2018, 23, x; doi: FOR PEER REVIEW www.mdpi.com/journal/molecules Molecules 2018, 23, x; doi: FOR PEER REVIEW www.mdpi.com/journal/molecules Molecules 2018,, 23,, 2639x FOR PEER REVIEW 1212 of of 21 2.8. GC Methods for the Analysis of Volatile Compounds from Hemp 2.8. GC Methods for the Analysis of Volatile Compounds from Hemp Hemp volatile compounds extracted by HS-SPME were analysed by GC-FID and GC-MS and they Hempwere identified volatile compounds according extractedto both their by HS-SPME calculated were linear analysed retention by GC-FIDindex (LRI and) GC-MSand MS andspectra, they werewhich identified were compared according with to available both their data calculated in the literature linear retention and MS index spectra (LRIl libraries,) and MS respectively. spectra, which As wereshown compared in Figure with 12 availableand Table data 4, the in the GC literature analysis and allowed MS spectral us to identify libraries, 44 respectively. compounds As in shown hemp ininflorescences Figure 12 and. Table4, the GC analysis allowed us to identify 44 compounds in hemp inflorescences. In general, general, all all the the hemp hemp samples samples analysed analysed in this in this study study displayed displayed the same the same qualitative qualitative and semi and- semi-quantitativequantitative profile: profile: monoterpenes monoterpenes were the were most the represented most represented class of compounds class of compounds among volatiles, among volatiles,ranging between ranging 47% between and 89% 47% of and the 89% total of peak the totalarea. peakSample area. C5 Sample was the C5 only was exception the only to exception this trend, to thisas the trend, total as area the total of sesquiterpenes area of sesquiterpenes (52% of (52% the total of the area) total wasarea) found was found to be to high be higherer than than that that of ofmonoterpen monoterpenes.es. β--Myrcene,Myrcene, α- and β--pinene,pinene, and limonene represented the most abundant compounds among monoterpenes. It is worth worthyy of notice that the head space of sample C2 had a remarkably high relative content of α--pinene,pinene, while samples C4 and C6 displayed a notably notably high high relative relative amount amount of of limonene. limonene. As to the sesquiterpenes, β--caryophyllenecaryophyllene was the most abundant compound in all the samplessamples analysed, followed followed by byα -humulene. α-humulene. Curiously, Curiously, sample sample C5 was C5 thewas one the with one the with higher the relativehigher relative content ofcontent caryophyllene of caryophyllene oxide, followed oxide, followed by sample by C2. sample It should C2. It be should pointed be outpointed that samplesout that samples C2 and C5 C2 were and theC5 were same the in that same had in atha cannabinoidt had a cannabinoid profile opposite profile to opposi that ofte fresh to that plant of fresh material, plant further material, suggesting further theirsuggesting aging ortheir non-optimal aging or non storage-optimal conditions. storage conditions. Nevertheless, the GC data of the hemp inflorescencesinflorescences analysed in this work are in accordance with the available one previously described in the literature relatingrelating to hemp essential oil or volatile fraction [[14,15,2714,15,27–29].]. pA 5 1600 1400 2 1200 1000 800 29 15 600 13 400 4 31 38 34 11 200 1 3 18 20 0 0 5 10 15 20 25 30 35 40 45 min Figure 12. Representative GC GC-FID-FID chromatogram obtained by using the HS-SPMEHS-SPME method optimised for volatile compounds from hemphemp inflorescencesinflorescences (sample(sample C3).C3). ForFor peakpeak numbering,numbering, seesee TableTable4 .4. Molecules 2018, 23, x; doi: FOR PEER REVIEW www.mdpi.com/journal/molecules Molecules 2018, 23, 2639 13 of 21 Table 4. Semi-quantitative data (% relative peak area values) for volatile compounds from hemp inflorescences a. % Relative Peak Area Peak Number Compound LRI Calc. LRI Lit. c C1 C2 C3 C4 C5 C6 1 α-Thujene 932 911 3.7 ± 1.1 1.4 ± 1.0 0.2 b 0.2 b 0.5 ± 0.1 - 2 α-Pinene 939 932 20.1 ± 0.5 40.1 ± 2.3 26.7 b 9.6 ± 0.4 15.9 ± 2.1 10.3 b 3 Camphene 952 957 0.3 b - 0.5 b 0.3 b 0.3 b 0.2 b 4 β-Pinene 980 978 5.7 b 9.3 ± 0.1 6.9 ± 0.1 2.9 ± 0.2 4.2 ± 0.3 3.7 b 5 β-Myrcene 1000 992 28.7 ± 0.1 12.9 ± 0.1 36.9 ± 0.2 32.1 ± 3.3 6.9 ± 0.5 34.1 ± 1.1 6 α-Phellandrene 1009 1007 0.1 b - 0.4 b 0.5 b - 0.3 b 7 ∆3-Carene 1014 1010 1.3 b 1.4 b 0.5 b 0.6 b - 0.3 b 8 α-Terpinene 1020 1018 - - 0.3 b 0.4 b - 0.3 ± 0.1 9 o-Cymene 1022 1021 0.1 b - 0.1 b 0.1 b 1.3 ± 0.1 - 10 p-Cymene 1028 1026 0.2 b - 0.1 b 0.5 ± 0.1 2.5 ± 0.1 0.8 ± 0.1 11 Limonene 1032 1035 2.4 b 2.3 ± 0.1 1.6 b 11.3 ± 0.5 3.5 ± 0.2 11.5 ± 0.3 12 cis-Ocimene 1042 1040 0.5 b 0.7 b 0.2 b 0.6 ± 0.1 0.4 ± 0.1 - 13 trans-Ocimene 1053 1097 7.1 ± 0.3 0.6 b 6.1 ± 0.2 5.2 ± 0.1 0.4 ± 0.1 0.5 b 14 γ-Terpinene 1062 1062 0.2 b - 0.3 b 0.4 b 0.3 b 0.5 b 15 α-Terpinolene 1093 1088 3.9 ± 0.1 - 7.9 ± 0.1 12.0 ± 0.8 1.4 ± 0.1 4.9 ± 0.1 16 Linalool 1104 1104 0.6 b - 0.1 b 0.3 b 0.8 ± 0.1 0.4 b 17 6-Camphenol 1106 1110 0.3 b -- 0.1 b 1.2 ± 0.1 - 18 neo-Alloocimene 1133 1143 0.4 b - 0.1 b 0.4 b 1.1 ± 0.2 - 19 Isopinocarveol 1145 1139 - 0.6 b - - 0.6 ± 0.1 - 20 Terpinen-4-ol 1181 1177 0.5 b - 0.2 b 0.3 b 3.3 ± 0.2 0.6 b 21 p-Cymen-8-ol 1183 1181 - - 0.1 b - 2.2 ± 0.2 - 22 α-Terpineol 1193 1195 0.1 b -- 0.1 b 0.7 ± 0.1 0.4 b 23 Eugenol 1371 1373 - - - 0.3 ± 0.1 - - 24 α-Copaene 1375 1376 - - - 0.8 ± 0.2 - 0.3 ± 0.1 25 Geranyl acetate 1379 1386 - - - 0.3 ± 0.1 - - 26 Ylangene 1389 1406 - - 0.1 b --- 27 Z,Z-α-Farnesene 1415 1462 0.2 b 0.7 b - 0.1 b 1.2 b 0.4 b 28 α-Santalene 1423 1420 - - 0.1 b 0.1 b 0.3 b - 29 β-Caryopyllene 1430 1428 13.4 ± 0.1 18.1 ± 0.5 5.2 ± 0.1 8.1 ± 2.0 22.6 ± 1.0 21.8 ± 0.7 30 Z,E-α-Farnesene 1437 1486 - - - 0.1 b 2.2 b - 31 α-Guaiene 1444 1439 0.2 b 0.5 b 0.4 b 0.9 ± 0.3 - - 32 Aromadendrene 1446 1449 0.4 ± 0.1 - - 0.3 ± 0.1 - - 33 β-Farnesene 1460 1454 0.1 b - - 1.8 ± 0.5 0.3 b - 34 α-Humulene 1464 1455 2.8 b 4.6 ± 0.3 1.3 b 2.1 ± 0.5 8.7 ± 0.6 5.6 ± 0.2 35 Alloaromadendrene 1471 1467 0.3 b 0.6 b 0.1 b 0.3 ± 0.1 1.4 ± 0.2 0.2 b Molecules 2018, 23, 2639 14 of 21 Table 4. Cont. % Relative Peak Area Peak Number Compound LRI Calc. LRI Lit. c C1 C2 C3 C4 C5 C6 36 Germacrene D 1489 1480 - - - 0.1 b 0.5 b - 37 β-Selinene 1493 1485 0.3 b 0.6 b - 0.2 b 0.5 ± 0.1 0.1 b 38 Valencene 1496 1496 0.5 b - 0.1 b 0.1 b 1.4 ± 0.1 0.3 b 39 α-Selinene 1505 1494 0.4 b 0.4 b 0.1 b 0.1 b 1.2 ± 0.1 0.2 b 40 γ-Cadinene 1515 1514 0.3 b - - 0.6 ± 0.2 0.4 b - 41 δ-Cadinene 1532 1530 - - 0.1 b 0.4 ± 0.1 0.4 b - 42 α-Calacorene 1547 1542 - - - 0.6 ± 0.1 - 0.2 b 43 Celina-3,7(11)-diene 1554 1550 - - 0.6 b - 0.3 b - 44 Caryophyllene oxide 1591 1583 - 0.6 b 0.2 b 0.1 b 2.6 ± 0.4 - Total 94.8 ± 0.9 95.4 ± 1.0 97.5 ± 1.3 95.3 ± 1.1 91.5 ± 1.6 97.9 ± 0.7 Experimental conditions as in Sections 3.7–3.9. a Data are expressed as mean (n = 3) ± SD. b SD < 0.05. c LRI data from NIST Chemistry WebBook, SRD 69, https://www.nist.gov. Molecules 2018, 23, 2639 15 of 21 3. Materials and Methods 3.1. Chemicals and Solvents Cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabigerol (CBG), and cannabidiol (CBD) standard solutions (1 mg/mL in methanol or acetonitrile) were purchased from Cerilliant (Round Rock, TX, USA). Chrysoeriol was purchased from Sigma-Aldrich (Milan, Italy), while canniprene was kindly provided by Dr. Federica Pollastro of the Department of Drug Sciences of the University of Piemonte Orientale (Novara, Italy). Formic acid (HCOOH) and HPLC-grade solvents, including EtOH, MeOH, ACN, acetone, EtOAc, n-hexane, DCM, ethyl ether and toluene, were from VWR (Milan, Italy). H2O was purified by using a Milli-Q Plus185 system from Millipore (Milford, MA, USA). 3.2. Hemp Plant Material Six samples of fibre-type hemp female inflorescences (indicated in the text as C1–C6, respectively) were analysed in this study. including Antal (C1), Carma (C2), Carmagnola (C3), China (C4), Codimono (C5), and Fibrante (C6). These samples (about 100–500 g dry material each), belonging to different breeding lines, were cultivated under the same agronomic conditions; they were kindly provided by Dr. Gianpaolo Grassi of the research centre CREA-CIN (Rovigo, Italy) and they were certified for a content of ∆9-THC below 0.2% (w/w). All the samples considered in this study were approved for commercial use by the European Union [6]. The “Fibrante” sample (C6) was selected for the HPLC method development. The sample “Carmagnola” (C3) was used for the optimisation of the HS-SPME procedure; an essential oil sample of the same hemp variety, obtained by hydro-distillation, was also available for a comparison. For each sample, hemp inflorescences were manually separated from twigs and seeds. After this procedure, the samples were stored at +4.0 ◦C until required for chemical analysis. Hemp inflorescences were ground in a mortar before the extraction. 3.3. Sample Preparation 3.3.1. Extraction of Non-Psychoactive Cannabinoids Cannabinoids were extracted from hemp inflorescences by means of DM [9]. Briefly, a weighed amount of sample (0.25 g) was extracted with 10 mL of EtOH as the extraction solvent at room temperature for 15 min, under magnetic stirring. The solution was then paper filtered and the residue was extracted with the same procedure twice more with 10 and 5 mL of solvent, respectively. The filtrates of the three extractions were then combined and adjusted to 25 mL with the solvent in a volumetric flask. Before the injection in the HPLC system, the extracts were filtered by using a 0.45 µm PTFE filter into a HPLC vial. The extraction procedure was repeated twice for each sample. 3.3.2. Extraction of Flavonoids and Related Compounds A portion of 0.25 g of hemp inflorescences was treated with 10 mL of n-hexane at room temperature for 15 min, under magnetic stirring, in order to remove non-psychoactive cannabinoids. The solution was then filtered on a paper filter and the filtrate sent to waste. The same procedure was repeated twice more, by adding 10 and 5 mL of solvent to the residue, respectively. The residue was then let to dry and, subsequently, it was extracted with 10 mL of acetone. The solution was then filtered on a paper filter into a volumetric flask and the residue was extracted with the same procedure twice more with 10 and 5 mL of solvent, respectively. The filtrates of the three extractions were then pooled, concentrated under vacuum at 30 ◦C, and adjusted to a final volume of 1 mL with the extraction solvent. Before the injection into the HPLC system, the extracts were filtered by using a 0.45 µm PTFE filter into a HPLC vial. Molecules 2018, 23, 2639 16 of 21 The extraction procedure was repeated twice for each sample. 3.3.3. Extraction of Volatile Compounds HS-SPME was used for hemp volatiles. To do this, a manual holder and a 1 cm stable-flex 50/30 µm DVB/CAR/PDMS fibre were employed (Supelco, Bellefonte, PA, USA). Before GC analysis, the fibre was conditioned in the injector, according to the instructions provided by the manufacturer. A 400 mg amount of hemp inflorescences, previously ground, was placed in a 10 mL flat-bottom headspace vial sealed with a magnetic crimp cap and PTFE/silicone septa (Supelco). Under the optimized conditions, the sample was heated for 30 min during the equilibrium time in a thermostatic bath at 40 ◦C. The SPME device was then inserted into the sealed vial by manually penetrating the septum and the fibre was exposed to the headspace for 20 min during the extraction time. After sampling, the SPME fibre was immediately inserted into the GC injector and thermally desorbed. A desorption time of 5 min at 250 ◦C was used in the split-less mode. Before sampling, the fibre was reconditioned for 5 min in the GC injector port at 250 ◦C. The extraction procedure was repeated three times for each sample. 3.4. HPLC-UV/DAD Conditions The HPLC-UV/DAD analyses were performed on an Agilent Technologies (Waldbronn, Germany) modular model 1100 system, consisting of a vacuum degasser, a quaternary pump, an autosampler, a thermostated column compartment and a diode array detector (UV/DAD). Chromatograms were recorded by using an Agilent Chemstation for LC and LC-MS systems (Rev. B.01.03). 3.4.1. HPLC-UV/DAD Analysis of Non-Psychoactive Cannabinoids The chromatographic conditions for the analysis of non-psychoactive cannabinoids were set on the basis of a previous work [9]. HPLC analyses were carried out on an Ascentis Express C18 column (150 mm × 3.0 mm I.D., 2.7 µm, Supelco, Bellefonte, PA, USA), with a mobile phase composed of 0.1% HCOOH in both (A) H2O and (B) ACN. The gradient elution was modified as follows: 0–13 min 60% B, 13–17 min from 60% to 80% B, 17–22 min from 80% to 90% B. The post-running time was 15 min. The flow-rate was 0.4 mL/min. The column temperature was set at 30 ◦C. The sample injection volume was 3 µL. The UV/DAD acquisitions were carried out in the range 190–600 nm, while chromatograms were acquired at 210 nm (for decarboxylated cannabinoids) and at 220 nm (for cannabinoic acids) [9]. Three injections were performed for each sample. 3.4.2. HPLC-UV/DAD Analysis of Flavonoids and Related Compounds Regarding the analysis of flavonoids and related compounds, the chromatographic column and the mobile phase were the same as those used for the analysis of cannabinoids. Conversely, the gradient elution was optimized as follows: 0–5 min 40% B, 5–20 min from 40 to 80% B; 20–35 min from 80% to 90% B, which was held for 10 min. The post-running time was 10 min. The flow rate and the sample injection volume were kept at 0.4 mL/min and 3 µL, respectively and column was kept at room temperature. As regards detection, UV/DAD acquisitions were carried out in the range 190–600 nm, while chromatograms were acquired at 210 nm (for canniprene) and at 342 nm (for cannflavins). Three injections were performed for each sample. 3.5. HPLC-ESI-MS and MS2 Conditions The HPLC-ESI-MS and MS2 analyses were performed on an Agilent Technologies modular 1200 system, equipped with a vacuum degasser, a binary pump, a thermostated autosampler, a thermostated column compartment, and a 6310A ion trap mass analyser with an ESI ion source. The HPLC column and the applied chromatographic conditions were the same as those reported for the HPLC-UV/DAD Molecules 2018, 23, 2639 17 of 21 systems. In details, the chromatographic conditions described in Sections 3.4.1 and 3.4.2 were applied for the HPLC-ESI-MS and MS2 experiments of cannabinoids and polyphenols, respectively. The HPLC-ESI-MS system was operated both in the positive and in the negative ion mode. For the positive ion mode, the experimental parameters were set as follows: the capillary voltage was 3.5 kV, ◦ the nebulizer (N2) pressure was 32 psi, the drying gas temperature was 350 C, the drying gas flow was 10 L/min, and the skimmer voltage was 40 V. For the negative ion mode, the MS conditions were the same as described above. Data were acquired by Agilent 6300 Series Ion Trap LC/MS system software (version 6.2). The mass spectrometer was operated in the full-scan mode in the m/z range 200–1200. MS2 spectra were automatically performed with helium as the collision gas in the m/z range 50–1500, by using the SmartFrag function, which automatically selects the precursor ion to be fragmented and it eliminates the need for a time-consuming manual collision-induced dissociation (CID) voltage optimization [9]. 3.6. HPLC-UV/DAD Method Validation In this study, the validation of the HPLC-UV/DAD method for the determination of hemp flavonoids and related compounds was carried out, since the chromatographic method for the quantification of non-psychoactive cannabinoids was previously fully validated [9]. The validation was performed in agreement with the international guidelines for analytical techniques for the quality control of pharmaceuticals (ICH guidelines) [35]. As regards linearity, the stock standard solution of non-psychoactive cannabinoids (CBDA, CBGA, CBG and CBD) was prepared as follows: an accurately measured volume of reference solution (100–200 µL) was placed into a 1 mL volumetric flask; then, MeOH was added and the solution was diluted to volume with the same solvent. As to flavonoids, the stock standard solution of each compound (canniprene and chrysoeriol) was prepared by weighing an accurate amount of reference compound (0.9 mg) into a 1 mL volumetric flask; then, the solution was adjusted to volume with MeOH. The external standard calibration curve was generated by using six data points, covering the concentration ranges: 2.5–200.0 µg/mL for CBDA, CBGA and CBD; 1.3–100.0 µg/mL for CBG; 0.3–23.4 µg/mL for canniprene, and 1.3–43.0 µg/mL for chrysoeriol. Three µL aliquots of each standard solution were used for HPLC analysis. Injections were performed in triplicate for each concentration level. The calibration curve was obtained by plotting the peak area of the compound at each level versus the concentration of the sample. The quantification of non-psychoactive cannabinoids and canniprene was performed by using their calibration curves. The amount of cannflavins A and B in hemp samples was determined by using the calibration curve of chrysoeriol, having the same chromophore, and the content was corrected by using the molecular weight ratio. For reference compounds, the limit of detection (LOD) and the limit of quantification (LOQ) were experimentally determined by HPLC analysis of serial dilutions of a standard solution to reach a signal-to-noise (S/N) ratio of 3 and 10, respectively. The precision of the extraction technique was validated by repeating the extraction procedure on the inflorescence of the same hemp sample (C6) six times. An aliquot of each extract was then injected and quantified. The precision of the chromatographic system was tested by performing intra- and inter-day multiple injections of one extract from sample C6 and then checking the %RSD of retention times and peak areas. Six injections were performed each day for three consecutive days. 3.7. GC-FID Analysis of Volatile Compounds These analyses were performed on an Agilent Technologies 7820 gas chromatograph with a flame ionization detector (FID). Compounds were separated on an Agilent Technologies HP-5 cross-linked poly-5% diphenyl-95% dimethyl polysiloxane (30 m × 0.32 mm i.d., 0.25 µm film thickness) capillary column. The column temperature was initially set at 45 ◦C, then increased to 100 ◦C at a rate of 2 ◦C/min up, then raised to 250 ◦C at a rate of 5 ◦C/min, which was held for 5 min. Helium was used Molecules 2018, 23, 2639 18 of 21 as the carrier gas, at a flow rate of 1.0 mL/min. The injector and detector temperature was set at 250 and 300 ◦C, respectively. 3.8. GC-MS Analysis of Volatile Compounds These analyses were carried out on a 7890A gas chromatograph coupled with a 5975C network mass spectrometer (Agilent Technologies, Germany). Compounds were separated on an Agilent Technologies HP-5 MS cross-linked poly-5% diphenyl-95% dimethyl polysiloxane (30 m × 0.25 mm i.d., 1.00 µm film thickness) capillary column. The temperature program was the same as described above. Helium was used as the carrier gas, at a flow rate of 0.7 mL/min. The injector, transfer line and ion-source temperature was 250, 280, and 230 ◦C, respectively. MS detection was performed with electron ionization (EI) at 70 eV, operating in the full-scan acquisition mode in the m/z range 40–400. 3.9. Qualitative and Semi-Quantitative Analysis of Volatile Compounds Hemp volatile compounds were identified by comparing the retention times of the chromatographic peaks with those of authentic reference standards run under the same conditions and by comparing with the literature the linear retention index (LRI) relative to a mixture of n-alkanes (C8–C40) in n-hexane loaded onto the DVB-CAR-PDMS fibre and injected under the same conditions. Peak enrichment by co-injection with authentic reference compounds was also carried out. Comparison of the MS-fragmentation pattern of the target analytes with those of pure components was performed. A mass-spectrum database search was performed by using the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA) mass-spectral database (version 2.0d, 2005). The percentage relative amount of individual components was expressed as percent peak area relative to total peak area. 4. Conclusions A comprehensive analysis of the bioactive compounds in hemp inflorescences was carried out in this research work for the first time. The chemical classes considered in this study encompassed non-psychoactive cannabinoids, flavonoids and related compounds, and volatile compounds. In detail, the qualitative and quantitative profiling of non-psychoactive cannabinoids was performed by applying an HPLC method with UV/DAD, ESI-MS and MS2 detection. A new HPLC method with UV/DAD, ESI-MS and MS2 detection was developed and validated for the analysis of hemp-specific flavonoids, combined with a selective and simple extraction procedure. Finally, the characterisation of the hemp volatile fraction was achieved by optimising a new HS-SPME-GC procedure with FID and MS detection. All the analytical methods developed in the present work clearly demonstrated the provision of a comprehensive and reliable analysis of the bioactive compounds present in hemp inflorescences, which are known to be characterised by a very complex composition. Therefore, they can be applied for a detailed chemical characterisation of the plant material in order to monitor its composition and guarantee a better reproducibility of biological assays, since several compounds may act synergistically, and also in quality control to ensure the efficacy and safety of hemp-based products to be used in the pharmaceutical and nutraceutical fields. Supplementary Materials: The Supplementary Materials are available online. Table S1: linearity and sensitivity data for compounds used as hemp standards, Table S2: intra- and inter-day precision data for retention time (tR) and peak area of the main flavonoids in hemp extracts (sample C6), Table S3: intra- and inter-day precision data for the extraction of the main flavonoids from hemp (sample C6). Author Contributions: Conceptualization, F.P.; Methodology, F.P. and V.B.; Software, V.B, L.M. and D.B.; Validation, F.P., V.B. and J.S.; Formal Analysis, F.P. and V.B.; Investigation, F.B., V.B. and J.S.; Resources, S.B.; Data Curation, F.P. and V.B.; Writing—Original Draft Preparation, F.P. and V.B.; Writing—Review and Editing, F.P.; Visualization, F.P.; Supervision, F.P. and S.B.; Funding Acquisition, S.B. Molecules 2018, 23, 2639 19 of 21 Funding: This work was carried out thanks to: a research grant awarded to Johanna Sperlea for her master thesis at the Department of Life Sciences of the University of Modena and Reggio Emilia (Italy) within the Erasmus+ program, under the scientific supervision of Federica Pellati; a research grant (Fondo di Ateneo per la Ricerca, FAR 2017) awarded to Stefania Benvenuti from the Department of Life Sciences of the University of Modena and Reggio Emilia for the project entitled “Active compounds from Cannabis sativa L. for the prevention and complementary therapy of neurodegenerative diseases”. Acknowledgments: The authors are very grateful to Federica Pollastro of the Department of Drug Sciences of the University of Piemonte Orientale (Novara, Italy) for having kindly provided canniprene standard. Gianpaolo Grassi from CREA-CIN centre (Rovigo, Italy) is also acknowledged for having provided fibre-type hemp samples. 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