By using the Inverted Chirality Columns Approach (ICCA) we have developed an enantioselective UHPSFC method to determine the enantiomeric excess (ee) of (-)-Δ(9)-THC in medicinal marijuana (Bedrocan®). The ee was high (99.73%), but the concentration of the (+)-enantiomer (0.135%) was not negligible, and it is worth a systematic evaluation of bioactivity

Cavazzini, A

D'Acquarica, I

Gasparrini, F.

Ismail, O

Manzo, C

Mazzoccanti, G

Villani, C

Wilcox, M

English

By using the Inverted Chirality Columns Approach (ICCA) we have developed an enantioselective UHPSFC method to determine the enantiomeric excess (ee) of (-)-Δ9-THC in medicinal marijuana (Bedrocan®). The ee was high (99.73%), but the concentration of the (+)-enantiomer (0.135%) was not negligible, and it is worth a systematic evaluation of bioactivity

Giulia Mazzoccanti

Omar H. Ismail

Ilaria D’Acquarica

Claudio Villani

C. Manzo

M. Wilcox

Alberto Cavazzini

Francesco Gasparrini

Archivio istituzionale della ricerca - Università di Ferrara

Cannabis through the looking glass: Chemo- and enantio-selective separation of phytocannabinoids by enantioselective ultra high performance supercritical fluid chromatography

D'acquarica, I

Archivio della ricerca- Università di Roma La Sapienza

ChemCommChemical Communicationsrsc.li/chemcommISSN 1359-7345COMMUNICATIONI. D’Acquarica, F. Gasparrini et al.Cannabis through the looking glass: chemo- and enantio-selective separation of phytocannabinoids by enantioselective ultra high performance supercritical fluid chromatographyVolume 53 Number 91 25 November 2017 Pages 12247–1237412262 | Chem. Commun., 2017, 53, 12262--12265 This journal is©The Royal Society of Chemistry 2017Cite this:Chem. Commun., 2017,53, 12262Cannabis through the looking glass: chemo- andenantio-selective separation of phytocannabinoidsby enantioselective ultra high performancesupercritical fluid chromatography†G. Mazzoccanti, a O. H. Ismail, a I. D’Acquarica, *a C. Villani, a C. Manzo,bM. Wilcox,c A. Cavazzini d and F. Gasparrini *aBy using the Inverted Chirality Columns Approach (ICCA) we havedeveloped an enantioselective UHPSFC method to determine theenantiomeric excess (ee) of ()-D9-THC in medicinal marijuana(Bedrocans). The ee was high (99.73%), but the concentration ofthe (+)-enantiomer (0.135%) was not negligible, and it is worth asystematic evaluation of bioactivity.Cannabinoids are one of the constituents of marijuana, thecrude drug derived from the plant Cannabis sativa L. The termphytocannabinoids has been coined just to emphasize theirplant-derived origin1 with respect to synthetic cannabinoids(e.g., nabilone,2a dexanabinol,2b or ajulemic acid2c) and endogenouscannabinoid receptor ligands (namely, endocannabinoids).3 Themost abundant and psychologically active compound of the class is()-D9-trans-tetrahydrocannabinol (D9-trans-THC),4,5 which hasindeed been the subject of thousands of papers.6,7 Furthermore,we are currently witnessing a remarkable breakthrough inthe recreational use of cannabis, with respect to the mode ofconsumption, short-term effects, chronic health consequencesand cannabis use disorders.8Another fascinating feature of phytocannabinoids is that mostof them are chiral, and are produced, in the plant, in a single-enantiomer format, as typically occurs for natural products,with some exceptions.9 As a result, restricted stereochemicalrequirements are present for the interaction of cannabinoidswith the cannabinoid (CB) receptors,10 and this has led numerousgroups to investigate the possibility of separating the undesirablepsychotropic effects from the desirable effects by suitable changesin stereochemistry. For example, the ()-enantiomer of thesynthetic cannabinoid dexanabinol (HU-210) is one of the mostpotent psychotropically active cannabinoids known, while thecorresponding (+)-enantiomer (i.e., HU-211) is devoid of theTHC-like psychotropic effects.11 It becomes evident, therefore,the importance of the stereochemical efficiency of syntheticpathways to unnatural cannabinoid analogues, as well as theefficiency of the determination of the enantiomeric purity(namely, enantiomeric excess, ee) in naturally occurring samples,in both single and in more complex mixtures.We became interested in investigating the composition ofCannabis plant extracts from a stereochemical point of view; infact, while stringent rules have been established by the FDA forthe content determination of THC and cannabidiol (CBD) inmedicinal marijuana,12 the stereochemical features of suchmolecules and the determination of the enantiomeric purityhave not yet been put into the foreground by the agency.Furthermore, although the point of the chromatographicresolution of chiral cannabinoids13a has already been raised in1993 and more recently addressed for synthetic cannabinoids,13banalytical approaches to crude plant extracts have been developedby just taking into account the single cannabinoids, irrespectiveof their stereochemistry.14We started the investigation by focusing our attention on thephytocannabinoids collected in Fig. 1. Apart from the typicalthree-letter acronyms,4,5 we included a bold number for eachmolecular entity, for the sake of clarity in the identification of theenantiomerically correlated molecules. In fact, it is well knownthat all the naturally occurring cannabinoids exist in the single-enantiomer format, i.e., the (R,R)-form, which displays a negativeoptical rotation and a trans-configuration at the cyclohexenering. Nevertheless, we have designed, for each structure, its mirrorimage (i.e., the unnatural dextrorotatory enantiomer) to get a fullidea of any implicit stereochemical scenario in cannabinoids.15Cannabidivarin (CBDV, 1) and cannabidiol (2), which feature twochiral centres, belong to the CBD-type subgroup of cannabinoids,4the only difference between them being the length of the alkylside chain at C30 (propyl and pentyl, respectively). From thea Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Universita` di Roma,p.le A. Moro 5, 00185 Roma, Italy. E-mail: francesco.gasparrini@uniroma1.itb Avantech Group S.r.l. Via M. Salernitano 28, 84012 Angri, Italyc Regis Technologies, Inc, 8210 Austin Avenue, Morton Grove, IL 60053, USAd Dipartimento di Chimica e Scienze Farmaceutiche, Universita` degli Studi diFerrara, via L. Borsari 46, 44121 Ferrara, Italy† Electronic supplementary information (ESI) available: Chemo- and enantio-selective separation of seven phytocannabinoid standards by eUHPSFC on the(S,S)-Whelk-O1 column; simultaneous UV and CD detection for racemic CBC onthe (R,R)-Whelk-O1 column. See DOI: 10.1039/c7cc06999eReceived 6th September 2017,Accepted 17th October 2017DOI: 10.1039/c7cc06999ersc.li/chemcommChemCommCOMMUNICATIONThis journal is©The Royal Society of Chemistry 2017 Chem. Commun., 2017, 53, 12262--12265 | 12263THC-type subgroup of cannabinoids,4 we extracted both theD8-THC-type (3) and the D9-THC-type (4) cannabinoids, whichare double-bond position isomers of THC, the former being lesspsychologically potent than the latter. Another fascinatingchiral phytocannabinoid is cannabichromene (CBC, 5), thatpresents only one chiral centre, and it is typically reported as arare case of a natural racemate.4,5 This cannabinoid has nopsychotropic effect, but interesting pharmacological activitiessuch as anti-inflammatory, antifungal and antimicrobial pro-perties have been reported.16 We have included, in the inves-tigation, also two achiral cannabinoids, i.e., cannabigerol(CBG, 6), which is the biosynthetic precursor of CBC, andcannabinol (CBN, 7), whose concentration in Cannabis products(i.e., marijuana, hashish and hash oil) has been shown toincrease during the storage of these materials. Thus, it wasnecessary to develop an analytical method, which had to beenantio- and chemo-selective at the same time.The first problem we faced in stereoselective analysis ofCannabis plant extracts was that vegetable extracts are highlyenriched complex mixtures and often the minor enantiomers,or the racemates, are not available as reference samples. Toovercome this limitation, our group has previously developed amethod for the identification and accurate quantification of theminor enantiomer in trace analysis of natural products, namedthe ‘‘Inverted Chirality Columns Approach’’ (ICCA).17a,bThis method is based on the switching between two ChiralStationary Phases (CSPs) having the same bound selector withan opposite configuration, to reverse the elution order of agiven enantiomeric pair, according to the reciprocal principleof selectand–selector-systems.17c This technique is very usefulwhen the minor enantiomer follows the major one and it ispartially hidden by the tailing of the leading enantiomer: on theCSP with opposite configuration the trace enantiomer is elutedfirst, thus enabling a more precise and accurate quantificationby peak area integration.Pirkle-type CSPs (such as the DACH-DNB and the Whelk-O1)are the ideal instruments for the application of the ICCA approach,because they exist in both the enantiomeric versions.17a,b From2010, a clear trend towards the development of CSPs in thesub-2 mm format has been reported for the chromatographicseparation of chiral analytes to enhance both analysis speed andcolumn efficiency.18 This has opened new frontiers in the field ofenantioselective ‘‘e’’ Ultra-High Performance Liquid Chromato-graphy (eUHPLC)19 and enantioselective ‘‘e’’ Ultra-High PerformanceSupercritical Fluid Chromatography (eUHPSFC).20 SFC has indeedrecently undergone an outstanding revival in the enantio-separation of pharmaceuticals, particularly in the initial screeningstage of the drug discovery process.21In this work, we successfully applied, for the chemo- andenantio-selective separation of phytocannabinoids, the ICCAmethod using, as the inverted chirality columns, those basedon the (S,S)- and (R,R)-Whelk-O1 CSPs in the sub-2 mm format,under eUHPSFC conditions. It must be acknowledged thatUHPSFC technology proved recently to be an alternative and ortho-gonal solution for the separation of synthetic cannabinoids.13b,14The two UHPC-Whelk-O1 sub-2 mm CSPs were preparedaccording to a previously described procedure19c starting fromKromasil 1.8 mm silica particles, and slurry packed into 100 4.6 mm I.D. stainless steel columns. To evaluate the separationability of the Whelk-O1 CSP towards different cannabinoids, amixture containing seven standards (i.e., ()-1, ()-2, ()-3,()-4, ()-5, 6 and 7, whose structures are shown in Fig. 1)was prepared starting from the commercially available stocksolutions (0.1–1.0 mg ml1) in methanol. All the standards havebeen well separated within 12 minutes using CO2/MeOH, 98 : 2as the mobile phase, at a flow-rate of 3.5 ml min1, underisocratic and isoconfertic conditions (see Fig. S1 of the ESI†).This result was taken as a proof of the optimal chemoselectivityof the Whelk-O1 CSP, in particular for the couple D8-THC (3)and D9-THC (4), which are simply two positional isomers.Fig. 1 Chemical structures of the main naturally occurring chiral phytocannabinoids and of their mirror images. The picture also includes two achiralcannabinoids, namely CBG (6) and CBN (7).Communication ChemComm12264 | Chem. Commun., 2017, 53, 12262--12265 This journal is©The Royal Society of Chemistry 2017The analysis of a further standard mixture obtained by repla-cing the ()-4 standard with the corresponding commerciallyavailable racemate (i.e., ()-4) has provided evidence of theenantioselective capability of the phase towards ‘‘THC’’ as well,the (+)-enantiomer eluting before the naturally occurring ()-4on the (S,S)-Whelk-O1 column. Furthermore, the column provedto be capable of separating the two enantiomers of CBC(compound 5), which was added to the mixture of standardsas the racemate, and whose absolute configuration at C30 hasnot yet been determined.4 The identification of the two CBCenantiomers was made by checking their elution order in the(R,R)-Whelk-O1 column under standard HPLC conditions (seeFig. S2 of the ESI†) and simultaneous UV and CD detection at280 nm (the first eluting enantiomer is the negative peak).Afterwards, the two UHPC-Whelk-O1 sub-2 mm columnswere tested in order to check their full equivalence, both froma chemical (i.e., retention and selectivity) and a geometrical(i.e., packing efficiency) point of view, as required by the ICCAmethod.17a For this purpose, a six-component standard mixturewas analyzed for both the (S,S)- and (R,R)-Whelk-O1 columnsunder the same UHPSFC conditions (see Fig. 2), and we couldconfirm that just the enantiomerically correlated peaks havebeen inverted by switching from one column to the other,whereas the achiral molecules (i.e., 6 and 7) were not affectedby the variation, and eluted from the two columns with identicalretention behaviours (see Table S1 of the ESI†).By applying the ICCA protocol to the above-mentionedstandard mixture, we were able to simulate a virtual racematefor those compounds that were available just as single enantiomers(i.e., ()-1 and ()-2). The simulation (see dot lines in Fig. 2) hasallowed us to predict the retention factor (k0), the enantio-selectivity (a) and the resolution (Rs) for the virtual ()-1/(+)-1 and()-2/(+)-2 couples (see Table S1 of the ESI†).As a final investigation, we analyzed an ethanol extract fromBedrocans (medicinal marijuana from Cannabis sativa L. strains)which is claimed to contain ‘‘THC’’ as the major component(Fig. 3). Indeed, by expanding the chromatogram baseline, wehave detected, besides ‘‘THC’’, also ‘‘CBC’’, ‘‘CBG’’ and ‘‘CBN’’in smaller amounts (see Table S2 of the ESI†), together with achiral unknown impurity. Moreover, thanks to the ICCAmethod, we made it possible to elute the minor enantiomericimpurity (+)-4 before the major one (i.e., ()-4) and thus wewere able to measure an ee equal to 99.73% on the UHPC-(S,S)-Whelk-O1 column (see Table 1). Notably, for ‘‘CBC’’, we foundthat, instead of being a racemate, the ee is about 25% (equallymeasurable on both the columns), which means that it occursas a scalemic mixture.We can conclude that the chromatographic system we havedeveloped proved useful in the chemo- and enantio-selectiveseparation of all the tested cannabinoid samples, and it has beendemonstrated to be reliable for the determination of extreme eeof D9-THC in pharmaceutical formulations. Notably, it is the firsttime that the trace (+)-enantiomer of ‘‘THC’’ has been quantifiedin medicinal marijuana. Such a feature is fundamental both forfurther evaluation of the bioactivity and in the case of single-molecule cannabinoid pharmaceutical products (e.g., Marinols),whose stereochemistry must be accurately controlled.We thank financial support from Sapienza Universita` diRoma, Italy (Funds for Selected Research Topics 2017–2018).Fig. 2 Demonstration of the chemical equivalence of the two 1.8 mmUHPC-(R,R)- and (S,S)-Whelk-O1 columns (100  4.6 mm I.D.) uponseparation of a six-component cannabinoid standard mixture. The dottedlines represent the (+)-enantiomers of compounds 1 and 2 predicted by theICCA method. Mobile phase: CO2/MeOH = 98 : 2; flow-rate: 3.5 ml min1;T = 30 1C; ABPR = 1500 psi; detection: UV at 214 nm. For the chemicalstructures of 1–7, see Fig. 1.Fig. 3 A Bedrocans ethanol extract analyzed by applying the ICCAprotocol. For peak identification, the chromatogram of a six-componentcannabinoid standard mixture has been shown in the inset as a dotted linetrace. The asterisk denotes a chiral unknown impurity.Table 1 Determination of the enantiomeric excess (ee) for ‘‘THC’’ (4)and CBC (5) in the Bedrocans ethanol extract on the UHPC-Whelk-O1sub-2 mm columns under eUHPSFC conditionsCSP config. Peak name Area (mV s) EFa (%) ee (%)(R,R) ()-4 3 694 825 —b —b(+)-4 —b —b(S,S) (+)-4 4917 0.135 99.73()-4 3 642 648 99.865(R,R) [CD()280]-5c 18 766 37.42 25.16[CD(+)280]-5c 31 383 62.58(S,S) [CD(+)280]-5c 31 346 62.81 25.62[CD()280]-5c 18 559 37.19a EF = enantiomeric fraction. b On the (R,R)-Whelk-O1 column the (+)-4peak is eluting on the tailing of the major ()-4 component and thus itis not quantifiable. c Plus and minus signs refer to the signs of thecircular dichroism (CD) band at the indicated wavelength.ChemComm CommunicationThis journal is©The Royal Society of Chemistry 2017 Chem. Commun., 2017, 53, 12262--12265 | 12265We would also like to give special thanks to Prof. G. Appendino(Universita` del Piemonte Orientale, Novara, Italy) for hishelpful discussion on the nomenclature of chiral cannabinoids,to Dr E. Bianchini (University of Ferrara, Italy) for technicalsupport and Farmacia San Vittorio, Castenedolo, Brescia (Italy)for kindly providing an ethanol extract of Bedrocans.Conflicts of interestThere are no conflicts to declare.Notes and references1 (a) J. Gertsch, R. G. Pertwee and V. Di Marzo, Br. J. Pharmacol., 2010,160, 523; (b) A. Ligresti, L. De Petrocellis and V. Di Marzo, Physiol.Rev., 2016, 96, 1593.2 (a) A. Ward and B. Holmes, Drugs, 1985, 30, 127; (b) R. Mechoulam,N. Lander, A. Breuer and J. Zahalka, Tetrahedron: Asymmetry, 1990, 1, 315;(c) S. H. Burstein, C. A. Audette, A. Breuer, W. A. Devane, S. Colodner,S. A. Doyle and R. Mechoulam, J. Med. Chem., 1992, 35, 3135.3 (a) V. Di Marzo and A. Fontana, Prostaglandins, Leukotrienes Essent. FattyAcids, 1995, 53, 1; (b) V. Di Marzo, M. Bifulco and L. De Petrocellis, Nat.Rev. 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Cannabis through the looking glass: chemo- and enantio-selective separation of phytocannabinoids by enantioselective ultra high performance supercritical fluid chromatography

An extreme ee was measured for Δ9-THC in medicinal marijuana, by applying the ICCA method under eUHPSFC, using sub-2 μm Whelk-O1 columns.</p

G. Mazzoccanti

O. H. Ismail

I. D’Acquarica

C. Villani

A. Cavazzini

F. Gasparrini

Crossref

Chemical Communications

<i>Cannabis</i> through the looking glass: chemo- and enantio-selective separation of phytocannabinoids by enantioselective ultra high performance supercritical fluid chromatography

https://iris.uniroma1.it/bitstream/11573/1016698/2/Mazzoccanti_Cannabis_2017.pdf

Cannabis through the looking glass: chemo- and enantio-selective separation of phytocannabinoids by enantioselective ultra high performance supercritical fluid chromatography

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Archivio istituzionale della ricerca - Università di Ferrara

Archivio istituzionale della ricerca - Università di Ferrara

Archivio della ricerca- Università di Roma La Sapienza

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