Cannabis Sativa L. Chemotype Distinction Extract

7/26/2019 Cannabis Sativa L. Chemotype Distinction Extract

1/16

1H NMR and HPLC/DAD for Cannabis sativa L. chemotype distinction,extract proling and specication

Wieland Peschel n,1, Matteo Politi

Centre for Pharmacognosy and Phytotherapy, Department for Pharmaceutical and Biological Chemistry, The School of Pharmacy, University College London,

29-39 Brunswick Square, London WC1N 1AX, United Kingdom

a r t i c l e i n f o

Article history:

Received 20 September 2014Received in revised form

12 February 2015

Accepted 23 February 2015Available online 5 March 2015

Keywords:

Cannabis sativaL.

Extracts

THC

CBD

CBG

HPLC1H NMR

Analytical markers

a b s t r a c t

The medicinal use of different chemovars and extracts of Cannabis sativa L. requires standardization

beyond 9-tetrahydrocannabinol (THC) with complementing methods. We investigated the suitability of1H NMR key signals for distinction of four chemotypes measured in deuterated dimethylsulfoxide

together with two new validated HPLC/DAD methods used for identication and extract proling based

on the main pattern of cannabinoids and other phenolics alongside the assayed content of THC, can-

nabidiol (CBD), cannabigerol (CBG) their acidic counterparts (THCA, CBDA, CBGA), cannabinol (CBN) and

cannavin A and B. Effects on cell viability (MTT assay, HeLa) were tested. The dominant cannabinoid

pairs allowed chemotype recognition via assignment of selective proton signals and via HPLC even in

cannabinoid-low extracts from the THC, CBD and CBG type. Substantial concentrations of cannabinoid

acids in non-heated extracts suggest their consideration for total values in chemotype distinction and

specications of herbal drugs and extracts. Cannavin A/B are extracted and detected together with

cannabinoids but always subordinated, while other phenolics can be accumulated via fractionation and

detected in a wide ngerprint but may equally serve as qualitative marker only. Cell viability reduction in

HeLa was more determined by the total cannabinoid content than by the specic cannabinoid prole.

Therefore the analysis and labeling of total cannabinoids together with the content of THC and 2 4 lead

cannabinoids are considered essential. The suitability of analytical methods and the range of compound

groups summarized in group and ratio markers are discussed regarding plant classi

cation and phar-maceutical specication.

& 2015 Elsevier B.V. All rights reserved.

1. Introduction

Alongside the development of synthetic cannabinergics, the

authorized and the off-label medicinal use of cannabis regain

popularity [1]. The chemical and pharmacological complexity of

cannabis makes the pharmaceutical standardization challenging

and requires complementing identity, purity and assay methods to

characterize the starting material (plant/chemotype), the herbaldrug (Cannabisos) and the preparation (extract).

Approximately 70 phytocannabinoidsbesides 419 other com-

poundsare described for Cannabis sativa L.; classied chemically

into 10 major groups, the 9-trans-tetrahydrocannabinol (THC),

cannabidiol (CBD), cannabigerol (CBG), and cannabinol (CBN)-type

being the most abundant [2]. The psychotropic THC, with the

highest afnity to cannabinoid receptors (CB1, CB2), has been

manifold tested pharmacologically and clinically[3,4]. Meanwhile

other non-psychotropic, non-CB binding cannabinoids, mainly

cannabidiol (CBD) [5,6] but also cannabigerol (CBG) [7], are

increasingly investigated showing partly distinct effects. Moreover

activities are reported for minor non-cannabinoid co-constituents

such as the prenylated avone cannavin A[8] (CFL-A), common

avonoids [9] or terpenes [10,11]. Despite or because of the

complexity some authors advocate the advantage of the natural

mixtures with combinations of cannabis constituents[12,13] pri-

marily determined by the chemovar. Conventional plant classi-

cations as drug-, intermediate or ber type (hemp) are based on

the THC and CBD content [1416] while the nowadays available

spectrum includes varieties with other lead compounds such as

CBG. Within those plants and derived materials the total and

relative amount of main constituents can vary considerably.

Besides plant distinction cannabis analysis served historically for-

ensic/legal purposes to determine THC in biological uids and con-

scated material. Originally the plant synthesizes and accumulates

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/talanta

Talanta

http://dx.doi.org/10.1016/j.talanta.2015.02.040

0039-9140/&2015 Elsevier B.V. All rights reserved.

n Corresponding author.

E-mail address:[email protected](W. Peschel).1 Present address: European Medicines Agency, 30 Churchill Place, Canary

Wharf, London E14 5EU, United Kingdom.

Talanta 140 (2015) 150165

7/26/2019 Cannabis Sativa L. Chemotype Distinction Extract

2/16

carboxylated forms (e.g. 9-trans-tetrahydrocannabinolic acid-THCA)

which are converted into post-harvest neutralderivatives accelerated

by light and heat (e.g. THC) [17]. Otherssuch as CBNare only

degradation products of those derivatives. The common focus on

neutralcannabinoids can be explained by their activity, bioavailability

(traditional hot smoke inhalation), but also the heat conversion of the

acids when traditionally analyzed by GC. However, in case of cold

preparation, analysis and application, herbal starting materials and

derived extracts contain the original carboxylated cannabinoids. Theirseparate HPLC determination is reportedly more precise than total

THC values via GC or derivatisation before chromatography [18].

Limitations of all chromatographic methods encouraged also testing

other analytical methods including NMR spectroscopy[1922].

Characterization beyond the THC content became more rele-

vant with the increasing acceptance of medicinal use [23,24].

Specications based on analytical markers vary now according to

purpose i.e. not only to discriminate drug and non-drug but

guarantee identity and consistent quality of specic preparations.

Even more important is in view of the multiple effects from sev-

eral co-constituents the determination of prevailing active con-

stituent groups that may contribute to the activity.

We therefore used a new targeted 1H NMR proling method

and two newly developed and validated HPLC/DAD methods as

complementary tools to distinguish chemotypes and identify

extracts of different polarity. HPLC/DAD was further used to prole

extracts as regards main cannabinoid pattern aside more polar

constituents such as avonoids based on the quantication of

main cannabinoids (THC, CBD, CBG and CBN), the corresponding

acids, and the cannabis-characteristic prenylated avones CFL-A

and cannavin B (CFL-B). Group and ratio markers were derived

that are potentially useful in cannabis specications and their

variation determined according to starting material and extrac-

tion. As a simple activity test in relation to these markers we

checked exemplarily their effect to reduce cell viability in HeLa

cells.

2. Materials and methods

2.1. Reference standards

THC, CBD, CBG, CBN and THCA were purchased from THC

Pharm GmbH (Frankfurt, Germany) and stored in the dark at

20 C. CFL-A/CFL-B were kindly provided by Giovanni Appen-

dino, (Novarra, Italy). As phenolic standards we used the canna-

binoid precursor olivetol, as common phenolcarbonic acid

chlorogenic acid, and as avonoids the aglycons quercetin and

apigenin (all Sigma UK) and the glycosides orientin, homorientin,

vitexin, isovitexin (all Extrasynthese S.A. Co., Genay-Sedex,

France).

2.2. Plant material

Four C. sativa L. dry herbal drugs varied in composition (leaf-

ower ratio), genotype (THC-type, CBD-type, CBG-type, ber

(CBD)-type) and production parameters such as cultivation, drying

conditions and age. THC-type drug I , a standardized indoor culti-

var from controlled cultivation for medicinal use was provided by

TNO (Zeist, Netherlands) with specication certicate (18% THC

after heating, 0.8% CBD, o1% CBN). The pure Cannabis os drug

contained female ower tops without leaves and stalks from lower

plant parts. Samples from a CBD-rich (labeled II), a low-cannabi-

noid (III) and a CBG-rich (IV) variety were kindly provided by

Giampaolo Grassi (ISCI, Experimental Institute for Industrial Crops,

Rovigo, Italy). They originate from three outdoor lines with former

batches specication (GC analysis: II: 0.7% THC, 13.7% CBD, 1.0%

CBG; III: 0.01% THC, 0.68% CBD, 0.02% CBG; IV: 0.3% THC, 5.8% CBD,

25.2% CBG). The II (III, IV) drugs obtained from non-standardized

growing and drying conditions contained 38% (42%, 42%) owers,

44% (51%, 28%) leaves, 13% (4%, 23%) stalks larger than 2 mm dia-

meter and 5% (3%, 7%) seeds (all w/w), respectively. Stalks 43 mm

diameter were removed before extraction. The age of the materials

at time of extraction was 18 months (I, II and III) and 3 months

(IV).

2.3. Extraction and fractionation

The four drugs (I, II, III, IV) were extracted with ethyl acetate

and ethanol 40% using the classic drugsolvent ratio 1:10 for

tinctures. 10 g samples were macerated with 100 mL solvent in

two passages (24 h each) at room temperature in the dark under

agitation (aluminum foil covered Erlenmeyer ask on an auto-

mated shaker). After ltration, organic solvents were removed

under vacuum (o40 C) followed by freeze drying in case of the

ethanolic 40% extracts (Et40, EtOAc). A test passage with drugs I

and III analyzed separately showed that 8394% of cannabinoids

had been extracted. Another 10 g of each drug was defatted with

heptane in order to remove a major proportion of cannabinoids.

The defatted residues were dried at room temperature and

exhaustively extracted in four passages using methanol (2 6 h)

and methanol 70% (2 6 h) and ultrasonic treatment of 1 h. After

reduction under vacuum or freeze drying extracts were dried

under nitrogen until a constant weight had been reached (Me70).

Et40 and Me70 extracts were further fractionated in a liquid/

liquid system between water and organic solvents in several steps,rst with dichloromethane, and secondly with ethyl acetate (Et40-

diclo, Et40-etoac, Et40-wat, Me70-diclo, Me70-etoac, Me70-wat).

EtOAc extracts were fractionated into a hexane (EtOAc-hex), and

an aqueous (8% methanol) fraction (EtOAc-wat). All unied frac-

tions were reduced and dried as described above. All extracts were

stored at 20 C in the dark.

2.4. 1H NMR procedures

Stock solution with 100 mg/mL of dry extracts was prepared

with deuterated dimethylsulfoxide (99.8% DMSO-d6, Sigma UK). In

some cases a minor amount of D2O (maximum 10%, Goss Scientic,

UK) was added. Then 150 L of this stock solution was dilutedwith 1350 L DMSO-d6and ltered through a 0.45 mm Acrodiscs

syringe lter (Fisher Scientic, Loughborough, UK) and stored at

4 C. For analysis the 10 mg/mL solutions were thawed and 0.6 mL

lled into WG-5 mm NMR tubes (Wilmad). 1H NMR spectra of

samples were obtained on Bruker AVANCE 400 MHz instruments

equipped with a multinuclear probehead with z-gradient. The

Xwin-NMR 3.5 software was used for spectra acquisition and

processing. The size of all 1D spectra was 65 K and number of

transients varied for different type of spectra. The standard 1D 1H

NMR spectra are acquired with 30o pulse length, relaxation delayof 2 s. The numbers of scans were 128 or 64. The spectra were

recorded at 300 K.

2.5. 1H NMR experiments and analysis

We measured pure reference standards (at minimum three

concentrations usually 100, 10 and 1 mg/mL adapted to the natural

occurrence in cannabis extracts) and combinations of reference

substances in different ratios. Spectra were recorded between

0 and 14 ppm chemical shift (). After baseline correction, the

calibration was carried out on the residual DMSO-d6solvent peak

in terms of the chemical shift (2.5 ppm) and intensity (set as

normalized basis for integration). The ngerprint 0 - 11 ppm and

enlarged and expanded sections e.g. between 4 and 9 ppm were

W. Peschel, M. Politi / Talanta 140 (2015) 150165 151

7/26/2019 Cannabis Sativa L. Chemotype Distinction Extract

3/16

selected as suitable areas for extract identication. Signal alloca-

tion (DMSO-d6) was performed by comparison with reference

standards, extracts with known HPLC prole and previously pub-

lished assignments in deuterochloroform or deuteromethanol

(deuteroacetone for cannavins)[1921,25].

THC: (1H NMR, 400 MHz in DMSO-d6,, all in ppm; in bold dis-

tinguishable diverse signals of THCA in extracts): 0.85 (H-5, 3H,t), 0.98 (H-9, 3H,s; THCA1.03), 1.25 (H-3, H-4,m), 1.30 (H-5,m,

THCA1.38), 1.31 (H-10, 3H,s), 1.48 (H-2, 2H,m), 1.60-1 (H-6, H-7,3H,s), 1.85 (H-5, 1H,m), 2.08 (H-4, 2H,m), 2.34 (H-1, 2H,m, THCA

2.742.85), 3.08 (H-1, 1H, dm; THCA3.15), 6.01 (H-3, 1H, m),

6.15 (H-5,1H,m), 6.37 (H-2, 1H,m;THCA6.31), 9.20 (2-OH 1H,s)

CBD: (1H NMR, 400 MHz in DMSO-d6,, all in ppm; in bold

distinguishable diverse signals of CBDA in extracts): 0.86 (H-53H, t), 1.25 (H-3, H-44H, m), 1.48 (H-2, 2H, q), 1.60-61 (H-7,

H-9, 3H,s), 1.67 (H-5,m;CBDA:1.72), 1.93 (H-4, 1H,m), 2.09 (H-4,

1H,m), 2.30 (H-1, 2H, t, CBDA2.75),3.04 (H-6, 1H, t), 3.82 (H-1,1H,d), 4.42 (H-10, 1H, s), 4.49 (H-10, 1H, s), 5.08 (H-2, 1H, s), 6.01

(H-3, H-5, 2H, s; CBDA H-5 6.13), 8.62 (2-OH)

CBG: (1H NMR, 400 MHz in DMSO-d6, all in ppm; in bold dis-

tinguishable diverse signals of CBGA in extracts): 0.85 (H-5, 3H,t), 1.25 (H-3 and H-4, 4H,m), 1.48 (H-2, 2H, m), 1.52 (H-10, 3H,

s), 1.59 (H-9, 3H,s), 1.69 (H-7, 3H,s), 1.89 (H-5, 2H,m,CBGA1.99),

1.99 (H-4, 2H, m, CBGA: 2.17), 2.32 (H-1, 2H, t; CBGA: 2.78),

5.04 (H-6, 1H, m), 5.15 (H-2, 1H,m), 6.08 (H-3 H-5, 2H,s, CBGA

H-5: 6.22), 8.86 (2-OH, 2H, s)CFL-A: (1H NMR, 400 MHz in DMSO-d6, all in ppm): 1.51 (H-

8, 3H, s), 1.58 (H-10, 3H, s), 1.73 (H-9, 3H, s), 1.92 (H-4, 2H, t),

2.0 (H-5(2H,t), 3.23 (H-1(2H,m), 3.89 (O-Me, 3H, s), 5.03 (H-6,

t), 5.19 (H-2, 1H,t), 6.55 (H-10, 1H,s), 6.89 (H-3, 1H,s), 6.94 (H-5,

1H, d), 7.55 (H-2and H-6, 2H,m), 13.21 (5-OH, 1H, s)

2.6. HPLC

2.6.1. Fingerprint

A HPLC Waters

system 900, with a Waters 996 PDA detectorand a Waters 717plus auto sampler device and EmPower soft-

ware equipped with an Aces 5 Phenyl (25 cm x 4.6 mm) column

(ACT, Aberdeen, UK) and a Nova-Paks C8 Guard Column

3.9 20 mm, 2/pkg (Waters UK, Elstree, UK) was used. Conditions

were as follows: column temperature 25 C, auto sampler 8 C,

ow 0.9 mL/min, running time 80 min including a polar pre-phase

and a lipophilic washing post-phase (solvent C 63-71 min 100%);

solvent A water (TFA 0.1%), solvent B water-acetonitrile (65:35, TFA

0.1%) solvent C acetonitrile; gradient: solvent A 0 min 70%, 10 min

60%, 38 min 40%, 40 min 5%, 55 min 0%, 74 min 70%. For identi -

cation a triple ngerprint was recorded near absorbance maxima

of avonoids (254 nm) cannabinoids (275 nm), and avonoids/

phenolcarbonic acids/cannabinoid acids (324). Detection at214 nm provided the best levelled information for all compounds

and was used for quantication. Samples (starting concentration

extracts 10 mg/mL, standards 1 mg/mL) were diluted in methanol,

methanol/water mixtures and, prior to use, ltered through a

0.45 mm Acrodiscs syringelter (Fisher Scientic, Loughborough,

UK) before injection (10 L or 30 L).

2.6.2. Cannabinoid prole

The same equipment and methodology as described for the

ngerprint were used for cannabinoid analysis with modications

in column (Agilent Zorbax RX-C18 column, 5 mm 4.6 250 mm

Highchrom, UK), solvents (only solvent B and C), running time and

gradient (55 min, solvent B: 0 min 70%, 30 min 35%, 43 min 5%,

48 min 70%).

2.6.3. Identication and qualication

THC, THCA, CBD, CBG, CBN, CFL-A/B (Fig. 1), avonoids and phe-

nolcarbonic acids were identied using reference standards (retention

times inTable 1). The characteristic DAD-UV spectra (210400 nm) of

the standards and literature reports [24] allowed a classication of

compounds into neutral cannabinoids (THC/CBD/CBG pattern and

CBN/CBC pattern), cannabinoid acids (THCA/CBDA/CBGA pattern or

CBDA: R = COOH

CH2

OH

CH3

CH3

OH

CH3

R

CBD: R = H

1

2

3

4

5

6

7

9

10

1 2

3

4

5

6

1

2

3

4

5

CBDA: R = COOH

CH2

OH

CH3

CH3

OH

CH3

R

CBD: R = H

1

2

3

4

5

6

7

9

10

1 2

3

4

5

6

1

2

3

4

5

CBGA: R = COOH

CBG: R = H

CH3

OH

CH3

CH3

OH

CH3

R

1

234

56

7

910

12

3

4

5

6

1

2

3

4

5

CBGA: R = COOH

CBG: R = H

CH3

OH

CH3

CH3

OH

CH3

R

1

234

56

7

910

12

3

4

5

6

1

2

3

4

5

O

CH3

CH3

CH3

OH

CH3

R1

2

3

4

56

7

8

9

10

12

3

4

5

61

2

3

4

5

THC: R = HTHCA: R = COOH

(1)

(2)

(4)

(10)

O

CH3

CH3

CH3

OH

CH3

R1

2

3

4

56

7

8

9

10

12

3

4

5

61

2

3

4

5

THC: R = HTHCA: R = COOH

(1)

(2)

(4)

(10)

CH3

CH3

OH

OH

CH3O

OH

O

CH3

2

34

5

6

7

8

9

10

1

2

3

4

5

6

1

2

3

4

56

7

8

9

10

CH3

CH3

OH

OH

CH3O

OH

O

CH3

2

34

5

6

7

8

9

10

1

2

3

4

5

6

1

2

3

4

56

7

8

9

10

Fig. 1. (A) THC and THCA, CBD and CBDA, CBG and CBGA. The numbering follows

adjusted p-cymene based monoterpene nomenclature of CBD after Choi et al. [19]

for direct comparison in this study. For THC the other common nomenclature

(dibenzopyran system) is indicated at key positions (in brackets). (B) cannavin A.

W. Peschel, M. Politi / Talanta 140 (2015) 150165152

7/26/2019 Cannabis Sativa L. Chemotype Distinction Extract

4/16

cannabinolic acid (CBNA)/cannabichromenic acid (CBCA) pattern),

avonoids and phenolcarbonic acids (Fig. A1). CBDA and CBGA were

identied as dominant peaks in extracts from CBD-type and CBG-type

drugs and their conversion to their neutral forms. Unidentied peaks

were qualied by the PDA spectrum (Fig. A1) as avonoid, phe-

nolcarbonic acid, cannabinoid, and cannabinoid acid (Tables 1and2).

2.6.4. Assay and marker calculation

Standard curves reference substances as external standards

were established and standard mixtures injected with each ana-

lytical run. For extract proling all main peaks (above 0.05% of the

total peak area) were qualied according to their spectrum usually

resulting in 1535 major peaks per extract. Peaks with a minimum

of 0.2% of the total peak area were integrated corresponding

approximately to a 3:1 signal to noise ratio for the limit of

detection and 10:1 for the limit of quantication. Identied and

qualied peaks were summarized to group markers total THC

(THCtotTHCTHCACBN), CBG(A) ( CBGCBGA) and CBD(A)

( CBDCBDA), neutral cannabinoids (CAN), cannabinoid acids

(CANA), total cannabinoids (CANtot), and total phenolics (TPC).

From these group markers three ratio markers described as che-

motype marker (THC(A)/(CBG(A) CBD(A)), decarboxilation

marker (CANA/CAN) and a polarity marker (CANtot/TPC) were

calculated (Table 2).

Table 1

Retention times for standard references in two applied HPLC methods and their classication.

Reference standard tRFPa (min) tRCP

b (min) UV max (nm) Classication for inclusion in group markers

chlorogenic acid 9.27 240325 TPC

homorientin 21.78 256348 TPC

orientin 22.12 256348 TPC

isovitexin 25.62 268337 TPC

vitexin 26.05 268337 TPC

quercetin 31.42 255372 TPCapigenin 37.65 267338 TPC

olivetol 44.19 5.98 274

cannavin B (CFL-B) 54.54 11.49 273242 CFL

cannavin A (CFL-A) 59.18 20.17 274342 CFL

cannabidiolic acid (CBDA) 59.54 20.36 269307 CANA, CBD(A), CANtotcannabigerolic acid (CBGA) 59.87 20.93 267309 CANA, CBG(A), CANtotcannabigerol (CBG) 60.19 21.57 272 CAN, CBG(A), CANtotcannabidiol (CBD) 60.39 22.21 273 CAN, CBD(A), CANtotcannabinol (CBN) 61.52 28.78 283 CAN, THC(A), THCtot, CANtot9-tetrahydrocannabinol (THC) 62.37 32.95 276 CAN, THC(A), THCtot, CANtottetrahydrocannabinolic acid A (THCA) 62.67 41.26 270304 CANA, THC(A), THCtot, CANtot

a FP: ngerprint (80 min).b CP: cannabinoid prole (55 min).

Table 2Group markers and ratio markers for cannabis extract characterization. Description, relevance and determination from HPLC cannabinoid pro le (CP) or ngerprint (FP).

Calculation Calculated as Description/Relevance

THCtot THC, THCA, CBN THC, THCA, CBN (CP/FP) Total THC (potency marker), dominant in classical plants and preparations,

constituent with highest CB1/CB2 receptor activity plus its instable parent

and main degradation product, psychotropic legal/forensic importance

THC(A) THC, THCA THC, THCA (CP/FP) Sum of THC and THCA

CBD(A) CBD, CBDA CBD (CP) Sum of C BD a nd C BDA, d omina nt i n some ch emotyp es (in termedi ate, hemp),

main constituents without CB1/CB2 receptor afnity, non-psychotropic,

specic pharmacological effects

CBG(A) CBG, CBGA CBG (CP) Sum of CBG and CBGA , dominant in some chemoty pes, main const ituents

without CB1/CB2 receptor afnity, non-psychotropic, specic pharmacologi-

cal effects

CAN neutral cannabinoids THC, CBD, CBG, CBN (CP/FP) neutral (decarboxylated) cannabinoids - dominant in heated, aged and

excessively dried drugs

CANA a ci dic c an na binoid s T HC , C BD, C BG, C BN (CP/FP) acidic (carboxylated) cannabinoids -dominant in fresh plants andpreparations without heating

CANtot neutral and acidic cannabi-

noids, CAN CANA

THC, CBD, CBG, CBN (CP/FP) total cannabinoid content

oCAN CANtot (THCtotCBD(A)

CBG(A))

THC (in THC-type) CBD (in CBD-

type) CBG (in CBG-type) (CP)

other cannabinoids than those usually found as main cannabinoids in

common chemotypes, (i.e. THCtot, CBD(A), CBG(A))

CFL CFL-A CFL-B CFL-A, CFL-B(CP) cannavins-cannabis specic prenylated avones

TPC avonoids and phenolcarbonic

acids

vitexin, chlorogenic acid (FP) total phenolic content (without CFL)

THCtot/ (CBD(A)

CBG(A))

THC, THCA, CBN / CBD(A),

CBG(A)

as for THC(A), CBG(A), CBD(A)

(CP/FP)

chemotype marker, ratio main constituents in common chemotypes, ratio

main CB1/CB2 active vs. inactive constituents (plus acidic pro-drug), ratio

main psychotropic/ non-psychotropic constituents (plus acidic pro-drug),

legal/forensic importance

CANA / CAN CANA/ CAN as for CANA, CAN (CP/FP) decarboxilation marker, ratio acidic /neutral cannabinoids, indicator for

drug and extract quality and age

CANtot/ TPC CANtot/ TPC as for CANtot, TPC (FP) polarity marker, indicator for extract/solvent polarity, indicator for leaf and

ower portions, potential inuence of phenolics on activity

W. Peschel, M. Politi / Talanta 140 (2015) 150165 153

7/26/2019 Cannabis Sativa L. Chemotype Distinction Extract

5/16

2.7. HPLCngerprint method validation

The ngerprint covers cannabinoids and more polar con-stituents (phenolcarbonic acids, avonoid glycosides) separating

THC, THCA and CBN from other cannabinoids and closed avo-

noids previously described from each other such as vitexin/iso-

vitexin and orientin/homorientin.

2.7.1. Selectivity and specicity

THC/THCA, CBD/CBDA or CBG/CBGA were always the promi-

nent peaks within the cannabinoids (50 and 65 min) range largely

separated from common phenolic co-constituents (840 min)

(Table 1, Fig. 2). Spiking of extracts conrmed peak allocation

(example Fig. 2D). Despite overlapping of minor peaks qualica-

tion via UV spectra allowed the summation of AUC for calculation

of CANA, CAN, THCtot, CANtotand TPC.

2.7.2. Linearity and range

In addition to common standard curves for single reference

standards (not shown), we tested linearity and range for the morecomplex group markers. Comparable results were obtained for

different extract types in the range between 1 mg/mL and

25 mg/mL extract (example Fig. 3A). The relative standard devia-

tion of the mean (RSD) was maximum 38% for group markers and

615% for ratio markers.

2.7.3. Accuracy

The accuracy for group markers was checked via the recovery

rate of added reference standards (theoretical amount versus

found amount). We tested combinations of pure standards, com-

binations of one extract with different standards, and combina-

tions of three benchmark extracts (low avonoid/cannabinoid,

high-cannabinoid and high-phenolic) with the same standard

mixture (Table A1). The recovery rate for THCtot, CBD(A) CBG(A),

10.00 20.00 30.00 40.00 50.00 60.00 70.0010.00 20.00 30.00 40.00 50.00 60.00 70.00

THCA

THC

CBGA

CFL-A

CFL-B^

^

vitexin

*

10.00 20.00 30.00 40.00 50.00 60.00 70.0010.00 20.00 30.00 40.00 50.00 60.00 70.00

THCA

THC

CBGA

CFL-A

CFL-B^

^

vitexin

*

10.00 20.00 30.00 40.00 50.00 60.00 70.0010.00 20.00 30.00 40.00 50.00 60.00 70.0010.00 20.00 30.00 40.00 50.00 60.00 70.0010.0 30.00 40.00 50.00 60.00 70.00

** * * * **

*

CBD

CBDA

THC

THCA

10.00 20.00 30.00 40.00 50.00 60.00 70.0010.00 20.00 30.00 40.00 50.00 60.00 70.0010.00 20.00 30.00 40.00 50.00 60.00 70.0010.0 30.00 40.00 50.00 60.00 70.00

** * * * **

*

CBD

CBDA

THC

THCA

Minutes

10.00 20.00 30.00 40.00 50.00 60.00 70.00

THC

CBN

CBG

CFL-

A

CFL-

B

olive

tolo

rien

tin

chlorogen

icac

id

Minutes

10.00 20.00 30.00 40.00 50.00 60.00 70.00

THC

CBN

CBG

CFL-

A

CFL-

B

olive

tolo

rien

tin

chlorogen

icac

id

Minutes

10.00 20.00 30.00 40.00 50.00 60.00 70.00

*+or

ien

tin

R

S

*+v

itex

inR

S **

^^+

CFL-

BRS

+CFL-A

RS

CBDA

CBD

^c

hlorogen

icac

idRS

* ** *

*

^^

^^^

Minutes

10.00 20.00 30.00 40.00 50.00 60.00 70.00

*+or

ien

tin

R

S

*+v

itex

inR

S **

^^+

CFL-

BRS

+CFL-A

RS

CBDA

CBD

^c

hlorogen

icac

idRS

* ** *

*

^^

^^^

Fig. 2. HPLC ngerprint. (A) selected standard references (: 254 nm), (B) hydroethanolic crude extract from THC-type, and (C) CBD-type drugs, (D) extract fraction from

ber-type drug spiked with reference substances (RS) (all : 214 nm). Non-identied qualied peaks marked as: n avonoid, nn phenolcarbonic acid, ^ cannabinoid,

^^cannabinoid acid.

W. Peschel, M. Politi / Talanta 140 (2015) 150165154

7/26/2019 Cannabis Sativa L. Chemotype Distinction Extract

6/16

CANtotand TPC was between 89.9% and 106.9% (mixed standards),

79.8% and 126.4% (one extract plus different standards), and 92.8%

and 123.4% (different extracts plus one standard mix). Results

were considered acceptable for ngerprint sum parameters.

2.8. HPLC cannabinoid prole method validation

For cannabinoid and cannavin assay, a 55 min HPLC analysis

was applied (Fig. 4).

2.8.1. Selectivity and specicity

THC, CBD, CBG (as well as THCA, CBDA, CBGA) share compar-

able UV spectra with minor differences in the maximum absorp-

tion (265285 nm). Distinct UV spectra are obtained with CBN and

CBNA (equal to CBC and CBCA, Fig. A1)[24]. In extracts from THC-,

CBD- and CBG-type starting materials, the three main neutral

constituents and acidic forms were separated (Fig. 4B-E). Spiking

of different extracts did conrm peak allocations (exampleFig. 4F).

Both the THC and THCA peak covered in some extracts a minor

y = 112.92x + 57.828

R = 0.9988

y = 51.924x + 17.725

R = 0.9984

y = 36.99x + 23.56

R = 0.9969

y = 31.663x - 5.9281

R = 0.9963

y = 21.111x + 17.156

R = 0.983

y = 2.0963x + 0.9869

R = 0.9962

0

500

1000

1500

2000

2500

3000

3500

0 10 20 30

sample conc. (mg/mL)

mg

THC(A) CBx(A) CAN

CANA CANtot TPC

y = 217.17x + 76.659

R = 0.9994

y = 191.45x - 38.998

R = 0.9998

0

1000

2000

3000

4000

5000

6000

0 10 20 30

sample conc. (mg/mL)

mg

THCA THC

y = 7.2496x + 6.4821

R = 0.9949

y = 1.3577x - 1.2049

R = 0.9693

y = 0.8425x + 0.7867

R = 0.9921

y = 13.969x + 1.2058

R = 0.9998y = 8.3066x + 1.2675

R = 0.9999

0

100

200

300

400

0 10 20 30

sample conc. (mg/mL)

mg

CBN CBDA CBD CBGA CBG

Fig. 3. Examples for HPLC linearity tests. (A) Group markers calculated from the ngerprint of a CBD-type extract fraction, (B and C) cannabinoids calculated from can-

nabinoid prole of a THC-type extract. Samples were injected at 5 different concentrations between 1 and 25 mg/mL.

Fig. 4. HPLC cannabinoid prole (: 214 nm). (A) selected references standards; and extracts from (B) THC-type, (C) CBD-type, (D) CBG-type, (E) ber-type extract alone and

(F) spiked with reference standards THC, CBD and CBG.

W. Peschel, M. Politi / Talanta 140 (2015) 150165 155

7/26/2019 Cannabis Sativa L. Chemotype Distinction Extract

7/16

peak with a similar spectrum-visible via a shoulder at low THC

(A) concentrations.

2.8.2. Linearity and range

After establishing standard curves for pure substances, the

linearity was determined in different extracts.Fig. 3B and C shows

exemplarily the calculation of main cannabinoids in a THC-type

extract at ve concentrations. The results were consistent at con-

centrations 525 mg/mL with a relative condence interval for

single constituents between 1.4% and 5.7%. As only peaks with a

minimum signal to noise ratio of 1:10 (about 0.2% of the total peak

area) were integrated, the limit of quantication was approxi-

mately 0.5 mg in 1 g extract corresponding to about 0.25% of the

amount of the lead cannabinoid in cannabis preparations domi-

nated by one compound pair. Satisfactory peak separation

alongside detection of minor peaks was found with 10 mg/mL

sample concentrations.

2.8.3. Accuracy

We tested the recovery of single compounds when added

combined to different types of extracts. THC-type extracts were

spiked with THC, CBD or CFL-A and three CBD-type extracts with

3 reference standards. Recovery rates were between 93.6104.8%

(standard mixtures) and 70.8129.5% (in extracts) for cannabi-

noids and 91.6118.6% (standard mixtures) and 77.393.2% (in

extracts) for cannavins, respectively. Percentage deviations

higher than 15% resulted only from very low concentrated sub-

stances. CFL-A determination was partially hampered when com-

bined with a CBDA-rich extract and recovery rates of 60.5

88.2%

Fig. 5. 1H NMR spectra of cannabis constituents (DMSO-d6). THC and mixtures of THC CBD (2:3), THC CBD (6:1) and CBD CFL-A (10:1) with proton signal assignments

for THC, CBD and CFL-A (in brackets).

W. Peschel, M. Politi / Talanta 140 (2015) 150165156

7/26/2019 Cannabis Sativa L. Chemotype Distinction Extract

8/16

suggest that the method is not suitable when cannavins are the

main focus of analysis for CBD-type extracts.

2.8.4. Precision

In addition to the instrument precision for single compounds

(e.g. RSD THCA 2.11%, THC 4.72%, CBN 1.75%, CBG 6.24%; n 6) we

tested the intra-assay precision when analyzing extracts over

several hours. To detect whether mixtures decompose or instru-

ment parameters shift, ve times the same extract was injected atthe beginning, the middle and the end of a 36 h analytical run. We

obtained a satisfying precision with RSD below 4%. No trends were

observed. The inter-assay precision tested in different extract

types (measurement in duplicate at day 1, day 7 and day 12) waso5% RSD for compounds 420 mg/g extract concentration, o10%

for compounds between 5 and 20 mg/g in the extract and o20%

for compounds between LoD and 5 mg/g in the extract. A trend

was observed regarding the conversion of acids into neutral forms

within 12 days.

2.9. Cell culture and cell viability assay

HeLa-IL-6 (HeLaluc) cells originating from Dr. M. L. Lienhard

Schmitz (University of Giessen, Germany) were maintained in

DMEM (Invitrogen, UK) supplemented with 10% fetal bovine

serum and antibiotics at 37 C in a 5% CO2humidied atmosphere

and split when conuent. Cells were allowed to grow in media to

6080% conuence before harvesting. For the MTT assay cell sus-

pension was adjusted to 7.5 104 cells/mL and 96-well plates

(200 L per well) were incubated for 1824 h at 37 C with the 5%CO2, 95% humidity.

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide] assay was performed as previously described [26]. Samples

were primarily dissolved in DMSO or methanol and diluted in media

for a starting concentration of 200 g/mL (Et40 extracts) or 50 g/mL(EtOAc extracts). Absorbance values were measured with an Anthos

Lucy 1 luminometer at 570 nm (reference lter at 620 nm, ASYS,

Eugendorf, Austria). Absorbance values were converted into % growth

values in comparison to the non-treated control. Toxic effects wereexpressed as maximum non-toxic concentration (MNTC85% of

control) and as IC50value.

2.10. Statistical analysis

After HPLC validation (see above) samples were injected in

duplicate and results expressed as mean7SD. MTT assay: Samples

were tested in duplicate per plate and the median values from

three independent experiments were used to calculate the mean

(n 3)7SEM. Correlation between single group and ratio markers

of cannabis extracts and cell viability reduction in HeLa cells (IC50and MNTC) were tested using Pearson's correlation test.

3. Results and discussion

3.1. 1H NMR identication of cannabis extracts in DMSO-d6

After qualitative peak assignments of major cannabinoids and

cannavins using 1H, 13C, 1H-1H COSY and HMBC [19], 1H NMR

was favored to differentiate between chemovarieties followed by

proposals to quantify constituents in extracts [20,21] and to use

metabolomics for chemovar distinction [30]. A direct analysis of

tinctures with suppression of water signals using standard deut-

erated solvents for extraction was previously reported by our

group [22]. Here, in contrast to commonly used chloroform,

methanol or water, the DMSO-d6ngerprintallows for extracts of

distinctive polarities a single sample preparation in only one non-

volatile solvent for direct comparison. The same samples can also

be used for further dilution in other solvents for chromatographic

analysis or in cell assays. Of advantage is also the use of the solvent

signal for calibration and normalization (integration) without need

for internal standards. Most cannabinoid proton signals in DMSO-d6were found comparable to those assignments previously made

for deuterochloroform (with a general downward shift) and also

previous assignments with the protic solvent deuteromethanol

[19](Figs.1 and5). While the discrimination of THC, CBD, CBG andtheir carboxylic counterparts in the aliphatic area (04 ppm), is

often hampered by overlaps of close signals, the aromatic areain

particular the H-5position and the two olenic methins (H-2 and

H-6) in CBD and CBG between 4 and 6.5 ppm are more suitable.

The hydroxyl groups provide additionally distinguishable signals

for main cannabinoids such as in 2 position for THC at 9.2 ppm,

for CBD at 8.62 ppm, for CBG at 8.86 ppm, although somewhat

unreliable due to temperature dependence as previously reported

[19,21]. We compared mixtures of pure compounds to estimate

the range of suitable ratios for simultaneous identication in

extracts. Fig. 5 illustrates the distinction of THC and CBD in two

combinations (011 ppm) as well as appearance of avonoid

proton signals when CBD is combined with CFL-A. Fig. 6 shows

extracts from the four chemovars (49 ppm) demonstrating that in

non-heated cannabis extracts usually two main substances have to

be considered. In some cases, extracts with a balanced ratio

between two pairs, even four signal sets have to be taken into

account. Nonetheless the per seselectivity of NMR by suppressing

less intense signals allows in principle an easy identication of the

chemotype. The difference between benchmark extracts (canna-

binoid-rich versus practically cannabinoid-devoid but containing

other phenolics) as well as the use of selected proton signals to

allow the distinction between neutral and acidic forms is shown in

Fig. 7.Addressing both supports identication, indicates the level

of decarboxilation but also illustrates possible differences in the

NMRngerprint despite equal content in the prevailing pair such

as CBG(A). With our conditions, 1:10 ratios were found feasible to

recognize patterns of the lower concentrated compound in pure

compound mixtures. For extracts, only a maximum 1:5 ratio to thehighest concentrated compound may still allow to address com-

fortably the subordinated compound. Below that, it depends case

by case on the actual concentrations and numbers of main co-

constituents (e.g. THC plus THCA or THC plus THCA plus CBD plus

CBDA etc.).

3.2. 1H NMR (DMSO-d6) key signals for chemotype distinction and

herbal drug identication

Spectra were manually checked for specic signals (qualitative

marker), for overlapping and selectivity (identication without

interference, potential for integration) and for intensity (detect-

able also at lower concentrations). We summarize in the following

signals for THC(A), CBD(A), CBG(A) differentiation and demarca-tion between the acidic and neutral forms.

3.2.1. THC(A)

44 ppm: For THC(A) the 6.15 ppm signal of the 5 proton is the

best recognizable one to distinguish from CBD(A)/CBG(A) with

CBDA (H-5) at 6.13 usually well separated. The THC 6.01 signal of

H-3 (shared with CBD) is the most selective peak versus THCA,

although too weak in low concentrated extracts. For both demar-

cations, the H-2 signal (THC 6.37, THCA 6.31 ppm) represents the

marker ofrst choice.

o4 ppm: The THC-characteristic H-9 signal at 0.98 (THCA:

1.03) is in mixtures within a large bulk of adjacent peaks. Although

both are not always completely separated, they may serve for

demarcation to CBD(A)/CBG(A). Following signals are not shared

W. Peschel, M. Politi / Talanta 140 (2015) 150165 157

7/26/2019 Cannabis Sativa L. Chemotype Distinction Extract

9/16

but can partially overlap with close signals from other cannabi-

noids, i.e. useful for identication but less so for quantication:

1.31 (H-5; THCA 1.38), 2.34 (H-1, CBD 2.31, CBG 2.32) and 3.08 (H-1, CBD 3.05). THCA-specic split H-1signals at 2.74 and 2.85 (THC

single at 2.34) are well detectable in THC-type extracts. However,

when less concentrated in extracts of other chemotypes, they are

not sharp and too close to e.g. CBDA H-1(2.75) for reliable iden-

tication. Also differences in the H-1 (THC 3.08, THCA 3.13-3.18)

may be disturbed by signals from other cannabinoids.

3.2.2. CBD(A):

44 ppm: Most characteristic are the split CBD H-10 proton sig-

nals appearing at 4.42 and 4.49 ppm. Both peaks are shared with

CBDA (4.41 and 4.47), but there are no THC(A) or CBG(A) signals

interfering. The prominent 6.01 signal of CBD is not only caused by H-

3 as in THC but also by H-5, complicating its use for eventual

quantication of neutral cannabinoids. H-5of CBDA in contrast can

be found at 6.13 closed to THC(A) (6.15). The 2-OH signal at 8.62 may

help distinguishing from THC and CBG. Only in extracts containing

CBDA appeared another OH-group signal at 5.32 obviously due tohydrogen bonding of the carboxyl group. A sharp and intense signal

is exhibited by the H-2 proton at 5.08, not interfering with

THC(A) but eventually with the 5.04 peak of CBG(A).

o4 ppm: The CBD-specic not very sharp 3.82 peak (H-1) is

often too weak within mixtures. CBD characteristic signals at 2.30

(H-1, CBDA 2.75) and 3.05 (H-6) can overlap with corresponding

THC signals (2.34, 3.01). The CBDA-characteristic H-1 signal at

2.75 distinguishes from CBD (2.3) but partially overlaps with THCA

H-1(2.75 2.85) in extracts with substantial THCA content or

with the corresponding CBGA signal at 2.78.

3.2.3. CBG(A):

44 ppm: The most distinguishable to THC(A) and CBD(A) are

the H-5signals at 6.08 ppm (CBG) and 6.22 (CBGA), that were also

Fig. 6. 1H NMR spectra of extracts from four chemotypes (DMSO-d6, 10 mg/mL, 49 ppm, amplied 4 ). Extracts (Et40-EtOAc) from THC-type (A), CBD-type (B), Fiber-type

(C) and CBG-type (D) drugs with key proton signals for identication of main cannabinoids.

W. Peschel, M. Politi / Talanta 140 (2015) 150165158

7/26/2019 Cannabis Sativa L. Chemotype Distinction Extract

10/16

recognizable as minor peaks in THC-type extracts with a CBG

(A) content around 1:10 vis--vis THC(A). CBG H-5 overlaps with

the H-3signal. Equally to THC and CBD, the 2-OH signal is specic

and usually well recognizable at 8.82. The two CBG(A) olenic

methin protons H-6 and H-2 at 5.04 and 5.15 are characteristic as

well as selective and sufciently intense in most cases yet prone to

overlaps with the CBD(A) 5.08 signal (H-2) in case of balanced

mixtures. Equal to CBDA, a signal assigned to 6-OH at 5.31 appears

only in CBGA containing mixtures.

o4 ppm: Some signals of CBG(A) dominated extracts in the

aliphatic area are slightly distinct from THC(A) and CBD(A) but

hampered by overlaps or low intensity such as those of H-4 / H-5

or H-9/H-10. The H-9 signal at 1.59 is useful in CBG(A) dominant

materials but may overlap with the 1.61 signal of other

Fig. 7. 1H NMR spectra of selected extracts in DMSO- d6.(all 10 mg/mL). (A) cannabinoid-rich extract (THC(A) 364.2 mg/g, CBG(A) 20.2 mg/g, HPLC; CANA/CAN ratio: 0.83)

with key signals to distinguish THC and THCA, (B) cannabinoid-reduced extract: THC(A) o1 mg/g, CBD(A) o1 mg/g, CBG(A) o1 mg/g, (C1) extract from CBG-type drug

(CBG(A) 193.3 mg/g, CANA/CAN ratio 0.82), with key signals to distinguish CBG and CBGA (C2) extract from the same CBG-type drug with comparable CBG(A) content

(189.0 mg/g) but different CANA/CAN ratio (0.28).

Table 3Key proton signals (in ppm) in DMSO-d6for identication of characteristic compounds/groups in THC(A), CBD(A) or CBG(A) dominant cannabis chemotypes.

Specic signals for identicationa Most selective in extractsb

THC 0.98 (H-9, s), 2.34 (H-1, m), 3.08 (H-1, dm), 6.37 (H-2, m), 9.21 (2-OH,s) 6.37, 9.21

THCA 1.03 (H-9, s), 1.38 (H-5, m), 2.74/2.85 (H-1, m), 6.31 (H-2, dm) 6.31, 2.74/2.85

THC(A) 1.85 (H-5, m), 6.15 (H-5, m) 6.15, 0.98/1.03

(shared signals THC and THCA not shared with CBD(A) or CBG(A))

CBD 1.93/2.09 (H-4, m), 2.28 (H-1, t), 3.05 (H-6, t), 3.82 (H-1, d), 5.08 (H-2, s), 8.62 (2-OH,s) 5.08, 8.62

CBDA 2.18/2.27 (H-4, m), 2.75 (H-1, m), 3.91 (H-1, d)

CBD(A) 4.41-4.42/4.47-9 (H-10) (shared signals CBD and CBDA not shared with THC(A) or CBG(A)) 4.414.49

CBG 1.89 (H-5, m), 2.32 (H-1, t), 6.08 (H-5, s), 8.82 (2-OH,s) 6.08, 8.82

CBGA 2.78 (H-1, t), 6.22 (H-5, s) 6.22

CBG(A) 1.69 (H-7, s), 5.04/ 5.15 (H-6, m and H-2, m), (shared signals CBG and CBGA not shared with THC(A) or CBG(A)) 5.04/ 5.15

CAN 2.28-2.34 (H-1) 2.282.34

(shared signals THC, CBG, CBD not shared with THCA, CBGA, CBDA)

CANA 2.75-2.78 (H-1) 2.752.78

(shared signals THCA, CBGA, CBDA not shared with THC, CBG, CBD)

CFL-A 1.73 (H-9, s), 6.55 (H-10, s), 6.89 (H-3, s), 6.95 (H-5, d), 7.55 (H-2, H-6, m), 13.21 (5-OH, s) 6.55, 6.89, 7.55

a Specic signals of the respective constituent not shared with other main cannabinoids in THC-type, CBD, type, ber type, and CBG-type derived cannabis extracts.b Consideration of intensity and overlaps with other main compounds in THC-type, CBD, type, ber type, and CBG-type derived cannabis extracts.

W. Peschel, M. Politi / Talanta 140 (2015) 150165 159

7/26/2019 Cannabis Sativa L. Chemotype Distinction Extract

11/16

cannabinoids if CBGA is a minor constituent. The CBG H-1triplet

at 2.32 is only slightly different to CBD (2.29) but useful to dis-

tinguish from CBGA (2.78) that may be also identied by some

minor differences in methylene groups H-4 and H-5.

3.2.4. CFL-A:

44 ppm: CFL-A exhibits some signals typical for the avonoidstructure and the prenyl moiety which are distinctive from main

phytocannabinoids. The aromatic protons at 7.55, 6.94, 6.89, and

6.55 may not be selective in mixtures with other avonoids but

quite specic for more lipophilic cannabis extracts where non-

prenylated avonoids are unlikely to be found. The rather broad

prenyl peaks of the H-6 and H-2 position at 5.03 and 5.19 are

interfering with CBG(A) peaks.

o4 ppm: The most specic prenyl peak is the singlet at 1.73

(H-9). The methoxy peak at 3.89 may be shared by other avo-

noids but is distinguishable from main cannabinoids. Overall,

despite the possibility for identication, cannavin detection in

cannabis extracts by 1H NMR appears limited due to low con-

centration and signal intensity in relation to predominant canna-

binoids. The same applies to lower concentrated common avo-noids. Exemplarily we had tested quercetin addition to cannabis

extracts (up to 20%), where typical signals such as at 1.90, 3.80,

4.04 where not intense enough while minor peaks can be identi-

ed in the less crowded area 6.58.0 ppm (data not shown).

3.2.5. Shared signals of THC(A), CBD(A), CBG(A):

Usually a standard cannabinoid pattern of the pentyl group

signals is recognizable with approximate shifts of 0.85 (H-5), 1.25

(H-3/4), 1.48 (H-2), whereof the signal at 0.85 interferes least

with others. Also H-1 is largely shared between THC, CBD and

CBG around 2.282.34, but the inuence of the neighboring car-

boxyl group makes it an important marker to distinguish from the

genuine acids (around 2.75). Another mutual signal of all main

acids (THCA, CBDA, CBGA) distinct to their neutral counterparts

was not identied. Signals most inuenced by the carboxyl group

are mainly the aromatic H-5, and to certain extent the hydroxyl-

and the methylen groups (H-1, H-5).

An overview of suitable signals to identify THC-, CBD-, and

CBG-type extracts is compiled in Table 3. Because NMR identi-

cation of multicompound mixtures is challenging due to over-

lapping peaks of similar structures[27]it is usually combined withmultivariate pattern recognition and principal component analysis

(PCA) for metabolomic analysis but also industrial quality control

[28,29]. The targeted analysis of key signals as performed here, can

amend PCA approaches in quality control because it provides in

contrast to the mathematical data output of PCA a set of specic

signal allocation to tick boxes for few compounds in particular

combined with HPLC information on major cannabinoids present.

Using the most selective signal in the aromatic and aliphatic area

for each of the key compounds, a pattern of a few key signals can

be compiled for fast identication as demonstrated in Table 4for

extracts of different polarity. It allows simplication avoiding the

need to analyze the complete proton signal set of complex mix-

tures. An extension beyond THC, CBD and CBG type distinction

may be tested in with other chemovarieties.

3.3. HPLC/DADngerprint and cannabinoid prole

Cannabis HPLC ngerprinting and quantitative analysis has

developed over years to improve the separation of very similar

cannabinoid structures via specic columns, detection (e.g. ame/

electro ionization) or hyphenation with MS [23,3133]. Compar-

able to a more recent validated method, we show that conven-

tional HPLC/DAD provides valuable information for routine quality

control despite limits for the simultaneous cannabinoid analysis

[34]. The ngerprint signals the main cannabinoids and presence

of more polar co-constituents and allows summarizing into groups

of qualied peaks for main extract pattern (Fig. 2). The advantage

of a broad range is made on expense of baseline separation e.g.

Table 4

Recognizable1H NMR pattern of main cannabis constituents in cannabis extracts.Selective proton signals for discrimination of chemotypes for reference standards and THC-,

CBD,- and CBG-type extracts (all 10 mg/mL in DMSO- d6), strong signal, weak signal, no detected signal.

W. Peschel, M. Politi / Talanta 140 (2015) 150165160

7/26/2019 Cannabis Sativa L. Chemotype Distinction Extract

12/16

CFL-A/B or CBD/CBG versus CBDA/CBGA. The cannabinoid prole

method allows in shorter time quantication of the main neutral

and acidic cannabinoids to THC, CBD and CBG type drugs plus CFL,

the only non-cannabinoids that appeared within the cannabinoid

range (Fig. 4). Limits are baseline separation of minor cannabinoids

and in some cases overlapping peaks for excessive concentrations

of one compound in the range 2022 min (e.g. CBDA or CBGA)

which can be solved by sample dilution. Extension to other rele-

vant cannabinoids of less common chemotypes such as 8 THC,CBC/CBCA or tetrahydrocannabivarin (THCV) may be tested in the

future.

3.4. HPLC based key markers for chemotype and herbal drug

identication

Main pattern for chemotype comparison and herbal drug

characteristics of non-heated material are presented as (1) abso-

lute values of main compound groups, (2) ratios of key compound

groups, and (3) relative proportion of cannabinoids (Fig. 8): (1) In

line with traditionally important identication of drug-typethe

absolute content in THC (plus THCA and CBN) is shown in relation

the content of common main cannabinoids CBD(A) and CBG(A),

accompanying minor cannabinoids and the total phenolic content

(Fig. 8A). (2)Fig. 8B provides at one glance the information can-

nabinoid-rich versus cannabinoid-low plant (CANtot) alongside

indicators whether THC(A) prevail over non-psychotropic canna-

binoids, how fresh the material is (degree of decarboxilation) and

to which extent cannabinoids prevail over accompanying phe-

nolics. The THCtot/ CBD(A) CBG(A) ratio indicates for I THC-type

independent from absolute values. Even if very low amounts

would be found, e.g. in old material with low THC but high CBN

values, any upwards directed bar signals THC-type origin. The

CANA/CAN ratio agged progressed decarboxylation in all four

drugs; most advanced in II. The CANtot/TPC ratio is inuenced by

the cannabinoid content (e.g. low in III) but also other drug char-

acteristics, e.g. the leave portion higher in II and III than in I and IV

leading to higher TPC values. (3) Fig. 8C, the cannabinoid prole,

focuses on the ratio of main cannabinoids to each other inde-

pendent of total values. It illustrates in a different way the pre-

vailing cannabinoid pair, the ratio between the main acidic and

neutral cannabinoids and additionally CBN (conrming the

advanced age of I) and cannavins (measurable amounts only in I:

0.79 mg/g). Notably, I contains more CBG(A) than CBD(A) which

conrms other reports for THC-type drugs and raising doubts on

the suitability of the conventional focus on THC and CBD only[34].

3.5. Chemotype distinction

HPLC analysis of the four varieties conrmed THC-type (I),

CBD-type (II), CBD-type cannabinoid-low (III) and CBG-type (IV,

rst described by Fournier et al. in 1987) [35]several CBG strains

are available[34]). In contrast, traditional terms may be used with

caution when characterizing dry herbal drugs. Small and Beck-

stead had rst differentiated between a chemotype withTHC40.3%/CBDo0.5%, an intermediate type with dominant CBD

but THC also present, and a particularly THC-low type [14]. The

still common classication drug type (THC42%, CBD 0%), ber

type (THCo0.3%, CBD40.5%, THC/CBD ratioo0.1) and an inter-

mediate type (THC40.5%, CBD40.5%, THC/CBD ratio40.5)

reects the importance distinguishing between Cannabis os for

recreational use (often425% THC) and low-THC industrial hemp

without considering other possible inherited biosynthesis pattern

in plant populations[16,3638]. CBD forming an essential part of

those classications was supposed to be partially overestimated

due to non-selective methods, which became more relevant with

medicinal applications using plants optimized on CBD [3,13,39].

For IIV neither provider specication nor our results (EtOAc

extracts from specic batches of dry drugs of certain age) allows

Fig. 8. Comparison of 40% ethanol (A D) and ethyl acetate (E H) extracts from four drugs. (I THC type, II CBD type, III CBD ber type, IV CBG-type) (A E) Main compound

groups THCtot, CBx(A) ( CBG(A) CBD(A)), oCAN and TPC, (B F) Total cannabinoid content and ratio markers, (C G) cannabinoid prole, (D H) Effect on cell viability in

HeLa (MTT assay, mean7SEM, n 3).

W. Peschel, M. Politi / Talanta 140 (2015) 150165 161

7/26/2019 Cannabis Sativa L. Chemotype Distinction Extract

13/16

unambiguous allocation (Table 5): the declared CBD-value of I

would not match conventional drug-type denition, II does nei-

ther t into intermediate type nor ber type denition, and for

extracts derived from III the CBD-values were too low to comply

with ber type criteria, which, however, CBG-type IV would

match. Nor does the three-type system consider varieties with

prevailing cannabichromene (CBC), enhanced 8-THC instead of9-THC, or the propyl homologs instead of the usual pentyl can-nabinoids (e.g. tetrahydrocannabivarin / tetrahydrocannabivarenic

acid dominant clones often of South African origin)[4042]. THC

or CBD values (HPLC) alone without consideration of the acids

would not only be too low but also unreliable due to decarbox-ylation according to the age and storage. As actual THC/ CBD ratio

to indicate intermediate or ber type we obtained 4529.6, 0.03,

0.11 and 0.25, for IIV respectively. The more real ratio

THCtot/CBx(A), independent from the actual main co-cannabinoids,

the decarboxilation progress, and the THC to CBN conversion, was:

7.5, 0.03, 0.04 and 0.04 for I IV, respectively. CBx(A) summarizes

the main non-CB1/CB2-active constituents: here CBD(A) and CBG

(A), in other cases eventually CBC plus CBCA or may even include

all other cannabinoids (oCAN) to obtain information on the por-

tion of psychotropic THCtot. In addition, such data are only com-

parable together with specication of the plant part (such as os,

folium or resinum), limits for admixtures (such as seeds, stalks,

foreign matter), and the analytical method. It is assumed that

traditional THC and CBD values are based on assumed exhaustive

extraction of not always well dened plant samples and total

conversion of the acids such as via GC. Yet materials/extracts may

differ and nowadays methods with separate acid detection prevail.

3.6. HPLC-based extract proling range and enrichment of com-

pound groups

The prole of different extracts was investigated for the pos-

sibility to identify the original herbal drug and the extent com-

pound groups are extracted, which determines their relevance as

potential markers for specication. Standard solvents to exhaus-

tively extract cannabinoids are traditionally chloroform orchloroform/methanol mixtures. We used EtOAc to macerate the

main part of the cannabinoids without enrichment of more lipo-

philic substances (e.g. terpenoids, fatty acids). EtOAc extracts were

compared with Et40 extracts (moderate cannabinoid with more

polar co-constituents), Me70 extracts of defatted material (low

cannabinoid) and extract fractions (Fig. 8, Table 6).

Et40 extracts:Et40 extracts yielded only 6.2% (I), 16.6% (II), 15.3%

(III) and 20.4% (IV) of CANtot extracted with EtOAc (Fig. 8E). TPC

values varied between 4.3 and 33.2 mg/g in line with the portion

of leaves in the drugs. Contrary, the relative chemotype marker

(Fig. 8F 1st bar) indicates the chemotype solvent-independently.

For I, a reduced extraction of THC(A) in relation to CBG(A) when

using polar solvents instead of EtOAc is indicated by an only two-

fold THCtot value compared to CBx(A). The CANA/CAN ratio

Table 5

Classication of four cannabis chemotypes and proposed specication of herbal drugs and extracts.

I II III IV

Provider specication ofCannabis osa:

THC (%) 18 (after heating) 0.7 0.01 0.3

CBD (%) 0.8 13.7 0.68 5.8

CBG (%) n.s. 1.0 0.02 25.2

THC/ CBD ratio 22.5 0.05 0.015 0.01

Conventional classication of plants (based on exhaustive analysis ofCannabis os)b:

drug type? intermediate type? ber type intermediate type?

THC (%) 42 40.5 o0.3 40.5

CBD (%) 0 40.5 40.5 40.5

CBG (%) n.s. n.s. n.s. n.s.

THC/ CBD ratio n.s. 40.5 o0.1 40.5

Proposed specication ofCannabis osc:

THC-type CBD-type CBD-type, low cannabinoid CBG-type

CANtot(%) 18.94 10.64 1.31 19.34

THCtot(%) 14.68 0.33 0.06 0.72

CBD(A) (%) o0.04 9.72 0.61 2.92

CBG(A) (%) 1.97 0.40 0.02 16.66

THCtot /CBx(A)d 7.5 0.03 0.04 0.04

Solvent/Anal. Meth: (EtOAc/HPLC) (EtOAc/HPLC) (EtOAc/HPLC) (EtOAc/HPLC)

or

CANtot(mg/g) 448.8 273.5 120.6 344.2

THC(A)/CBx(A)d 7.5 0.03 0.04 0.04CANA/CAN 0.47 0.23 0.63 0.64

CANtot/TPC 192.1 6.6 4.3 29.0

Solvent/Anal. Meth (EtOAc/HPLC) (EtOAc/HPLC) (EtOAc/HPLC) (EtOAc/HPLC)

Proposed specication of cannabis extracts:

Drug/extract ratio 2.4:1 2.6:1 9.2:1 1.7:1

Solvent EtOAc EtOAc EtOAc EtOAc

CANtot(mg/g) 448.8 273.5 120.6 344.2

THCtot(mg/g) 347.8 8.5 5.1 11.9

CBD(A) (mg/g) o1 249.7 56.3 48.2

CBG(A) (mg/g) 46.6 10.3 1.8 274.9

[TPC (mg/g)]e [2.3] [16.8] [13.4] [12.0]

a As provided by the supplier at time of delivery I (HPLC), II-IV (GC).b Ref. [14].c Based on EtOAc extraction of specic batches available in this study.d CBx(A) CBD(A) CBG(A).e Optional such as in case of hydroethanolic extracts from Cannabis folium.

W. Peschel, M. Politi / Talanta 140 (2015) 150165162

7/26/2019 Cannabis Sativa L. Chemotype Distinction Extract

14/16

(Fig. 8F 2nd bar) proved to be not only material but also extract-

dependent. The CANtot /TPC ratio (Fig. 8F 3rd bar) revealed that

Et40 extraction lowers the cannabinoid content more (10-fold

lower for the Et40 vs. EtOAc) than it accumulates phenolic co-

constituents (doubled). The relative cannabinoid prole (Fig. 8G)

conrmed the genotype without substantial differences between

Et40 and EtOAc. In I Et40, however, the portion of other com-

pounds than the dominant cannabinoids were found relatively

increased compared to EtOAc.

Exhaustive methanol/methanol 70% extraction from defatted

material: Four-fold TPC values were obtained with exhaustive

Me70 extracts (I 4.3 and 19.4 mg/g, II 33.2 and 150.8 mg/g, III 20.4and 85.2 mg/g, respectively) compared to Et40. Despite removal of

the major part of cannabinoids they remained more concentrated

than phenolics in extracts from cannabinoid-rich material

(CANtot/TPC ratios: 6.2 (in I), 5.1 (in IV)). The cannabinoid prole

still allowed the information about the original chemotype. The

enrichment of cannavins in relation to cannabinoids could hardly

be achieved.

Recognition of the chemotype in extract fractions: Despite vari-

able quantity of cannabinoids and their proportions the

THCtot/CBxA ratio allowed for all fractions the identication of the

dominant cannabinoid pair, as long as cannabinoids could still be

detected. The THCtot/ CBx(A) ratio varied between 0.7 and 22 for

THC-type, 0.03-0.09 for CBD-type, 0.01-0.05 for CBG-type, and

0.07-0.12 for the ber type derived extracts (Table 6).

Enrichment of cannabinoids: Fractionation allowed further

concentration of total cannabinoids and affected slightly group

ratios such as THCtot/ CBG(A). Highest amounts in cannabinoids

per extract where obtained in most cases with high lipophilic

fractions (dichloromethane) following polar rst extraction (Et40).

Lipophilic extraction throughout such as hexane fractions of EtOAc

extracts gave overall higher yields but did not substantially

increase CANtotcompared to the crude extract. Although two-step

extraction allows modifying the ratio of particular cannabinoidgroups, purication of main compound pairs by simple solvent

based fractionation (e.g. separation of CBG(A) from accompanying

THC(A) or CBD(A)) was not possible.

Enrichment of phenolics: We obtained fractions with a more

levelled content of avonoids and cannabinoids via removal of

major parts of the cannabinoids. Et40-etoac/Me70-etoac and

EtOAc-wat fractions had CANtot/TPC values between 1.44.8, 0.2-

1.8, 0.41.2, and 0.2-20.9 for varieties I, II, III and IV, respectively.

Cannavins were often below the limit of quantication, did never

reach more than 8 mg/g extract and remained usually minor

constituents in relation to cannabinoids or other phenolic

compounds.

In summary, fractionation of crude cannabis extracts inuenced

the CANtot

/TPC ratios but less so THCtot

/CBx(A) ratios. Cannabi-

noids are chemically too similar to enrich separately via solvent

polarities without more sophisticated techniques. Thus chemotype

identication is possible solvent-independently. On the other hand

it shows that mixing of specic herbal drugs may provide a better

path of tailoring preparations to a prole that differs substantially

from the natural cannabinoid ratio in one chemovar.

3.7. Effect on the cell viability in HeLa

IC50 and MNTC levels were between 2.5-8.0 g/mL and 0.3-2.2 g/mL for EtOAc extracts compared to 6.125.2 g/mL and 2.012.7 g/mL for Et40 extracts, respectively (Fig. 8D and H). With anassumed co-inuence of the quantitative and the qualitative

composition, the cytotoxic effect in HeLa cells was less determined

by the dominant cannabinoids e.g. the THC(A) content but by theextraction solvent and the resulting CANtotvalue. For instance Et40

extracts of II, III, and IV with a 50-fold, 12.5-fold and 33-fold higher

CBD(A) or CBG(A) content compared to THC(A) affected cell via-

bility with an IC50value that was maximum 2.5 times higher (Et40

extract of III) or even similar (Et40 extract of IV) to the THC(A)-rich

Et40 extract of I. This was conrmed by testing additionally the

fractions from all crude extracts. Over the complete matrix no

clear correlation could be found between single constituents such

as THC, group or the dened ratio markers. The most likely rela-

tionship between cannabis extract composition and resulting

toxicity in the HeLa cells was between CANtotand log IC50 values

from the MTT assay (Fig. 9).

3.8. Markers for standardization of herbal drugs and extracts

For adequate specication of non-heated drugs (e.g. Cannabis

os, C. resinum, C. folium) and drug preparations (including

extraction solvent and drug extract ratio) for pharmaceutical

purposes [43], the preferred specication of absolute values of

CANtot, THCtot, and CBD(A) and other relevant main cannabinoids

can be rationalized as follows (Table 5):

CANtot: Due to the increased pharmacological knowledge on

non-CB receptor mediated and non-THC caused effects, the overall

amount of cannabinoids represents useful basic information which

should be further specied with regard to the main constituents.

Relevant specic markers may be different to classic main phyto-

cannabinoids[44,45]. The content of specic cannabinoids needs

to be put into context, i.e. the relevance of 20 mg/g THC is different

Table 6

Group markers and ratio markers for six extract fractions from four cannabis

varieties. Marker maxima as obtained per chemotype are indicated in bold, minima

underlined.

EtOAc Ethanol 40% Methanol 70%

Hexane Watera CH2Cl2 E tOAc CH2Cl2 EtOAc

(I)THC-type

THC(A) (mg/g) 372.5 14.0 245.5 87.4 33.3 1.2

CBD(A) (mg/g) nd nd 5.3 nd nd ndCBG( A) (mg/ g) 20.8 19.5 28.4 11.1 1.5 0.5

CFL (mg/g) nd 3.7 nd 7.4 2.8 2.7

TPC (mg/g) nd 30.0 nd 94.9 nd 27.4

THCtot/CBxA 17.9 0.72 7.2 7.9 21.9 3.1

CANA/CAN 0.83 0.46 0.89 0.27 0.31 0.36

CANtot/TPC 4846 4.8 4786 1.4 4172 1.6

(II) CBD-type

THC(A) 10.7 nd 14.0 1.1 nd nd

CBD(A) 122.7 14.6 137.9 14.6 12.7 nd

CBG(A) nd nd 9.1 1.1 1.6 nd

CFL nd nd nd nd 4.5 nd

TPC nd 17.8 nd 168.3 125.1 290.4

THCtot/CBxA 0.09 o0.03 0.09 0.07 o0.04

CANA/CAN 0.51 0.78 0.75 0.34 1.46

CANtot/TPC 4164 1.78 4686 0.25 0.33 0.08

(III) Fiber-type

THC(A) 12.2 nd 14.9 10.2 4.1 ndCBD(A) 166.3 78.3 183.1 81.7 50.0 nd

CBG(A) 20.0 nd 16.3 1.4 nd nd

CFL nd nd nd 5.2 nd 5.1

TPC nd 118.9 45.1 86.1 5.6 55.4

THCtot/CBxA 0.07 0.01 0.08 0.12 0.08

CANA/CAN 2.40 1.78 1.24 2.20 1.79 0.62

CANtot/TPC 4384 1.24 3.32 0.80 8.69 0.45

(IV) CBG-type

THC(A) 13.8 10.1 1.6 nd

CBD(A) 69.1 41.8 21.8 5.2

CBG(A) 219.0 193.4 163.9 35.1

CFL nd 4.3 3.8 2.3

TPC nd 14.7 6.6 24.3

THCtot/CBxA 0.05 0 .04 0.01 o0.01

CANA/CAN 0.41 1.01 1.57 1.12

CANtot/TPC 4744 20.9 18.9 0.24

ndo 0.5 mg/g (LoQ).a Water containing 8% methanol.

W. Peschel, M. Politi / Talanta 140 (2015) 150165 163

7/26/2019 Cannabis Sativa L. Chemotype Distinction Extract

15/16

with overall amounts of 25 mg/g CANtot or 300 mg/g CANtot. Our

cell viability test showed that effects did not correlate with THC

but with CANtotwhen not only THC dominant samples are used.

THC, THCA, CBN versus THCtot: Fresh material contains mainly

THCA, if not converted via heating completely into THC. It has been

previously shown that both substances should be analyzed sepa-

rately and summarized[18]. Thus in non-heated herbal drugs and

cold extracts, THCtot including THCA and CBN instead of THC

alone determines the putative strength and pharmacological

effects. CBN has a low to moderate CB1 afnity and psychotropic

effect[6]. The separated specication of CBN, THC and THCA may

be useful in stability tests. Here, CBN was only detectable atadvanced age in extracts from the THC-type.

CBD, CBDA, CBG, CBGA: In analogy to THCA/THC, the pro-drug

CBDA should be detected and added to CBD values in non-heated

preparations from fresh material. The explicit determination and

labeling of CBG(A) is suggested because of increasingly more CBG-

type plants on the market (identication), their importance as

main co-constituents in THC-type extracts, and own pharmacolo-

gical effects[7].

CANA/CAN: Values between 0.27 and 2.4 in our extracts

demonstrate a considerable amount of acids in all extracts when

(relatively) fresh prone to be inuenced by age, light and tem-

perature. Apart from the analytical importance, rather pharma-

cokinetic than pharmacodynamic differences might be expected

for the oral use of extracts, although differences between acids andneutral cannabinoids have been reported from in-vitro tests [46].

This stability indicating parameter may be simplied to the

dominant cannabinoid pair e.g. CBGA/CBG.

TPC: Phenolic co-constituents including orientin/ vitexin

reached at highest 16.8 mg/g in crude extracts with CAN tot /TPC

ratios of 4.3 (i.e. maximum 23% of cannabinoids), while in polar

fractions maximum 290 mg/g were obtained mainly via removal of

cannabinoids. Although possible to manufacture extracts with a

signicant portion of phenolics, TPC and CANtot /TPC ratio speci-

cation may not be necessary for more common lipophilic

extracts. A check for phenolics in ngerprints may be useful to

indicate admixtures in Cannabis osherbal drugs.

CFL: CFL values reached at maximum 2.6% of the CANtotvalues

in Et40 and EtOAc crude extracts, 13.9% in cannabinoid-reduced

Me70 extracts, and 12% in fractions with two exceptions (I Me70-

etoac; II Me70-diclo) in which, however, other phenolics were at

least 10 fold more concentrated. Enrichment without parallel

concentration of cannabinoids (or other phenolics) may only be

achieved with more sophisticated fractionation and chromato-

graphic techniques. Despite some interesting biological activities

when applied alone8), cannavins may play no direct role in the

activity of conventional extracts and serve as indicator for identity

without need for specication.

4. Conclusions

We newly developed one 1H NMR and two HPLC methods that

combined are useful to distinguish THC, CBD and CBG dominant

cannabis chemotypes and indicate phenolic co-constituents. A set

of potentially relevant markers was dened, their range detected

in a variety of extracts and discussed vis--vis plant classication

(e.g. with consequences for cultivation eligibility) and relevance

for pharmaceutical specication. If the focus is on the putative

psychotropic strength of drug-type material like in conventional

chemotype distinction, for non-heated samples (CANA detection)

THC may be replaced by THCtot and THC/CBD ratios byTHCtot/CBx(A) with CBx(A) summarizing main non-CB1/CB2-active

neutral and acidic cannabinoids. For pharmaceutical extract spe-

cication, CANtotand THCtottogether with other lead cannabinoids

that may vary according to purpose and preparation should be

declared alongside a dened herbal substance, solvent and ana-

lytical method. The obtained data set as starting point for specic

drug specication requires conrmation of applicability with an

extended sample set and may potentially be extended to other and

less common phytocannabinoids.

Abbreviations

For abbreviations of cannabis constituents and summarizedgroup markers seeTables 1and 2.

Acknowledgments

We thank Jose Maria Prieto for the scientic and technical

support as well as Keith Helliwell (Ransom), Michael Heinrich and

Andrew Constanti for the possibility to do this work and to use the

facilities at the School of Pharmacy London, the organizational

framework of the European research project COOP-CT-2004-

512696 and partial funding by Ransom (Hitchin, UK). We thank

also for the samples from the cannabis collection in Rovigo (IT) by

Giampaolo Grassi and the provision of isolated cannavins by

Giovanni Appendino (Novarra, IT).

Appendix A. Supplementary material

Supplementary data associated with this article can be found in

the online version at http://dx.doi.org/10.1016/j.talanta.2015.02.

040.

References

[1] M. Summers, Independent (2010) 3435.[2] M.A. ElSohly, D. Slade, Life Sci. 78 (2005) 539548.[3] R. Mechoulam, L. Hanus, Chem. Phys. Lipids 108 (2000) 1 13.

[4] F. Grotenthermen, Curr. Drug Targets CNS Neurol. Disord. 4 (2005) 507530.

Fig. 9. Correlation between CANtot(mg/g) values in cannabis extract fractions and

cell viability reduction in HeLa cells (MTT assay, IC507SEM, n 3).

W. Peschel, M. Politi / Talanta 140 (2015) 150165164

7/26/2019 Cannabis Sativa L. Chemotype Distinction Extract

16/16

[5] G. Esposito, D. De Filippis, M.C. Maiuri, D. De Stefano, R. Carnuccio, T. Iuvone,Neurosci. Lett. 399 (2006) 9195.

[6] R.G. Pertwee, Br. J. Pharmacol. 15 (2008) 199215.[7] F. Borrelli, I. Fasolino, B. Romano, R. Capasso, F. Maiello, D. Coppola, et al.,

Biochem. Pharmacol. 85 (2013) 13061316.[8] M.L. Barrett, D. Gordon, F.J. Evans, Biochem. Pharmacol. 34 (1985) 20192024.[9] G. Vanhoenacker, R.P. Van, K.D. De, P. Sandra, Nat. Prod. Lett. 16 (2002) 57 63.

[10] K.W. Hillig, Biochem. Syst. Ecol. 32 (2004) 875891.[11] J. Gertsch, M. Leonti, S. Raduner, I. Racz, J.Z. Chen, X.Q. Xie, K.H. Altmann,

K.M. Karsak, A. Zimmer, Proc. Natl. Acad. Sci. U.S.A 105 (2008) 90999104.[12] E.M. Williamson, Phytomedicine 8 (2001) 401409.

[13] E. Russo, G.W. Guy, Med. Hypotheses 66 (2006) 234246.[14] E. Small, H.D. Beckstead, Lloydia 36 (1973) 144165.[15] C. Rustichelli, V. Ferioli, M. Baraldi, P. Zanoli, G. Gamberini, Chromatographia

47 (1998) 215222.[16] E.P.M. De Meijer, M. Bagatta, A. Carboni, P. Crucitti, V.M.C. Moliterni, P. Ranalli,

G. Mandolino, Genetics 163 (2003) 335346.[17] M. Fellermeier, W. Eisenreich, A. Bacher, M.H. Zenk, Eur. J. Biochem. 268

(2001) 15961604.[18] F.E. Dussy, C. Hamberg, M. Luginbuhl, T. Schwerzmann, T.A. Briellmann, For-

ensic Sci. Int. 149 (2005) 310.[19] Y.H. Choi, A. Hazekamp, A.M.G. Peltenburg-Looman, M. Frederich, C. Erkelens,

A.W.M. Lefeber, R. Verpoorte, Phytochem. Anal. 15 (2004) 345354.[20] Y.H. Choi, H.K. Kim, A. Hazekamp, C. Erkelens, A.W.M. Lefeber, R. Verpoorte,

J. Nat. Prod. 67 ( 2004) 953957.[21] A. Hazekamp, Y.H. Choi, R. Verpoorte, Chem. Pharm. Bull. 52 (2004) 718 721.[22] M. Politi, W. Peschel, N. Wilson, M. Zloh, J.M. Prieto, M. Heinrich, Phy-

tochemistry 69 (2008) 562570.[23] T. Lehmann, R. Brenneisen, J. Liq. Chromatogr. 18 (1995) 689 700.

[24] A. Hazekamp, A. Peltenburg, R. Verpoorte, C. Giroud, J. Liq. Chromatogr. Relat.Technol. 28 (2005) 23612382.

[25] F. Taura, S. Morimoto, Y. Shoyama, Phytochemistry 39 (1995) 457458.[26] W. Peschel, A. Kump, J.M. Prieto, J. Pharm. Pharmacol. 63 (2011) 1483 1494.[27] G.F. Pauli, B.U. Jaki, D.C. Lankin, J. Nat. Prod. 68 (2005) 133149.[28] M. Defernez, I.J. Colquhoun, Phytochemistry 62 (2003) 1009 1017.

[29] U. Holzgrabe, R. Deubner, C. Schollmayer, B. Waibel, J. Pharm. Biomed. Anal. 38(2005) 806812.

[30] J.T. Fischedick, A. Hazekamp, A. Erkelens, J.H. Choi, R. Verpoorte, Phytochem-istry 71 (2010) 20582073.

[31] A.K. Kovar, H. Lindner, Archiv der Pharmazie 324 (1991) 329 333.[32] V. Ferioli, C. Rustichelli, G. Pavesi, G. Gamberini, Chromatographia 52 (2000)

3944.[33] O. Zoller, P. Rhyn, B. Zimmerli, J. Chromatogr. A 872 (2000) 101110.[34] B. De Backer, B. Debrus, P. Lebrun, L. Theunis, N. Dubois, L. Decock, et al., Anal.

Technol. Biomed. Life Sci. 877 (2009) 4115 4124.[35] G. Fournier, C. Richez-Dumanois, J. Duvezin, J.P. Mathieu, M. Paris, Planta med.

53 (1987) 277280.[36] J.A. Beutler, A.H. DerMarderosian, Econ. Bot. 32 (1978) 387 394.[37] V.G. Virovets, J. Internat., J. Int. Hemp Assoc. 3 (1996) 13 15.[38] E. Small, D. Marcus, Econ. Bot. 57 (2003) 545558.[39] C. Slijkhuis, R. Hoving, L. Blok-Tip, D. de Kaste, [Kwaliteitsnormen Medicinale

Cannabis] Quality Standards for Medicinal Cannabis, 2004.[40] J.H. Holley, K.W. Hadley, C.E. Turner, J. Pharm. Sci. 64 (1975) 892 894.[41] P.B. Baker, T.A. Gough, S.I. Johncock, B.J. Taylor, L.T. Wyles, Bull. Narc. 34 (1982)

101108.[42] J. McPartland, Online Proceedings of the 3rd International Symposium, Bior-

esource Hemp, Wolfsburg (Germany), 1316 September 2000, Available at:www.Nova-institut.de.

[43] European Medicines Agency, Guideline on Declaration of Herbal Substancesand Herbal Preparations in Herbal Medicinal Products/Traditional HerbalMedicinal Products (EMA/HMPC/CHMP/CVMP/287539/2005 Rev.1), 11 March2010, Available at: http://www.ema.europa.eu.

[44] M.A. ElSohly, H. deWit, S.R. Wachtel, S. Feng, T.P. Murphy, J. Anal. Toxicol. 25(2001) 565571.

[45] A.J. Hill, S.E. Weston, N. Jones, I. Smith, S. Bevan, E.M. Williamson,G.J. Stephens, C.M. Williams, B.J. Whalley, Epilepsia 51 (2010) 15221532.[46] K.C.M. Verhoeckx, H.A.A.J. Korthout, A.P. van Meeteren-Kreikamp, K.A. Ehlert,

M. Wang, J. van der Greef, R.J.T. Rodenburgh, R.F. Witkamp, Int. Immunopharmacol.6 (2006) 656665.

W. Peschel, M. Politi / Talanta 140 (2015) 150165 165