Talanta 140 (2015) 150165

Contents lists available at ScienceDirect

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

H 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

art ic l e i nf o

a b s t r a c t

Article history:
Received 20 September 2014
Received in revised form
12 February 2015
Accepted 23 February 2015
Available online 5 March 2015

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 of
1
H 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, cannabidiol (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 24 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 classication and pharmaceutical specication.
& 2015 Elsevier B.V. All rights reserved.

Keywords:
Cannabis sativa L.
Extracts
THC
CBD
CBG
HPLC
1
H NMR
Analytical markers

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 herbal
drug (Cannabis os) and the preparation (extract).
Approximately 70 phytocannabinoidsbesides 419 other compoundsare 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
n

Corresponding author.
E-mail address: wieland.peschel@ema.europa.eu (W. Peschel).
1
Present address: European Medicines Agency, 30 Churchill Place, Canary
Wharf, London E14 5EU, United Kingdom.
http://dx.doi.org/10.1016/j.talanta.2015.02.040
0039-9140/& 2015 Elsevier B.V. All rights reserved.

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] primarily determined by the chemovar. Conventional plant classications 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 forensic/legal purposes to determine THC in biological uids and conscated material. Originally the plant synthesizes and accumulates

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

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

which are converted into post-harvest neutral derivatives accelerated
by light and heat (e.g. THC) [17]. Otherssuch as CBNare only
degradation products of those derivatives. The common focus on
neutral cannabinoids 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. Their
separate 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 relevant 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 several co-constituents the determination of prevailing active constituent 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 extraction. As a simple activity test in relation to these markers we
checked exemplarily their effect to reduce cell viability in HeLa
cells.

151

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 diameter 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 automated 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 (Et40diclo, 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 fractions were reduced and dried as described above. All extracts were
stored at
20 C in the dark.
2.4.

H NMR procedures

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 Appendino, (Novarra, Italy). As phenolic standards we used the cannabinoid 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 (leafower 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 cultivar from controlled cultivation for medicinal use was provided by
TNO (Zeist, Netherlands) with specication certicate (18% THC
after heating, 0.8% CBD, o 1% 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-cannabinoid (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%

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 diluted
with 1350 L DMSO-d6 and 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 delay
of 2 s. The numbers of scans were 128 or 64. The spectra were
recorded at 300 K.
2.5.

H 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-d6 solvent 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

152

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

selected as suitable areas for extract identication. Signal allocation (DMSO-d6) was performed by comparison with reference
standards, extracts with known HPLC prole and previously published assignments in deuterochloroform or deuteromethanol
(deuteroacetone for cannavins) [1921,25].
THC: (1H NMR, 400 MHz in DMSO-d6,, all in ppm; in bold distinguishable diverse signals of THCA in extracts) : 0.85 (H-5, 3H,
t), 0.98 (H-9, 3H, s; THCA 1.03), 1.25 (H-3, H-4, m), 1.30 (H-5, m,
THCA 1.38), 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
2.74 2.85), 3.08 (H-1, 1H, dm; THCA 3.15), 6.01 (H-3, 1H, m),
6.15 (H-5, 1H, m), 6.37 (H-2, 1H, m; THCA 6.31), 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 (H53H, 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, CBDA 2.75), 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 distinguishable 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, CBGA 1.99),
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 (H8, 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-2 and H-6, 2H, m), 13.21 (5-OH, 1H, s)

2.6.3. Identication and qualication

THC, THCA, CBD, CBG, CBN, CFL-A/B (Fig. 1), avonoids and phenolcarbonic acids were identied using reference standards (retention
times in Table 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

CH3
3

2 (10)

4
5

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%).

OH
2 (1)

R
3 (2)

H3C

10

H3C

CH3

5(4)

4
5

THC: R = H
THCA: R = COOH
7

CH3
3

OH

R
3

H2C

10

HO

H3C

CH3

CBD: R = H
CBDA: R = COOH

2.6. HPLC
2.6.1. Fingerprint
A HPLC Waters system 900, with a Waters 996 PDA detector
and a Waters 717plus auto sampler device and EmPower software 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 identication a triple ngerprint was recorded near absorbance maxima
of avonoids (254 nm) cannabinoids (275 nm), and avonoids/
phenolcarbonic acids/cannabinoid acids (324). Detection at
214 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 syringe lter (Fisher Scientic, Loughborough,
UK) before injection (10 L or 30 L).

CH3

OH

R
3

HO

H3C

CH3

CBG: R = H
CBGA: R = COOH

CH3

10

CH3

O
3
8

HO

7
6
5

H3C

10

OH

OH

2
3
5

H3C

9
8

CH3
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) 150165

153

Table 1
Retention times for standard references in two applied HPLC methods and their classication.
Reference standard

tR FPa (min)

tR CPb (min)

UV max (nm)

Classication for inclusion in group markers

chlorogenic acid
homorientin
orientin
isovitexin
vitexin
quercetin
apigenin
olivetol
cannavin B (CFL-B)
cannavin A (CFL-A)
cannabidiolic acid (CBDA)
cannabigerolic acid (CBGA)
cannabigerol (CBG)
cannabidiol (CBD)
cannabinol (CBN)
9-tetrahydrocannabinol (THC)
tetrahydrocannabinolic acid A (THCA)

9.27
21.78
22.12
25.62
26.05
31.42
37.65
44.19
54.54
59.18
59.54
59.87
60.19
60.39
61.52
62.37
62.67

5.98
11.49
20.17
20.36
20.93
21.57
22.21
28.78
32.95
41.26

240325
256348
256348
268337
268337
255372
267338
274
273242
274342
269307
267309
272
273
283
276
270304

TPC
TPC
TPC
TPC
TPC
TPC
TPC

CFL
CFL
CANA, CBD(A), CANtot
CANA, CBG(A), CANtot
CAN, CBG(A), CANtot
CAN, CBD(A), CANtot
CAN, THC(A), THCtot, CANtot
CAN, THC(A), THCtot, CANtot
CANA, THC(A), THCtot, CANtot

a
b

FP: ngerprint (80 min).

CP: cannabinoid prole (55 min).

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

Calculated as

Description/Relevance

THCtot

THC, THCA, CBN

THC, THCA, CBN (CP/FP)

THC(A)
CBD(A)

THC, THCA
CBD, CBDA

THC, THCA (CP/FP)

CBD (CP)

CBG(A)

CBG, CBGA

CBG (CP)

CAN

neutral cannabinoids

THC, CBD, CBG, CBN (CP/FP)

CANA

acidic cannabinoids

THC, CBD, CBG, CBN (CP/FP)

CANtot

THC, CBD, CBG, CBN (CP/FP)

THC (in THC-type) CBD (in CBDtype) CBG (in CBG-type) (CP)
CFL-A, CFL-B(CP)
vitexin, chlorogenic acid (FP)

other cannabinoids than those usually found as main cannabinoids in

common chemotypes, (i.e. THCtot, CBD(A), CBG(A))
cannavins-cannabis specic prenylated avones
total phenolic content (without CFL)

THCtot/ (CBD(A)
CBG(A))

neutral and acidic cannabinoids, CAN CANA

CANtot ( THCtot CBD(A)
CBG(A))
CFL-A CFL-B
avonoids and phenolcarbonic
acids
THC, THCA, CBN / CBD(A),
CBG(A)

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
Sum of THC and THCA
Sum of CBD and CBDA, dominant in some chemotypes (intermediate, hemp),
main constituents without CB1/CB2 receptor afnity, non-psychotropic,
specic pharmacological effects
Sum of CBG and CBGA, dominant in some chemotypes, main constituents
without CB1/CB2 receptor afnity, non-psychotropic, specic pharmacological effects
neutral (decarboxylated) cannabinoids - dominant in heated, aged and
excessively dried drugs
acidic (carboxylated) cannabinoids -dominant in fresh plants and
preparations without heating
total cannabinoid content

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

(CP/FP)

CANA / CAN

CANA/ CAN

as for CANA, CAN (CP/FP)

CANtot / TPC

CANtot / TPC

as for CANtot, TPC (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
decarboxilation marker, ratio acidic /neutral cannabinoids, indicator for
drug and extract quality and age
polarity marker, indicator for extract/solvent polarity, indicator for leaf and
ower portions, potential inuence of phenolics on activity

oCAN
CFL
TPC

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, phenolcarbonic acid, cannabinoid, and cannabinoid acid (Tables 1 and 2).
2.6.4. Assay and marker calculation
Standard curves reference substances as external standards
were established and standard mixtures injected with each analytical 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
(THCtot THC THCA CBN), CBG(A) ( CBG CBGA) and CBD(A)
(CBD CBDA), neutral cannabinoids (CAN), cannabinoid acids
(CANA), total cannabinoids (CANtot), and total phenolics (TPC).
From these group markers three ratio markers described as chemotype marker (THC(A)/(CBG(A) CBD(A)), decarboxilation
marker (CANA/CAN) and a polarity marker (CANtot/TPC) were
calculated (Table 2).

30.00

40.00
Minutes

50.00

THC

60.00

70.00

20.00

CFL-A
CBGA

CFL-B

30.00

40.00

50.00

60.00

40.00

50.00

60.00

70.00

10.00

THC
THCA

30.00

40.00
Minutes

50.00

70.00

60.00

+ CFL-A RS

**

**

* + orientin RS
*
* + vitexin RS
20.00

^^
^^
+ CFL-B RS
^
^^

30.00

**

20.00

chlorogenic acid RS

10.00
10.0

CBDA
CBD

*
**

*
*
*

CBDA
CBD

10.00

vitexin

^^

THCA

THC

20.00

CFL-A
CBG

olivetol

orientin

chlorogenic acid
10.00

CBN

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

CFL-B

154

70.00

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.

2.7. HPLC ngerprint method validation

The ngerprint covers cannabinoids and more polar constituents (phenolcarbonic acids, avonoid glycosides) separating
THC, THCA and CBN from other cannabinoids and closed avonoids previously described from each other such as vitexin/isovitexin and orientin/homorientin.
2.7.1. Selectivity and specicity
THC/THCA, CBD/CBDA or CBG/CBGA were always the prominent 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 qualication via UV spectra allowed the summation of AUC for calculation
of CANA, CAN, THCtot, CANtot and 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 more
complex 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 deviation 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, combinations of one extract with different standards, and combinations 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),

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

3500

THC(A)

CBx(A)

CAN

CANA

CANtot

TPC

6000
THCA

3000

400

THC

CBN

CBDA

CBD

CBGA

CBG

5000
y = 217.17x + 76.659
R = 0.9994

y = 112.92x + 57.828
R = 0.9988

y = 36.99x + 23.56
R = 0.9969

1500

y = 51.924x + 17.725
y = 31.663x - 5.9281
R = 0.9984
R = 0.9963
y = 21.111x + 17.156
R = 0.983

500

y = 2.0963x + 0.9869
R = 0.9962

0
0

10

y = 13.969x + 1.2058
R = 0.9998

20

30

sample conc. (mg/mL)

y = 191.45x - 38.998
R = 0.9998

3000

y = 8.3066x + 1.2675
R = 0.9999

mg

2000

1000

300

4000

mg

2500

mg

155

200
y = 7.2496x + 6.4821
R = 0.9949

2000

y = 1.3577x - 1.2049
R = 0.9693

100
1000

y = 0.8425x + 0.7867
R = 0.9921

0
0

10

20

sample conc. (mg/mL)

30

10

20

30

sample conc. (mg/mL)

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 cannabinoid 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.

CANtot and 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 comparable UV spectra with minor differences in the maximum absorption (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 (example Fig. 4F).
Both the THC and THCA peak covered in some extracts a minor

156

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

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).

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

(A) concentrations.

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

sample 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 concentrations 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 approximately 0.5 mg in 1 g extract corresponding to about 0.25% of the
amount of the lead cannabinoid in cannabis preparations dominated by one compound pair. Satisfactory peak separation

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 cannabinoids 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 substances. CFL-A determination was partially hampered when combined with a CBDA-rich extract and recovery rates of 60.588.2%

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

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 instrument parameters shift, ve times the same extract was injected at
the 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) was
o5% RSD for compounds 4 20 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% CO2 humidied atmosphere
and split when conuent. Cells were allowed to grow in media to
6080% conuence before harvesting. For the MTT assay cell suspension 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 were
expressed as maximum non-toxic concentration (MNTC85% of
control) and as IC50 value.
2.10. Statistical analysis
After HPLC validation (see above) samples were injected in
duplicate and results expressed as mean 7SD. 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 (IC50
and MNTC) were tested using Pearson's correlation test.

3. Results and discussion

3.1.

H 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 deuterated solvents for extraction was previously reported by our
group [22]. Here, in contrast to commonly used chloroform,
methanol or water, the DMSO-d6 ngerprint allows for extracts of
distinctive polarities a single sample preparation in only one non-

157

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 DMSOd6 were 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 and 5). While the discrimination of THC, CBD, CBG and
their carboxylic counterparts in the aliphatic area (04 ppm), is
often hampered by overlaps of close signals, the aromatic area in
particular the H-5 position 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 se selectivity of NMR by suppressing
less intense signals allows in principle an easy identication of the
chemotype. The difference between benchmark extracts (cannabinoid-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
NMR ngerprint 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 the
highest concentrated compound may still allow to address comfortably the subordinated compound. Below that, it depends case
by case on the actual concentrations and numbers of main coconstituents (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 (detectable also at lower concentrations). We summarize in the following
signals for THC(A), CBD(A), CBG(A) differentiation and demarcation 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 demarcations, the H-2 signal (THC 6.37, THCA 6.31 ppm) represents the
marker of rst 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

158

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

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.

but can partially overlap with close signals from other cannabinoids, 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 (H1, CBD 3.05). THCA-specic split H-1 signals 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 identication. 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 signals 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 H3 as in THC but also by H-5, complicating its use for eventual
quantication of neutral cannabinoids. H-5 of 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 to
hydrogen 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-5 signals at 6.08 ppm (CBG) and 6.22 (CBGA), that were also

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

159

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) o 1 mg/g, CBD(A) o 1 mg/g, CBG(A) o 1 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 3
Key proton signals ( in ppm) in DMSO-d6 for 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
THCA
THC(A)

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)
1.03 (H-9, s), 1.38 (H-5, m), 2.74/2.85 (H-1, m), 6.31 (H-2, dm)
1.85 (H-5, m), 6.15 (H-5, m)
(shared signals THC and THCA not shared with CBD(A) or CBG(A))

6.37, 9.21
6.31, 2.74/2.85
6.15, 0.98/1.03

CBD
CBDA
CBD(A)

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)
2.18/2.27 (H-4, m), 2.75 (H-1, m), 3.91 (H-1, d)
4.41-4.42/4.47-9 (H-10) (shared signals CBD and CBDA not shared with THC(A) or CBG(A))

5.08, 8.62

CBG
CBGA
CBG(A)

1.89 (H-5, m), 2.32 (H-1, t), 6.08 (H-5, s), 8.82 (2-OH, s)
2.78 (H-1, t), 6.22 (H-5, s)
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))

6.08, 8.82
6.22
5.04/ 5.15

CAN

2.28-2.34 (H-1)
(shared signals THC, CBG, CBD not shared with THCA, CBGA, CBDA)
2.75-2.78 (H-1)
(shared signals THCA, CBGA, CBDA not shared with THC, CBG, CBD)
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)

2.282.34

CANA
CFL-A
a
b

4.414.49

2.752.78
6.55, 6.89, 7.55

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.
Consideration of intensity and overlaps with other main compounds in THC-type, CBD, type, ber type, and CBG-type derived cannabis extracts.

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-3 signal. 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

160

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

Table 4
Recognizable 1H 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.

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

at 2.32 is only slightly different to CBD (2.29) but useful to distinguish 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 avonoid
structure 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 nonprenylated 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 avonoids 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 concentration and signal intensity in relation to predominant cannabinoids. The same applies to lower concentrated common avonoids. 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 identied 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 carboxyl 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 hydroxyland 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 identication of multicompound mixtures is challenging due to overlapping peaks of similar structures [27] it is usually combined with
multivariate 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 4 for
extracts of different polarity. It allows simplication avoiding the
need to analyze the complete proton signal set of complex mixtures. An extension beyond THC, CBD and CBG type distinction
may be tested in with other chemovarieties.
3.3. HPLC/DAD ngerprint 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]. Comparable to a more recent validated method, we show that conventional 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.

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

161

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, mean 7 SEM, n 3).

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

the cannabinoid content (e.g. low in III) but also other drug characteristics, 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 independent of total values. It illustrates in a different way the prevailing 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].

3.4. HPLC based key markers for chemotype and herbal drug
identication

3.5. Chemotype distinction

Main pattern for chemotype comparison and herbal drug

characteristics of non-heated material are presented as (1) absolute 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-type the
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 cannabinoid-rich versus cannabinoid-low plant (CANtot) alongside
indicators whether THC(A) prevail over non-psychotropic cannabinoids, how fresh the material is (degree of decarboxilation) and
to which extent cannabinoids prevail over accompanying phenolics. 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

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 Beckstead had rst differentiated between a chemotype with
THC4 0.3%/CBD o0.5%, an intermediate type with dominant CBD
but THC also present, and a particularly THC-low type [14]. The
still common classication drug type (THC 42%, CBD 0%), ber
type (THC o 0.3%, CBD 4 0.5%, THC/CBD ratioo0.1) and an intermediate type (THC 4 0.5%, CBD 40.5%, THC/CBD ratio 4 0.5)
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

162

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

Table 5
Classication of four cannabis chemotypes and proposed specication of herbal drugs and extracts.
I
Provider specication of Cannabis osa:
THC (%)
18 (after heating)
CBD (%)
0.8
CBG (%)
n.s.
THC/ CBD ratio
22.5

II

III

IV

0.7
13.7
1.0
0.05

0.01
0.68
0.02
0.015

0.3
5.8
25.2
0.01

ber type
o0.3
40.5
n.s.
o0.1

intermediate type?
40.5
40.5
n.s.
40.5

CBD-type
10.64
0.33
9.72
0.40
0.03
(EtOAc/HPLC)

CBD-type, low cannabinoid

1.31
0.06
0.61
0.02
0.04
(EtOAc/HPLC)

CBG-type
19.34
0.72
2.92
16.66
0.04
(EtOAc/HPLC)

273.5
0.03
0.23
6.6
(EtOAc/HPLC)

120.6
0.04
0.63
4.3
(EtOAc/HPLC)

344.2
0.04
0.64
29.0
(EtOAc/HPLC)

2.6:1
EtOAc
273.5
8.5
249.7
10.3
[16.8]

9.2:1
EtOAc
120.6
5.1
56.3
1.8
[13.4]

1.7:1
EtOAc
344.2
11.9
48.2
274.9
[12.0]

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

drug type?
intermediate type?
THC (%)
42
40.5
CBD (%)
0
40.5
CBG (%)
n.s.
n.s.
THC/ CBD ratio
n.s.
40.5
Proposed specication of Cannabis osc:
THC-type
CANtot (%)
18.94
14.68
THCtot (%)
CBD(A) (%)
o0.04
CBG(A) (%)
1.97
d
THCtot /CBx(A)
7.5
Solvent/Anal. Meth:
(EtOAc/HPLC)
or
CANtot (mg/g)
448.8
THC(A)/CBx(A)d
7.5
CANA/CAN
0.47
CANtot/TPC
192.1
Solvent/Anal. Meth
(EtOAc/HPLC)
Proposed specication of cannabis extracts:
Drug/extract ratio
2.4:1
Solvent
EtOAc
CANtot (mg/g)
448.8
THCtot (mg/g)
347.8
CBD(A) (mg/g)
o1
CBG(A) (mg/g)
46.6
[TPC (mg/g)]e
[2.3]
a

As provided by the supplier at time of delivery I (HPLC), II-IV (GC).

Ref. [14].
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.
b
c

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

would not match conventional drug-type denition, II does neither 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 of
9-THC, or the propyl homologs instead of the usual pentyl cannabinoids (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 decarboxylation 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 IIV, 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 portion of psychotropic THCtot. In addition, such data are only comparable 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 compound groups
The prole of different extracts was investigated for the possibility to identify the original herbal drug and the extent compound groups are extracted, which determines their relevance as
potential markers for specication. Standard solvents to exhaustively extract cannabinoids are traditionally chloroform or
chloroform/methanol mixtures. We used EtOAc to macerate the
main part of the cannabinoids without enrichment of more lipophilic 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 twofold THCtot value compared to CBx(A). The CANA/CAN ratio

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

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.

(I)THC-type
THC(A) (mg/g)
CBD(A) (mg/g)
CBG(A) (mg/g)
CFL (mg/g)
TPC (mg/g)
THCtot/CBxA
CANA/CAN
CANtot/TPC
(II) CBD-type
THC(A)
CBD(A)
CBG(A)
CFL
TPC
THCtot/CBxA
CANA/CAN
CANtot/TPC
(III) Fiber-type
THC(A)
CBD(A)
CBG(A)
CFL
TPC
THCtot/CBxA
CANA/CAN
CANtot/TPC
(IV) CBG-type
THC(A)
CBD(A)
CBG(A)
CFL
TPC
THCtot/CBxA
CANA/CAN
CANtot/TPC

EtOAc
Hexane

Watera

Ethanol 40%
CH2Cl2
EtOAc

Methanol 70%
CH2Cl2
EtOAc

372.5
nd
20.8
nd
nd
17.9
0.83
4 846

14.0
nd
19.5
3.7
30.0
0.72
0.46
4.8

245.5
5.3
28.4
nd
nd
7.2
0.89
4 786

87.4
nd
11.1
7.4
94.9
7.9
0.27
1.4

33.3
nd
1.5
2.8
nd
21.9
0.31
4172

1.2
nd
0.5
2.7
27.4
3.1
0.36
1.6

10.7
122.7
nd
nd
nd
0.09
0.51
4 164

nd
14.6
nd
nd
17.8
o 0.03
0.78
1.78

14.0
137.9
9.1
nd
nd
0.09
0.75
4 686

1.1
14.6
1.1
nd
168.3
0.07
0.34
0.25

nd
12.7
1.6
4.5
125.1
o 0.04
1.46
0.33

nd
nd
nd
nd
290.4




0.08

12.2
166.3
20.0
nd
nd
0.07
2.40
4 384

nd
78.3
nd
nd
118.9
0.01
1.78
1.24

14.9
183.1
16.3
nd
45.1
0.08
1.24
3.32

10.2
81.7
1.4
5.2
86.1
0.12
2.20
0.80

4.1
50.0
nd
nd
5.6
0.08
1.79
8.69

nd
nd
nd
5.1
55.4


0.62
0.45

13.8
69.1
219.0
nd
nd
0.05
0.41
4 744

10.1
41.8
193.4
4.3
14.7
0.04
1.01
20.9

1.6
21.8
163.9
3.8
6.6
0.01
1.57
18.9

nd
5.2
35.1
2.3
24.3
o 0.01
1.12
0.24

ndo 0.5 mg/g (LoQ).

a

Water containing 8% methanol.

(Fig. 8F 2nd bar) proved to be not only material but also extractdependent. 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 coconstituents (doubled). The relative cannabinoid prole (Fig. 8 G)
conrmed the genotype without substantial differences between
Et40 and EtOAc. In I Et40, however, the portion of other compounds 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 20.4
and 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 variable 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).

163

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 CANtot compared to the crude extract. Although two-step
extraction allows modifying the ratio of particular cannabinoid
groups, 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, 0.21.8, 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. Cannabinoids 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 0.32.2 g/mL for EtOAc extracts compared to 6.125.2 g/mL and 2.0
12.7 g/mL for Et40 extracts, respectively (Fig. 8D and H). With an
assumed 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 the
extraction solvent and the resulting CANtot value. 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 viability with an IC50 value 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 relationship between cannabis extract composition and resulting
toxicity in the HeLa cells was between CANtot and 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 phytocannabinoids [44,45]. The content of specic cannabinoids needs
to be put into context, i.e. the relevance of 20 mg/g THC is different

164

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

Me70 extracts, and 12% in fractions with two exceptions (I Me70etoac; 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 chromatographic 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

Fig. 9. Correlation between CANtot (mg/g) values in cannabis extract fractions and
cell viability reduction in HeLa cells (MTT assay, IC50 7 SEM, n 3).

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 CANtot when 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 separately 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 at
advanced 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 CBGtype plants on the market (identication), their importance as
main co-constituents in THC-type extracts, and own pharmacological 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 temperature. Apart from the analytical importance, rather pharmacokinetic than pharmacodynamic differences might be expected
for the oral use of extracts, although differences between acids and
neutral 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 CANtot /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 specication may not be necessary for more common lipophilic
extracts. A check for phenolics in ngerprints may be useful to
indicate admixtures in Cannabis os herbal drugs.
CFL: CFL values reached at maximum 2.6% of the CANtot values
in Et40 and EtOAc crude extracts, 13.9% in cannabinoid-reduced

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 by
THCtot/CBx(A) with CBx(A) summarizing main non-CB1/CB2-active
neutral and acidic cannabinoids. For pharmaceutical extract specication, CANtot and THCtot together with other lead cannabinoids
that may vary according to purpose and preparation should be
declared alongside a dened herbal substance, solvent and analytical 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 summarized
group markers see Tables 1 and 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-2004512696 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]
[2]
[3]
[4]

M. Summers, Independent (2010) 3435.

M.A. ElSohly, D. Slade, Life Sci. 78 (2005) 539548.
R. Mechoulam, L. Hanus, Chem. Phys. Lipids 108 (2000) 113.
F. Grotenthermen, Curr. Drug Targets CNS Neurol. Disord. 4 (2005) 507530.

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

[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) 5763.
[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, Forensic 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) 718721.
[22] M. Politi, W. Peschel, N. Wilson, M. Zloh, J.M. Prieto, M. Heinrich, Phytochemistry 69 (2008) 562570.
[23] T. Lehmann, R. Brenneisen, J. Liq. Chromatogr. 18 (1995) 689700.
[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) 14831494.
[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) 10091017.

165

[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, Phytochemistry 71 (2010) 20582073.
[31] A.K. Kovar, H. Lindner, Archiv der Pharmazie 324 (1991) 329333.
[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) 41154124.
[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) 387394.
[37] V.G. Virovets, J. Internat., J. Int. Hemp Assoc. 3 (1996) 1315.
[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) 892894.
[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, Bioresource Hemp, Wolfsburg (Germany), 1316 September 2000, Available at:
www.Nova-institut.de.
[43] European Medicines Agency, Guideline on Declaration of Herbal Substances
and Herbal Preparations in Herbal Medicinal Products/Traditional Herbal
Medicinal Products (EMA/HMPC/CHMP/CVMP/287539/2005 Rev.1), 11 March
2010, 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.