Talanta 140 (2015) 150–165

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

1
H NMR and HPLC/DAD for Cannabis sativa L. chemotype distinction,
extract profiling and specification
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: The medicinal use of different chemovars and extracts of Cannabis sativa L. requires standardization
Received 20 September 2014 beyond Δ9-tetrahydrocannabinol (THC) with complementing methods. We investigated the suitability of
Received in revised form 1
H NMR key signals for distinction of four chemotypes measured in deuterated dimethylsulfoxide
12 February 2015
together with two new validated HPLC/DAD methods used for identification and extract profiling based
Accepted 23 February 2015
on the main pattern of cannabinoids and other phenolics alongside the assayed content of THC, can-
Available online 5 March 2015
nabidiol (CBD), cannabigerol (CBG) their acidic counterparts (THCA, CBDA, CBGA), cannabinol (CBN) and
Keywords: cannflavin A and B. Effects on cell viability (MTT assay, HeLa) were tested. The dominant cannabinoid
Cannabis sativa L. pairs allowed chemotype recognition via assignment of selective proton signals and via HPLC even in
Extracts
cannabinoid-low extracts from the THC, CBD and CBG type. Substantial concentrations of cannabinoid
THC
acids in non-heated extracts suggest their consideration for total values in chemotype distinction and
CBD
CBG specifications of herbal drugs and extracts. Cannflavin A/B are extracted and detected together with
HPLC cannabinoids but always subordinated, while other phenolics can be accumulated via fractionation and
1
H NMR detected in a wide fingerprint but may equally serve as qualitative marker only. Cell viability reduction in
Analytical markers HeLa was more determined by the total cannabinoid content than by the specific cannabinoid profile.
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 classification and phar-
maceutical specification.
& 2015 Elsevier B.V. All rights reserved.

1. Introduction highest affinity to cannabinoid receptors (CB1, CB2), has been

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 flos) and the preparation (extract).
Approximately 70 phytocannabinoids—besides 419 other com-
pounds—are described for Cannabis sativa L.; classified chemically
into 10 major groups, the Δ9-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.
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 flavone cannflavin A [8] (CFL-A), common
flavonoids [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 classifi-
cations as drug-, intermediate or fiber type (hemp) are based on
the THC and CBD content [14–16] 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 fluids and con-
fiscated material. Originally the plant synthesizes and accumulates

http://dx.doi.org/10.1016/j.talanta.2015.02.040
0039-9140/& 2015 Elsevier B.V. All rights reserved.
 W. Peschel, M. Politi / Talanta 140 (2015) 150–165
carboxylated forms (e.g. Δ9-trans-tetrahydrocannabinolic acid-THCA)
which are converted into post-harvest ‘neutral’ derivatives accelerated
by light and heat (e.g. THC) [17]. Others—such as CBN—are 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 [19–22].
Characterization beyond the THC content became more rele-
vant with the increasing acceptance of medicinal use [23,24].
Specifications 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 specific 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 profiling 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 profile
extracts as regards main cannabinoid pattern aside more polar
constituents such as flavonoids based on the quantification of
main cannabinoids (THC, CBD, CBG and CBN), the corresponding
acids, and the cannabis-characteristic prenylated flavones CFL-A
and cannflavin B (CFL-B). Group and ratio markers were derived
that are potentially useful in cannabis specifications 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 flavonoids 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-
flower ratio), genotype (THC-type, CBD-type, CBG-type, fiber
(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 specification certificate (18% THC
after heating, 0.8% CBD, o 1% CBN). The pure Cannabis flos drug
contained female flower 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 specification (GC analysis: II: 0.7% THC, 13.7% CBD, 1.0%
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%) flowers,
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 drug–solvent 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 flask on an auto-
mated shaker). After filtration, 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 83–94% 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,
first 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 unified frac-
tions were reduced and dried as described above. All extracts were
stored at
20 °C in the dark.
1
2.4. H 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 Scientific,
UK) was added. Then 150 μL of this stock solution was diluted
with 1350 μL DMSO-d6 and filtered through a 0.45 mm Acrodiscs
syringe filter (Fisher Scientific, Loughborough, UK) and stored at
4 °C. For analysis the 10 mg/mL solutions were thawed and 0.6 mL
filled 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.
1
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 (δ). After baseline correction, the
calibration was carried out on the residual DMSO-d6 solvent peak
in terms of the chemical shift (δ ¼ 2.5 ppm) and intensity (set as
normalized basis for integration). The fingerprint 0 - 11 ppm and
enlarged and expanded sections e.g. between 4 and 9 ppm were
152 W. Peschel, M. Politi / Talanta 140 (2015) 150–165

selected as suitable areas for extract identification. Signal alloca- 2.6.3. Identification and qualification
tion (DMSO-d6) was performed by comparison with reference THC, THCA, CBD, CBG, CBN, CFL-A/B (Fig. 1), flavonoids and phe-
standards, extracts with known HPLC profile and previously pub- nolcarbonic acids were identified using reference standards (retention
lished assignments in deuterochloroform or deuteromethanol times in Table 1). The characteristic DAD-UV spectra (210–400 nm) of
(deuteroacetone for cannflavins) [19–21,25]. the standards and literature reports [24] allowed a classification of
THC: (1H NMR, 400 MHz in DMSO-d6,, all in ppm; in bold dis- compounds into neutral cannabinoids (THC/CBD/CBG pattern and
tinguishable diverse signals of THCA in extracts) δ: 0.85 (H-5”, 3H, CBN/CBC pattern), cannabinoid acids (THCA/CBDA/CBGA pattern or
t), 0.98 (H-9, 3H, s; THCA δ1.03), 1.25 (H-3”, H-4”, m), 1.30 (H-5, m,
THCA δ1.38), 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 7
CH3
δ2.74 þ2.85), 3.08 (H-1, 1H, dm; THCA δ3.15), 6.01 (H-3’, 1H, m), 3
6.15 (H-5’, 1H, m), 6.37 (H-2, 1H, m; THCA δ6.31), 9.20 (2’-OH 1H, s) 4 2 (10)
CBD: (1H NMR, 400 MHz in DMSO-d6,, all in ppm; in bold OH
1 2’ (1)
distinguishable diverse signals of CBDA in extracts) δ: 0.86 (H- 5
6 1’ R
5”3H, t), 1.25 (H-3”, H-4”4H, m), 1.48 (H-2”, 2H, q), 1.60-61 (H-7, 3’ (2)
H3C
H-9, 3H, s), 1.67 (H-5, m; CBDA: δ1.72), 1.93 (H-4, 1H, m), 2.09 (H-4, 10 4’ 2’’ 4’’
8 6’
1H, m), 2.30 (H-1”, 2H, t, CBDA δ2.75), 3.04 (H-6, 1H, t), 3.82 (H-1, H3C O 5’(4) 1’’ 3’’
CH3
9 5’’
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’ δ6.13), 8.62 (2’-OH) THC: R = H
CBG: (1H NMR, 400 MHz in DMSO-d6, all in ppm; in bold dis- THCA: R = COOH
tinguishable diverse signals of CBGA in extracts) δ: 0.85 (H-5”, 3H,
7
t), 1.25 (H-3” and H-4”, 4H, m), 1.48 (H-2”, 2H, m), 1.52 (H-10, 3H, CH3
s), 1.59 (H-9, 3H, s), 1.69 (H-7, 3H, s), 1.89 (H-5, 2H, m, CBGA δ1.99), 3

1.99 (H-4, 2H, m, CBGA: δ2.17), 2.32 (H-1”, 2H, t; CBGA: δ2.78), 4 2
OH
5.04 (H-6, 1H, m), 5.15 (H-2, 1H, m), 6.08 (H-3’ þH-5’, 2H, s, CBGA 5 1 2’
1’ R
H-5’: δ6.22), 8.86 (2’-OH, 2H, s) 6
3’
CFL-A: (1H NMR, 400 MHz in DMSO-d6, all in ppm) δ: 1.51 (H- H2C 2’’
4’ 4’’
8”, 3H, s), 1.58 (H-10”, 3H, s), 1.73 (H-9”, 3H, s), 1.92 (H-4”, 2H, t), 10
H3C HO 6’ CH3
2.0 (H-5” (2H, t), 3.23 (H-1” (2H, m), 3.89 (O-Me, 3H, s), 5.03 (H-6”, 9 5’ 1’’ 3’’ 5’’
t), 5.19 (H-2”, 1H, t), 6.55 (H-10, 1H, s), 6.89 (H-3, 1H, s), 6.94 (H-5’, CBD: R = H
1H, d), 7.55 (H-2’ and H-6’, 2H, m), 13.21 (5-OH, 1H, s) CBDA: R = COOH

2.6. HPLC 7
CH3 OH
1
2.6.1. Fingerprint 1’
2’
R
3
A HPLC Waters™ system 900, with a Waters™ 996 PDA detector 4
2 3’
and a Waters™ 717plus auto sampler device and EmPower soft- 4’ 2’’ 4’’
5
ware equipped with an Aces 5 Phenyl (25 cm x 4.6 mm) column 6
HO 6’ CH3
5’
(ACT, Aberdeen, UK) and a Nova-Paks C8 Guard Column 1’’ 3’’ 5’’

3.9  20 mm, 2/pkg (Waters UK, Elstree, UK) was used. Conditions CBG: R = H
H3C CH3 CBGA: R = COOH
were as follows: column temperature 25 °C, auto sampler 8 °C, 10 9
flow 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
CH3
60%, 38 min 40%, 40 min 5%, 55 min 0%, 74 min 70%. For identifi- O
cation a triple fingerprint was recorded near absorbance maxima 3’
OH
of flavonoids (254 nm) cannabinoids (275 nm), and flavonoids/ 2’ 4’
8
1’ 5’
phenolcarbonic acids/cannabinoid acids (324). Detection at HO 9 O
7 2
214 nm provided the best levelled information for all compounds 6’
6 3
and was used for quantification. Samples (starting concentration 1’’
4
10
extracts 10 mg/mL, standards 1 mg/mL) were diluted in methanol, 5
OH
methanol/water mixtures and, prior to use, filtered through a H3C
2’’
4’’
0.45 mm Acrodiscs syringe filter (Fisher Scientific, Loughborough, 3’’

UK) before injection (10 μL or 30 μL). 6’’ 5’’

H3C
2.6.2. Cannabinoid profile 9’’ 7’’
The same equipment and methodology as described for the 8’’
CH3
fingerprint were used for cannabinoid analysis with modifications
10’ ’
in column (Agilent Zorbax RX-C18 column, 5 mm 4.6  250 mm
Fig. 1. (A) THC and THCA, CBD and CBDA, CBG and CBGA. The numbering follows
Highchrom, UK), solvents (only solvent B and C), running time and
adjusted p-cymene based monoterpene nomenclature of CBD after Choi et al. [19]
gradient (55 min, solvent B: 0 min 70%, 30 min 35%, 43 min 5%, for direct comparison in this study. For THC the other common nomenclature
48 min 70%). (dibenzopyran system) is indicated at key positions (in brackets). (B) cannflavin A.
 W. Peschel, M. Politi / Talanta 140 (2015) 150–165 153

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

Reference standard tR FPa (min) tR CPb (min) UV λ max (nm) Classification for inclusion in group markers

chlorogenic acid 9.27 – 240–325 TPC

homorientin 21.78 – 256–348 TPC
orientin 22.12 – 256–348 TPC
isovitexin 25.62 – 268–337 TPC
vitexin 26.05 – 268–337 TPC
quercetin 31.42 – 255–372 TPC
apigenin 37.65 – 267–338 TPC
olivetol 44.19 5.98 274 –
cannflavin B (CFL-B) 54.54 11.49 273–242 CFL
cannflavin A (CFL-A) 59.18 20.17 274–342 CFL
cannabidiolic acid (CBDA) 59.54 20.36 269–307 CANA, CBD(A), CANtot
cannabigerolic acid (CBGA) 59.87 20.93 267–309 CANA, CBG(A), CANtot
cannabigerol (CBG) 60.19 21.57 272 CAN, CBG(A), CANtot
cannabidiol (CBD) 60.39 22.21 273 CAN, CBD(A), CANtot
cannabinol (CBN) 61.52 28.78 283 CAN, THC(A), THCtot, CANtot
Δ9-tetrahydrocannabinol (THC) 62.37 32.95 276 CAN, THC(A), THCtot, CANtot
tetrahydrocannabinolic acid A (THCA) 62.67 41.26 270–304 CANA, THC(A), THCtot, CANtot

a
FP: fingerprint (80 min).
b
CP: cannabinoid profile (55 min).

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

Calculation Calculated as Description/Relevance

THCtot Σ 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) Σ THC, THCA THC, THCA (CP/FP) Sum of THC and THCA
CBD(A) Σ CBD, CBDA CBD (CP) Sum of CBD and CBDA, dominant in some chemotypes (intermediate, hemp),
main constituents without CB1/CB2 receptor affinity, non-psychotropic,
specific pharmacological effects
CBG(A) Σ CBG, CBGA CBG (CP) Sum of CBG and CBGA, dominant in some chemotypes, main constituents
without CB1/CB2 receptor affinity, non-psychotropic, specific pharmacologi-
cal effects
CAN Σ neutral cannabinoids THC, CBD, CBG, CBN (CP/FP) neutral (decarboxylated) cannabinoids - dominant in heated, aged and
excessively dried drugs
CANA Σ acidic cannabinoids THC, CBD, CBG, CBN (CP/FP) acidic (carboxylated) cannabinoids -dominant in fresh plants and
preparations without heating
CANtot Σ neutral and acidic cannabi- THC, CBD, CBG, CBN (CP/FP) total cannabinoid content
noids, CAN þ CANA
oCAN CANtot – (Σ THCtot þCBD(A) þ THC (in THC-type) CBD (in CBD- other cannabinoids than those usually found as main cannabinoids in
CBG(A)) type) CBG (in CBG-type) (CP) common chemotypes, (i.e. THCtot, CBD(A), CBG(A))
CFL CFL-A þCFL-B CFL-A, CFL-B(CP) cannflavins-cannabis specific prenylated flavones
TPC Σ flavonoids and phenolcarbonic vitexin, chlorogenic acid (FP) total phenolic content (without CFL)
acids
THCtot/ (CBD(A) þ Σ THC, THCA, CBN / Σ CBD(A), as for THC(A), CBG(A), CBD(A) ‘chemotype marker’, ratio main constituents in common chemotypes, ratio
CBG(A)) CBG(A) (CP/FP) 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 Σ CANA/ Σ 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
flower portions, potential influence of phenolics on activity

cannabinolic acid (CBNA)/cannabichromenic acid (CBCA) pattern),
flavonoids and phenolcarbonic acids (Fig. A1). CBDA and CBGA were
identified as dominant peaks in extracts from CBD-type and CBG-type
drugs and their conversion to their neutral forms. Unidentified peaks
were qualified by the PDA spectrum (Fig. A1) as flavonoid, phe-
nolcarbonic 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 ana-
lytical run. For extract profiling all main peaks (above 0.05% of the
total peak area) were qualified according to their spectrum usually
resulting in 15–35 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 quantification. Identified and
qualified peaks were summarized to group markers ‘total THC’
(THCtot ¼THC þTHCA þCBN), CBG(A) ( ¼CBG þCBGA) and CBD(A)
(¼CBD þCBDA), 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) þCBD(A)), ‘decarboxilation
marker’ (CANA/CAN) and a ‘polarity marker’ (CANtot/TPC) were
calculated (Table 2).
154 W. Peschel, M. Politi / Talanta 140 (2015) 150–165

CFL-B

CBN
CFL-A
orientin
chlorogenic acid

THC
CBG
olivetol
10.00 20.00 30.00 40.00 50.00 60.00 70.00
Minutes

THC

THCA
CBGA
^^
CFL-B

CFL-A
vitexin

10.00 20.00 30.00 40.00 50.00 60.00 70.00

CBDA
CBD
*

THCA
**

THC
*
*
*

*
*

10.00
10.0 20.00 30.00 40.00 50.00 60.00 70.00
chlorogenic acid RS

+ CFL-A RS
**
* + vitexin RS
* + orientin RS

+ CFL-B RS

CBDA
CBD
^^

^^
^^
^
**

^
**
*

10.00 20.00 30.00 40.00 50.00 60.00 70.00

Minutes

Fig. 2. HPLC fingerprint. (A) selected standard references (λ: 254 nm), (B) hydroethanolic crude extract from THC-type, and (C) CBD-type drugs, (D) extract fraction from
fiber-type drug spiked with reference substances (RS) (all λ: 214 nm). Non-identified qualified peaks marked as: n flavonoid, nn phenolcarbonic acid, ^ cannabinoid,
^^ cannabinoid acid.

2.7. HPLC fingerprint – method validation 2.7.2. Linearity and range

In addition to common standard curves for single reference
The fingerprint covers cannabinoids and more polar con- standards (not shown), we tested linearity and range for the more
stituents (phenolcarbonic acids, flavonoid glycosides) separating complex group markers. Comparable results were obtained for
THC, THCA and CBN from other cannabinoids and closed flavo- different extract types in the range between 1 mg/mL and
noids previously described from each other such as vitexin/iso- 25 mg/mL extract (example Fig. 3A). The relative standard devia-
vitexin and orientin/homorientin. tion of the mean (RSD) was maximum 3–8% for group markers and
6–15% for ratio markers.

2.7.1. Selectivity and specificity 2.7.3. Accuracy

THC/THCA, CBD/CBDA or CBG/CBGA were always the promi- The accuracy for group markers was checked via the recovery
nent peaks within the cannabinoids (50 and 65 min) range largely rate of added reference standards (theoretical amount versus
separated from common phenolic co-constituents (8–40 min) found amount). We tested combinations of pure standards, com-
(Table 1, Fig. 2). Spiking of extracts confirmed peak allocation binations of one extract with different standards, and combina-
(example Fig. 2D). Despite overlapping of minor peaks qualifica- tions of three benchmark extracts (‘low flavonoid/cannabinoid’,
tion via UV spectra allowed the summation of AUC for calculation ‘high-cannabinoid’ and ‘high-phenolic’) with the same standard
of CANA, CAN, THCtot, CANtot and TPC. mixture (Table A1). The recovery rate for THCtot, CBD(A) þCBG(A),
 W. Peschel, M. Politi / Talanta 140 (2015) 150–165 155

3500 6000 400

THC(A) CBx(A) CAN THCA THC CBN CBDA CBD CBGA CBG

CANA CANtot TPC

3000
5000
y = 217.17x + 76.659
y = 112.92x + 57.828 300
2500 R = 0.9994
R = 0.9988 y = 13.969x + 1.2058
4000 R = 0.9998
y = 8.3066x + 1.2675
R = 0.9999
mg

mg

mg
2000
3000 y = 191.45x - 38.998 200
y = 36.99x + 23.56
R = 0.9969
R = 0.9998
1500
y = 7.2496x + 6.4821
y = 51.924x + 17.725 R = 0.9949
R = 0.9984 y = 31.663x - 5.9281 2000
1000 R = 0.9963 y = 1.3577x - 1.2049
100 R = 0.9693
y = 21.111x + 17.156
R = 0.983 1000 y = 0.8425x + 0.7867
500 R = 0.9921
y = 2.0963x + 0.9869
R = 0.9962
0 0 0
0 10 20 30 0 10 20 30 0 10 20 30

sample conc. (mg/mL) sample conc. (mg/mL) sample conc. (mg/mL)

Fig. 3. Examples for HPLC linearity tests. (A) Group markers calculated from the fingerprint of a CBD-type extract fraction, (B and C) cannabinoids calculated from can-
nabinoid profile of a THC-type extract. Samples were injected at 5 different concentrations between 1 and 25 mg/mL.

Fig. 4. HPLC cannabinoid profile (λ: 214 nm). (A) selected references standards; and extracts from (B) THC-type, (C) CBD-type, (D) CBG-type, (E) fiber-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 fingerprint sum parameters.
2.8. HPLC cannabinoid profile – method validation
For cannabinoid and cannflavin assay, a 55 min HPLC analysis
was applied (Fig. 4).
2.8.1. Selectivity and specificity
THC, CBD, CBG (as well as THCA, CBDA, CBGA) share compar-
able UV spectra with minor differences in the maximum absorp-
tion (265–285 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 confirm 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) 150–165

Fig. 5. 1H NMR spectra of cannabis constituents (DMSO-d6). THC and mixtures of THC þCBD (2:3), THCþ CBD (6:1) and CBDþ 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 alongside detection of minor peaks was found with 10 mg/mL
(A) concentrations. 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 five concentrations. The results were consistent at con-
centrations 5–25 mg/mL with a relative confidence 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 quantification 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
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 93.6–104.8%
(standard mixtures) and 70.8–129.5% (in extracts) for cannabi-
noids and 91.6–118.6% (standard mixtures) and 77.3–93.2% (in
extracts) for cannflavins, 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%
 W. Peschel, M. Politi / Talanta 140 (2015) 150–165
suggest that the method is not suitable when cannflavins 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 ¼6) we
tested the intra-assay precision when analyzing extracts over
several hours. To detect whether mixtures decompose or instru-
ment parameters shift, five 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 humidified atmosphere
and split when confluent. Cells were allowed to grow in media to
60–80% confluence before harvesting. For the MTT assay cell sus-
pension was adjusted to 7.5  104 cells/mL and 96-well plates
(200 μL per well) were incubated for 18–24 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 μg/mL (Et40 extracts) or 50 μg/mL
(EtOAc extracts). Absorbance values were measured with an Anthos
Lucy 1 luminometer at 570 nm (reference filter 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 (MNTC¼85% 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 ¼3) 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
1
3.1. H NMR identification of cannabis extracts in DMSO-d6
After qualitative peak assignments of major cannabinoids and
cannflavins 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-d6 fingerprint’ 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 DMSO-
d6 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’ (0–4 ppm), is
often hampered by overlaps of close signals, the ‘aromatic area’ in
particular the H-5′ position and the two olefinic 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 identification in
extracts. Fig. 5 illustrates the distinction of THC and CBD in two
combinations (0–11 ppm) as well as appearance of flavonoid
proton signals when CBD is combined with CFL-A. Fig. 6 shows
extracts from the four chemovars (4–9 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 identification 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 identification, indicates the level
of decarboxilation but also illustrates possible differences in the
NMR fingerprint 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 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 identification
Spectra were manually checked for specific signals (qualitative
marker), for overlapping and selectivity (identification 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 of first 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) 150–165

Fig. 6. 1H NMR spectra of extracts from four chemotypes (DMSO-d6, 10 mg/mL, 4–9 ppm, amplified 4  ). Extracts (Et40-EtOAc) from THC-type (A), CBD-type (B), Fiber-type
(C) and CBG-type (D) drugs with key proton signals for identification of main cannabinoids.

but can partially overlap with close signals from other cannabi-
noids, i.e. useful for identification but less so for quantification:
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-specific 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 iden-
tification. 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
quantification 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-specific 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 þ2.85) 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) 150–165 159

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 (δ in ppm) in DMSO-d6 for identification of characteristic compounds/groups in THC(A), CBD(A) or CBG(A) dominant cannabis chemotypes.

Specific signals for identificationa 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)) 4.41–4.49

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″) 2.28–2.34

(shared signals THC, CBG, CBD not shared with THCA, CBGA, CBDA)
CANA 2.75-2.78 (H-1″) 2.75–2.78
(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
Specific signals of the respective constituent not shared with other main cannabinoids in THC-type, CBD, type, fiber type, and CBG-type derived cannabis extracts.
b
Consideration of intensity and overlaps with other main compounds in THC-type, CBD, type, fiber type, and CBG-type derived cannabis extracts.

recognizable as minor peaks in THC-type extracts with a CBG
(A) content around 1:10 vis-à-vis THC(A). CBG H-5′ overlaps with
the H-3′ signal. Equally to THC and CBD, the 2′-OH signal is specific
and usually well recognizable at 8.82. The two CBG(A) olefinic
methin protons H-6 and H-2 at 5.04 and 5.15 are characteristic as
well as selective and sufficiently 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) 150–165
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), □ ¼ strong signal, □ ¼ weak signal, □¼ 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 dis-
tinguish from CBGA (2.78) that may be also identified 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 flavonoid
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 flavonoids but
quite specific for more lipophilic cannabis extracts where non-
prenylated flavonoids 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 specific prenyl peak is the singlet at 1.73
(H-9″). The methoxy peak at 3.89 may be shared by other flavo-
noids but is distinguishable from main cannabinoids. Overall,
despite the possibility for identification, cannflavin 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 flavo-
noids. Exemplarily we had tested quercetin addition to cannabis
extracts (up to 20%), where typical signals such as at δ 1.90, 3.80,
4.04 where not intense enough while minor peaks can be identi-
fied in the less crowded area 6.5–8.0 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 2.28–2.34, but the influence 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 identified. Signals most influenced 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 identifi-
cation of multicompound mixtures is challenging due to over-
lapping 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 specific
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 identification as demonstrated in Table 4 for
extracts of different polarity. It allows simplification 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/DAD fingerprint and cannabinoid profile
Cannabis HPLC fingerprinting and quantitative analysis has
developed over years to improve the separation of very similar
cannabinoid structures via specific columns, detection (e.g. flame/
electro ionization) or hyphenation with MS [23,31–33]. 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 fingerprint signals the main cannabinoids and presence
of more polar co-constituents and allows summarizing into groups
of qualified 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) 150–165
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 fiber type, IV CBG-type) (Aþ E) Main compound
groups THCtot, CBx(A) ( ¼ CBG(A)þ CBD(A)), oCAN and TPC, (Bþ F) Total cannabinoid content and ratio markers, (C þG) cannabinoid profile, (D þH) Effect on cell viability in
HeLa (MTT assay, mean 7 SEM, n¼ 3).
CFL-A/B or CBD/CBG versus CBDA/CBGA. The cannabinoid profile
method allows in shorter time quantification 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 20–22 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 Δ8 THC,
CBC/CBCA or tetrahydrocannabivarin (THCV) may be tested in the
future.
3.4. HPLC based key markers for chemotype and herbal drug
identification
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 identification 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 ‘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)þ 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 flagged progressed decarboxylation in all four
drugs; most advanced in II. The CANtot /TPC ratio is influenced by
161
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 profile,
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 (confirming the
advanced age of I) and cannflavins (measurable amounts only in I:
0.79 mg/g). Notably, I contains more CBG(A) than CBD(A) which
confirms 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 confirmed THC-type (I),
CBD-type (II), CBD-type cannabinoid-low (III) and CBG-type (IV,
first 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 first 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 classification drug type (THC 42%, CBD 0%), fiber
type (THC o 0.3%, CBD 4 0.5%, THC/CBD ratioo0.1) and an inter-
mediate type (THC 4 0.5%, CBD 40.5%, THC/CBD ratio 4 0.5)
reflects the importance distinguishing between Cannabis flos for
recreational use (often425% THC) and low-THC industrial hemp
without considering other possible inherited biosynthesis pattern
in plant populations [16,36–38]. CBD forming an essential part of
those classifications 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 I–IV neither provider specification nor our results (EtOAc
extracts from specific batches of dry drugs of certain age) allows
162 W. Peschel, M. Politi / Talanta 140 (2015) 150–165

Table 5
Classification of four cannabis chemotypes and proposed specification of herbal drugs and extracts.

I II III IV

Provider specification of Cannabis flosa:

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 classification of plants (based on exhaustive analysis of Cannabis flos)b:

‘drug type’? ‘intermediate type’? ‘fiber 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 specification of Cannabis flosc:

‘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
d
THCtot /CBx(A) 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 0.04
CANA/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 specification 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 specific batches available in this study.
d
CBx(A)¼ CBD(A) þ CBG(A).
e
Optional such as in case of hydroethanolic extracts from Cannabis folium.

unambiguous allocation (Table 5): the declared CBD-value of I
would not match conventional ‘drug-type’ definition, II does nei-
ther fit into ‘intermediate type’ nor ‘fiber type’ definition, and for
extracts derived from III the CBD-values were too low to comply
with fiber type criteria, which, however, CBG-type IV would
match. Nor does the three-type system consider varieties with
prevailing cannabichromene (CBC), enhanced Δ8-THC instead of
Δ9-THC, or the propyl homologs instead of the usual pentyl can-
nabinoids (e.g. tetrahydrocannabivarin / tetrahydrocannabivarenic
acid dominant clones often of South African origin) [40–42]. 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 fiber type we obtained 4529.6, 0.03,
0.11 and 0.25, for I–IV 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 specification of the plant part (such as flos,
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 defined 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 profiling – range and enrichment of com-
pound groups
The profile 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 specification. Standard solvents to exhaus-
tively extract cannabinoids are traditionally chloroform or
chloroform/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
 W. Peschel, M. Politi / Talanta 140 (2015) 150–165 163

Table 6 Enrichment of cannabinoids: Fractionation allowed further

Group markers and ratio markers for six extract fractions from four cannabis concentration of total cannabinoids and affected slightly group
varieties. Marker maxima as obtained per chemotype are indicated in bold, minima
ratios such as THCtot/ CBG(A). Highest amounts in cannabinoids
underlined.
per extract where obtained in most cases with high lipophilic
EtOAc Ethanol 40% Methanol 70% fractions (dichloromethane) following polar first extraction (Et40).
Hexane Watera CH2Cl2 EtOAc CH2Cl2 EtOAc Lipophilic extraction throughout such as hexane fractions of EtOAc
extracts gave overall higher yields but did not substantially
(I)THC-type
THC(A) (mg/g) 372.5 14.0 245.5 87.4 33.3 1.2
increase CANtot compared to the crude extract. Although two-step
CBD(A) (mg/g) nd nd 5.3 nd nd nd extraction allows modifying the ratio of particular cannabinoid
CBG(A) (mg/g) 20.8 19.5 28.4 11.1 1.5 0.5 groups, purification of main compound pairs by simple solvent
CFL (mg/g) nd 3.7 nd 7.4 2.8 2.7 based fractionation (e.g. separation of CBG(A) from accompanying
TPC (mg/g) nd 30.0 nd 94.9 nd 27.4
THC(A) or CBD(A)) was not possible.
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 Enrichment of phenolics: We obtained fractions with a more
CANtot/TPC 4 846 4.8 4 786 1.4 4172 1.6 levelled content of flavonoids and cannabinoids via removal of
(II) CBD-type major parts of the cannabinoids. Et40-etoac/Me70-etoac and
THC(A) 10.7 nd 14.0 1.1 nd nd EtOAc-wat fractions had CANtot/TPC values between 1.4–4.8, 0.2-
CBD(A) 122.7 14.6 137.9 14.6 12.7 nd
CBG(A) nd nd 9.1 1.1 1.6 nd
1.8, 0.4–1.2, and 0.2-20.9 for varieties I, II, III and IV, respectively.
CFL nd nd nd nd 4.5 nd Cannflavins were often below the limit of quantification, did never
TPC nd 17.8 nd 168.3 125.1 290.4 reach more than 8 mg/g extract and remained usually ‘minor
THCtot/CBxA 0.09 o 0.03 0.09 0.07 o 0.04
constituents’ in relation to cannabinoids or other phenolic
CANA/CAN 0.51 0.78 0.75 0.34 1.46

compounds.
CANtot/TPC 4 164 1.78 4 686 0.25 0.33 0.08
(III) Fiber-type In summary, fractionation of crude cannabis extracts influenced
THC(A) 12.2 nd 14.9 10.2 4.1 nd the CANtot/TPC ratios but less so THCtot/CBx(A) ratios. Cannabi-
CBD(A) 166.3 78.3 183.1 81.7 50.0 nd noids are chemically too similar to enrich separately via solvent
CBG(A) 20.0 nd 16.3 1.4 nd nd polarities without more sophisticated techniques. Thus chemotype
CFL nd nd nd 5.2 nd 5.1
TPC nd 118.9 45.1 86.1 5.6 55.4
identification is possible solvent-independently. On the other hand
THCtot/CBxA 0.07 0.01 0.08 0.12 0.08
it shows that mixing of specific herbal drugs may provide a better
CANA/CAN 2.40 1.78 1.24 2.20 1.79 0.62 path of tailoring preparations to a profile that differs substantially
CANtot/TPC 4 384 1.24 3.32 0.80 8.69 0.45 from the natural cannabinoid ratio in one chemovar.
(IV) CBG-type
THC(A)

13.8 10.1 1.6 nd
CBD(A)

69.1 41.8 21.8 5.2 3.7. Effect on the cell viability in HeLa
CBG(A)

219.0 193.4 163.9 35.1
CFL

nd 4.3 3.8 2.3 IC50 and MNTC levels were between 2.5-8.0 μg/mL and 0.3-
TPC

nd 14.7 6.6 24.3 2.2 μg/mL for EtOAc extracts compared to 6.1–25.2 μg/mL and 2.0–
THCtot/CBxA 0.05 0.04 0.01 o 0.01
CANA/CAN 0.41 1.01 1.57 1.12
12.7 μg/mL for Et40 extracts, respectively (Fig. 8D and H). With an
CANtot/TPC 4 744 20.9 18.9 0.24 assumed co-influence of the quantitative and the qualitative
composition, the cytotoxic effect in HeLa cells was less determined
ndo 0.5 mg/g (LoQ). by the dominant cannabinoids e.g. the THC(A) content but by the
a
Water containing 8% methanol. 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
(Fig. 8F 2nd bar) proved to be not only material but also extract- CBD(A) or CBG(A) content compared to THC(A) affected cell via-
dependent. The CANtot /TPC ratio (Fig. 8F 3rd bar) revealed that bility with an IC50 value that was maximum 2.5 times higher (Et40
Et40 extraction lowers the cannabinoid content more (10-fold extract of III) or even similar (Et40 extract of IV) to the THC(A)-rich
lower for the Et40 vs. EtOAc) than it accumulates phenolic co- Et40 extract of I. This was confirmed by testing additionally the
constituents (doubled). The relative cannabinoid profile (Fig. 8 G) fractions from all crude extracts. Over the complete matrix no
confirmed the genotype without substantial differences between clear correlation could be found between single constituents such
Et40 and EtOAc. In I Et40, however, the portion of other com- as THC, group or the defined ratio markers. The most likely rela-
pounds than the dominant cannabinoids were found relatively tionship between cannabis extract composition and resulting
increased compared to EtOAc. toxicity in the HeLa cells was between CANtot and log IC50 values
Exhaustive methanol/methanol 70% extraction from defatted from the MTT assay (Fig. 9).
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 3.8. Markers for standardization of herbal drugs and extracts
and 85.2 mg/g, respectively) compared to Et40. Despite removal of
the major part of cannabinoids they remained more concentrated For adequate specification of non-heated drugs (e.g. Cannabis
than phenolics in extracts from cannabinoid-rich material flos, C. resinum, C. folium) and drug preparations (including
(CANtot/TPC ratios: 6.2 (in I), 5.1 (in IV)). The cannabinoid profile extraction solvent and drug extract ratio) for pharmaceutical
still allowed the information about the original chemotype. The purposes [43], the preferred specification of absolute values of
enrichment of cannflavins in relation to cannabinoids could hardly CANtot, THCtot, and CBD(A) and other relevant main cannabinoids
be achieved. can be rationalized as follows (Table 5):
Recognition of the chemotype in extract fractions: Despite vari- CANtot: Due to the increased pharmacological knowledge on
able quantity of cannabinoids and their proportions the non-CB receptor mediated and non-THC caused effects, the overall
THCtot/CBxA ratio allowed for all fractions the identification of the amount of cannabinoids represents useful basic information which
dominant cannabinoid pair, as long as cannabinoids could still be should be further specified with regard to the main constituents.
detected. The THCtot/ CBx(A) ratio varied between 0.7 and 22 for Relevant specific markers may be different to classic main phyto-
THC-type, 0.03-0.09 for CBD-type, 0.01-0.05 for CBG-type, and cannabinoids [44,45]. The content of specific cannabinoids needs
0.07-0.12 for the fiber type derived extracts (Table 6). to be put into context, i.e. the relevance of 20 mg/g THC is different
164 W. Peschel, M. Politi / Talanta 140 (2015) 150–165

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), cannflavins may play no direct role in the
activity of conventional extracts and serve as indicator for identity
without need for specification.

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 defined, their range detected
in a variety of extracts and discussed vis-à-vis plant classification
(e.g. with consequences for cultivation eligibility) and relevance
for pharmaceutical specification. 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
Fig. 9. Correlation between CANtot (mg/g) values in cannabis extract fractions and THCtot/CBx(A) with CBx(A) summarizing main non-CB1/CB2-active
cell viability reduction in HeLa cells (MTT assay, IC50 7 SEM, n¼ 3).
neutral and acidic cannabinoids. For pharmaceutical extract spe-
cification, CANtot and THCtot together with other lead cannabinoids
with overall amounts of 25 mg/g CANtot or 300 mg/g CANtot. Our that may vary according to purpose and preparation should be
cell viability test showed that effects did not correlate with THC declared alongside a defined herbal substance, solvent and ana-
but with CANtot when not only THC dominant samples are used. lytical method. The obtained data set as starting point for specific
THC, THCA, CBN versus THCtot: Fresh material contains mainly drug specification requires confirmation of applicability with an
THCA, if not converted via heating completely into THC. It has been extended sample set and may potentially be extended to other and
previously shown that both substances should be analyzed sepa- less common phytocannabinoids.
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
Abbreviations
effects. CBN has a low to moderate CB1 affinity and psychotropic
effect [6]. The separated specification of CBN, THC and THCA may
For abbreviations of cannabis constituents and summarized
be useful in stability tests. Here, CBN was only detectable at
group markers see Tables 1 and 2.
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
Acknowledgments
preparations from fresh material. The explicit determination and
labeling of CBG(A) is suggested because of increasingly more CBG-
We thank Jose Maria Prieto for the scientific and technical
type plants on the market (identification), their importance as
support as well as Keith Helliwell (Ransom), Michael Heinrich and
main co-constituents in THC-type extracts, and own pharmacolo-
Andrew Constanti for the possibility to do this work and to use the
gical effects [7].
facilities at the School of Pharmacy London, the organizational
CANA/CAN: Values between 0.27 and 2.4 in our extracts
framework of the European research project COOP-CT-2004-
demonstrate a considerable amount of acids in all extracts when
512696 and partial funding by Ransom (Hitchin, UK). We thank
(relatively) fresh – prone to be influenced by age, light and tem-
also for the samples from the cannabis collection in Rovigo (IT) by
perature. Apart from the analytical importance, rather pharma-
Giampaolo Grassi and the provision of isolated cannflavins by
cokinetic than pharmacodynamic differences might be expected
Giovanni Appendino (Novarra, IT).
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 simplified to the
Appendix A. Supplementary material
dominant cannabinoid pair e.g. CBGA/CBG.
TPC: Phenolic co-constituents including orientin/ vitexin
Supplementary data associated with this article can be found in
reached at highest 16.8 mg/g in crude extracts with CANtot /TPC
the online version at http://dx.doi.org/10.1016/j.talanta.2015.02.
ratios of 4.3 (i.e. maximum 23% of cannabinoids), while in polar
040.
fractions maximum 290 mg/g were obtained mainly via removal of
cannabinoids. Although possible to manufacture extracts with a
significant portion of phenolics, TPC and CANtot /TPC ratio speci-
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