Talanta 140 (2015) 150–165

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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: 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 Δ9-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 identification and extract profiling 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 cannflavin 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 specifications of herbal drugs and extracts. Cannflavin 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 fingerprint but may equally serve as qualitative marker only. Cell viability reduction in 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 pharmaceutical specification. & 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 flos) and the preparation (extract). Approximately 70 phytocannabinoids—besides 419 other compounds—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: [email protected] (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 affinity 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 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] primarily determined by the chemovar. Conventional plant classifications 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 forensic/legal purposes to determine THC in biological fluids and confiscated material. Originally the plant synthesizes and accumulates

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 relevant 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 several co-constituents the determination of prevailing active constituent 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 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%) flowers, 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 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 automated 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 (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 unified fractions were reduced and dried as described above. All extracts were stored at  20 °C in the dark. 2.4.

1

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 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 (leafflower 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 cultivar 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-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 specification (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 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. 2.5.

1

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

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selected as suitable areas for extract identification. Signal allocation (DMSO-d6) was performed by comparison with reference standards, extracts with known HPLC profile and previously published assignments in deuterochloroform or deuteromethanol (deuteroacetone for cannflavins) [19–21,25]. THC: (1H NMR, 400 MHz in DMSO-d6,, all in ppm; in bold distinguishable diverse signals of THCA in extracts) δ: 0.85 (H-5”, 3H, 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 δ2.74 þ2.85), 3.08 (H-1, 1H, dm; THCA δ3.15), 6.01 (H-3’, 1H, m), 6.15 (H-5’, 1H, m), 6.37 (H-2, 1H, m; THCA δ6.31), 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) δ: 0.86 (H5”3H, t), 1.25 (H-3”, H-4”4H, m), 1.48 (H-2”, 2H, q), 1.60-61 (H-7, H-9, 3H, s), 1.67 (H-5, m; CBDA: δ1.72), 1.93 (H-4, 1H, m), 2.09 (H-4, 1H, m), 2.30 (H-1”, 2H, t, CBDA δ2.75), 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’ δ6.13), 8.62 (2’-OH) CBG: (1H NMR, 400 MHz in DMSO-d6, all in ppm; in bold distinguishable diverse signals of CBGA in extracts) δ: 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 δ1.99), 1.99 (H-4, 2H, m, CBGA: δ2.17), 2.32 (H-1”, 2H, t; CBGA: δ2.78), 5.04 (H-6, 1H, m), 5.15 (H-2, 1H, m), 6.08 (H-3’ þH-5’, 2H, s, CBGA H-5’: δ6.22), 8.86 (2’-OH, 2H, s) CFL-A: (1H NMR, 400 MHz in DMSO-d6, all in ppm) δ: 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. Identification and qualification THC, THCA, CBD, CBG, CBN, CFL-A/B (Fig. 1), flavonoids and phenolcarbonic acids were identified using reference standards (retention times in Table 1). The characteristic DAD-UV spectra (210–400 nm) of the standards and literature reports [24] allowed a classification of compounds into neutral cannabinoids (THC/CBD/CBG pattern and CBN/CBC pattern), cannabinoid acids (THCA/CBDA/CBGA pattern or

7

CH3 3

2 (10)

4 5

6

2.6.2. Cannabinoid profile The same equipment and methodology as described for the fingerprint were used for cannabinoid analysis with modifications 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)

1’

R 3’ (2)

H3C

10

8

H3C

2’’

4’

6’

O

9

CH3

3’’

1’’

5’(4)

4’’ 5’’

THC: R = H THCA: R = COOH 7

CH3 3

4

2

5

OH

1

6

2’

1’

R 3’

H2C

2’’

4’

10

6’

HO

H3C

5’

9

1’’

4’’

CH3

3’’

5’’

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, 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 60%, 38 min 40%, 40 min 5%, 55 min 0%, 74 min 70%. For identification a triple fingerprint was recorded near absorbance maxima of flavonoids (254 nm) cannabinoids (275 nm), and flavonoids/ phenolcarbonic acids/cannabinoid acids (324). Detection at 214 nm provided the best levelled information for all compounds and was used for quantification. Samples (starting concentration extracts 10 mg/mL, standards 1 mg/mL) were diluted in methanol, methanol/water mixtures and, prior to use, filtered through a 0.45 mm Acrodiscs syringe filter (Fisher Scientific, Loughborough, UK) before injection (10 μL or 30 μL).

1

7

CH3

OH

1

3

4

2’

1’

R 3’

2

5

2’’

4’

6

6’

HO

H3C

1’’

CH3

3’’

5’’

CBG: R = H CBGA: R = COOH

CH3

10

5’

4’’

9

CH3

O 3’ 8

HO

9

O

7 6 5

H3C

4’’

4’

1’

5’

2

1’’

10

4

OH

2’

3

6’

OH

2’’ 3’’ 5’’

6’’

H3C

7’’

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) 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 homorientin orientin isovitexin vitexin quercetin apigenin olivetol cannflavin B (CFL-B) cannflavin A (CFL-A) cannabidiolic acid (CBDA) cannabigerolic acid (CBGA) cannabigerol (CBG) cannabidiol (CBD) cannabinol (CBN) Δ9-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

240–325 256–348 256–348 268–337 268–337 255–372 267–338 274 273–242 274–342 269–307 267–309 272 273 283 276 270–304

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: fingerprint (80 min). 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)

THC(A) CBD(A)

Σ THC, THCA Σ CBD, CBDA

THC, THCA (CP/FP) CBD (CP)

CBG(A)

Σ CBG, CBGA

CBG (CP)

CAN

Σ neutral cannabinoids

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

CANA

Σ 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)) cannflavins-cannabis specific prenylated flavones total phenolic content (without CFL)

THCtot/ (CBD(A) þ CBG(A))

Σ neutral and acidic cannabinoids, CAN þ CANA CANtot – (Σ THCtot þCBD(A) þ CBG(A)) CFL-A þCFL-B Σ flavonoids and phenolcarbonic acids Σ THC, THCA, CBN / Σ 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 affinity, non-psychotropic, specific pharmacological effects Sum of CBG and CBGA, dominant in some chemotypes, main constituents without CB1/CB2 receptor affinity, non-psychotropic, specific 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

Σ CANA/ Σ 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 flower portions, potential influence of phenolics on activity

oCAN CFL TPC

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, 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 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 ‘chemotype marker’ (THC(A)/(CBG(A) þCBD(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) 150–165

CFL-B

154

70.00

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 The fingerprint covers cannabinoids and more polar constituents (phenolcarbonic acids, flavonoid glycosides) separating THC, THCA and CBN from other cannabinoids and closed flavonoids previously described from each other such as vitexin/isovitexin and orientin/homorientin. 2.7.1. Selectivity and specificity 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 (8–40 min) (Table 1, Fig. 2). Spiking of extracts confirmed peak allocation (example Fig. 2D). Despite overlapping of minor peaks qualification 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 3–8% for group markers and 6–15% 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 flavonoid/cannabinoid’, ‘high-cannabinoid’ and ‘high-phenolic’) with the same standard mixture (Table A1). The recovery rate for THCtot, CBD(A) þCBG(A),

W. Peschel, M. Politi / Talanta 140 (2015) 150–165

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 0

10

20

sample conc. (mg/mL)

30

0

10

20

30

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 cannabinoid 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 comparable UV spectra with minor differences in the maximum absorption (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

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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 (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 five concentrations. The results were consistent at concentrations 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 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 93.6–104.8% (standard mixtures) and 70.8–129.5% (in extracts) for cannabinoids 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 substances. CFL-A determination was partially hampered when combined with a CBDA-rich extract and recovery rates of 60.5–88.2%

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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 instrument 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 suspension 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 3.1.

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 deuterated 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-

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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’ (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 (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 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 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 identification Spectra were manually checked for specific signals (qualitative marker), for overlapping and selectivity (identification 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 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

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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 cannabinoids, 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 (H1, 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 identification. 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 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

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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 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)

2.28–2.34

CANA CFL-A a b

4.41–4.49

2.75–2.78 6.55, 6.89, 7.55

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. 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

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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 distinguish 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 nonprenylated 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 flavonoids 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 concentration and signal intensity in relation to predominant cannabinoids. The same applies to lower concentrated common flavonoids. 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 identified 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 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 identified. Signals most influenced 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 identification 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 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 mixtures. 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]. 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 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.

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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 relevant cannabinoids of less common chemotypes such as Δ8 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 profile, 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 (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].

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

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 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 ‘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)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

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 Beckstead 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 intermediate 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

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Table 5 Classification of four cannabis chemotypes and proposed specification of herbal drugs and extracts. I Provider specification of Cannabis flosa: 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

‘fiber 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 classification of plants (based on exhaustive analysis of Cannabis flos)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 specification of Cannabis flosc: ‘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 specification 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 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. b c

unambiguous allocation (Table 5): the declared CBD-value of I would not match conventional ‘drug-type’ definition, II does neither 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 cannabinoids (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 decarboxylation 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 portion of psychotropic THCtot. In addition, such data are only comparable 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 compound groups The profile 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 specification. 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) 150–165

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 profile (Fig. 8 G) confirmed 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 profile still allowed the information about the original chemotype. The enrichment of cannflavins 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 identification 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 fiber type derived extracts (Table 6).

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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 first 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, purification 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 flavonoids and cannabinoids via removal of major parts of the cannabinoids. Et40-etoac/Me70-etoac and EtOAc-wat fractions had CANtot/TPC values between 1.4–4.8, 0.21.8, 0.4–1.2, and 0.2-20.9 for varieties I, II, III and IV, respectively. Cannflavins were often below the limit of quantification, 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 influenced 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 identification is possible solvent-independently. On the other hand it shows that mixing of specific herbal drugs may provide a better path of tailoring preparations to a profile 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 μg/mL and 0.32.2 μg/mL for EtOAc extracts compared to 6.1–25.2 μg/mL and 2.0– 12.7 μg/mL for Et40 extracts, respectively (Fig. 8D and H). With an assumed co-influence 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 confirmed 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 defined 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 specification of non-heated drugs (e.g. Cannabis flos, C. resinum, C. folium) and drug preparations (including extraction solvent and drug extract ratio) for pharmaceutical purposes [43], the preferred specification 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 specified with regard to the main constituents. Relevant specific markers may be different to classic main phytocannabinoids [44,45]. The content of specific cannabinoids needs to be put into context, i.e. the relevance of 20 mg/g THC is different

164

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

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, n¼ 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 affinity and psychotropic effect [6]. The separated specification 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 (identification), 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 influenced 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 simplified 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 significant portion of phenolics, TPC and CANtot /TPC ratio specification may not be necessary for more common lipophilic extracts. A check for phenolics in fingerprints may be useful to indicate admixtures in Cannabis flos 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 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 THCtot/CBx(A) with CBx(A) summarizing main non-CB1/CB2-active neutral and acidic cannabinoids. For pharmaceutical extract specification, CANtot and THCtot together with other lead cannabinoids that may vary according to purpose and preparation should be declared alongside a defined herbal substance, solvent and analytical method. The obtained data set as starting point for specific drug specification requires confirmation 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 scientific 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 cannflavins 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) 34–35. M.A. ElSohly, D. Slade, Life Sci. 78 (2005) 539–548. R. Mechoulam, L. Hanus, Chem. Phys. Lipids 108 (2000) 1–13. F. Grotenthermen, Curr. Drug Targets CNS Neurol. Disord. 4 (2005) 507–530.

W. Peschel, M. Politi / Talanta 140 (2015) 150–165

[5] G. Esposito, D. De Filippis, M.C. Maiuri, D. De Stefano, R. Carnuccio, T. Iuvone, Neurosci. Lett. 399 (2006) 91–95. [6] R.G. Pertwee, Br. J. Pharmacol. 15 (2008) 199–215. [7] F. Borrelli, I. Fasolino, B. Romano, R. Capasso, F. Maiello, D. Coppola, et al., Biochem. Pharmacol. 85 (2013) 1306–1316. [8] M.L. Barrett, D. Gordon, F.J. Evans, Biochem. Pharmacol. 34 (1985) 2019–2024. [9] G. Vanhoenacker, R.P. Van, K.D. De, P. Sandra, Nat. Prod. Lett. 16 (2002) 57–63. [10] K.W. Hillig, Biochem. Syst. Ecol. 32 (2004) 875–891. [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) 9099–9104. [12] E.M. Williamson, Phytomedicine 8 (2001) 401–409. [13] E. Russo, G.W. Guy, Med. Hypotheses 66 (2006) 234–246. [14] E. Small, H.D. Beckstead, Lloydia 36 (1973) 144–165. [15] C. Rustichelli, V. Ferioli, M. Baraldi, P. Zanoli, G. Gamberini, Chromatographia 47 (1998) 215–222. [16] E.P.M. De Meijer, M. Bagatta, A. Carboni, P. Crucitti, V.M.C. Moliterni, P. Ranalli, G. Mandolino, Genetics 163 (2003) 335–346. [17] M. Fellermeier, W. Eisenreich, A. Bacher, M.H. Zenk, Eur. J. Biochem. 268 (2001) 1596–1604. [18] F.E. Dussy, C. Hamberg, M. Luginbuhl, T. Schwerzmann, T.A. Briellmann, Forensic Sci. Int. 149 (2005) 3–10. [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) 345–354. [20] Y.H. Choi, H.K. Kim, A. Hazekamp, C. Erkelens, A.W.M. Lefeber, R. Verpoorte, J. Nat. Prod. 67 (2004) 953–957. [21] A. Hazekamp, Y.H. Choi, R. Verpoorte, Chem. Pharm. Bull. 52 (2004) 718–721. [22] M. Politi, W. Peschel, N. Wilson, M. Zloh, J.M. Prieto, M. Heinrich, Phytochemistry 69 (2008) 562–570. [23] T. Lehmann, R. Brenneisen, J. Liq. Chromatogr. 18 (1995) 689–700. [24] A. Hazekamp, A. Peltenburg, R. Verpoorte, C. Giroud, J. Liq. Chromatogr. Relat. Technol. 28 (2005) 2361–2382. [25] F. Taura, S. Morimoto, Y. Shoyama, Phytochemistry 39 (1995) 457–458. [26] W. Peschel, A. Kump, J.M. Prieto, J. Pharm. Pharmacol. 63 (2011) 1483–1494. [27] G.F. Pauli, B.U. Jaki, D.C. Lankin, J. Nat. Prod. 68 (2005) 133–149. [28] M. Defernez, I.J. Colquhoun, Phytochemistry 62 (2003) 1009–1017.

165

[29] U. Holzgrabe, R. Deubner, C. Schollmayer, B. Waibel, J. Pharm. Biomed. Anal. 38 (2005) 806–812. [30] J.T. Fischedick, A. Hazekamp, A. Erkelens, J.H. Choi, R. Verpoorte, Phytochemistry 71 (2010) 2058–2073. [31] A.K. Kovar, H. Lindner, Archiv der Pharmazie 324 (1991) 329–333. [32] V. Ferioli, C. Rustichelli, G. Pavesi, G. Gamberini, Chromatographia 52 (2000) 39–44. [33] O. Zoller, P. Rhyn, B. Zimmerli, J. Chromatogr. A 872 (2000) 101–110. [34] B. De Backer, B. Debrus, P. Lebrun, L. Theunis, N. Dubois, L. Decock, et al., Anal. Technol. Biomed. Life Sci. 877 (2009) 4115–4124. [35] G. Fournier, C. Richez-Dumanois, J. Duvezin, J.P. Mathieu, M. Paris, Planta med. 53 (1987) 277–280. [36] J.A. Beutler, A.H. DerMarderosian, Econ. Bot. 32 (1978) 387–394. [37] V.G. Virovets, J. Internat., J. Int. Hemp Assoc. 3 (1996) 13–15. [38] E. Small, D. Marcus, Econ. Bot. 57 (2003) 545–558. [39] C. Slijkhuis, R. Hoving, L. Blok-Tip, D. de Kaste, [Kwaliteitsnormen Medicinale Cannabis] Quality Standards for Medicinal Cannabis, 2004. [40] J.H. Holley, K.W. Hadley, C.E. Turner, J. Pharm. Sci. 64 (1975) 892–894. [41] P.B. Baker, T.A. Gough, S.I. Johncock, B.J. Taylor, L.T. Wyles, Bull. Narc. 34 (1982) 101–108. [42] J. McPartland, Online Proceedings of the 3rd International Symposium, Bioresource Hemp, Wolfsburg (Germany), 13–16 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) 565–571. [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) 1522–1532. [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) 656–665.

DAD for Cannabis sativa L. chemotype distinction, extract profiling and specification.

The medicinal use of different chemovars and extracts of Cannabis sativa L. requires standardization beyond ∆9-tetrahydrocannabinol (THC) with complem...
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