1H NMR and HPLC/DAD for Cannabis sativa L. chemotype distinction, extract profiling and specificationby Wieland Peschel, Matteo Politi

Talanta

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Year
2015
DOI
10.1016/j.talanta.2015.02.040
Subject
Chemistry (all)

Text

iv ical

Article history:

Received 20 September 2014

Received in revised form

Available online 5 March 2015

Keywords:

Cannabis sativa L.

Extracts

THC

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 1H NMR key signals for distinction of four chemotypes measured in deuterated dimethylsulfoxide effects. Moreover co-constituents

CFL-A), common because of the e of the natural ents [12,13] prial plant classifip) are based on

CBG. Within those plants and derived materials the total and

Contents lists available at ScienceDirect journal homepage: www.els

Talan

Talanta 140 (2015) 150–165http://dx.doi.org/10.1016/j.talanta.2015.02.040relative 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 0039-9140/& 2015 Elsevier B.V. All rights reserved. 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.cannabidiol (CBD), cannabigerol (CBG), and cannabinol (CBN)-type being the most abundant [2]. The psychotropic THC, with the the THC and CBD content [14–16] while the nowadays available spectrum includes varieties with other lead compounds such aspopularity [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), increasingly investigated showing partly distinct activities are reported for minor non-cannabinoid such as the prenylated flavone cannflavin A [8] ( flavonoids [9] or terpenes [10,11]. Despite or complexity some authors advocate the advantag mixtures with combinations of cannabis constitu marily determined by the chemovar. Convention cations as drug-, intermediate or fiber type (hemAlongside the development of synthetic cannabinergics, the authorized and the off-label medicinal use of cannabis regain other non-psychotropic, non-CB binding cannabinoids, mainly cannabidiol (CBD) [5,6] but also cannabigerol (CBG) [7], areCBD

CBG

HPLC 1H NMR

Analytical markers 1. Introductiontogether 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. highest affinity to cannabinoid receptors (CB1, CB2), has been manifold tested pharmacologically and clinically [3,4]. Meanwhile12 February 2015

Accepted 23 February 20151H NMR and HPLC/DAD for Cannabis sat extract profiling and specification

Wieland Peschel n,1, Matteo Politi

Centre for Pharmacognosy and Phytotherapy, Department for Pharmaceutical and Biolog 29-39 Brunswick Square, London WC1N 1AX, United Kingdom a r t i c l e i n f o a b s t r a c ta L. chemotype distinction,

Chemistry, The School of Pharmacy, University College London, evier.com/locate/talanta ta acids, and the cannabis-characteristic prenylated flavones CFL-A and cannflavin B (CFL-B). Group and ratio markers were derived

W. Peschel, M. Politi / Talanta 140 (2015) 150–165 151that 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. 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