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Developing Robust Standardised Analytical Procedures for Cannabinoid Quantification: Laying the Foundations for an Emerging Cannabis-Based Pharmaceutical Industry


Abstract and Figures

The plant genus Cannabis is a prolific producer of unique pharmaceutically relevant metabolites, commonly referred to as cannabinoids. Robust and standardised methods for the quantification of cannabinoids within botanical and drug forms is a critical step forward for an emerging Cannabis-based pharmaceutical industry, which is poised for rapid expansion. Despite a growing body of analytical methods for the quantification of cannabinoids, few have been validated using internationally accredited guidelines. Moreover, standardised methods have yet to be developed for application at various stages of manufacture as well as for different levels of processing and refinement. Validation parameters for establishing robust standardised methods for cannabinoid quantification within Cannabis-based drug forms are critically discussed. Determining an appropriate level of specificity (discrimination) among heterogeneous botanical matrices as well as evaluating accuracy (recovery) and inter-laboratory precision (reproducibility) within strict and volatile regulatory environments are potential obstacles to the establishment of robust analytical procedures. We argue that while some of these challenges remain unique to Cannabis, others are common to botanical-based drug development and manufacture. In order to address potential barriers to analytical method standardisation, a collaborative research initiative inclusive of academic and commercial stakeholders is proposed.
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Basic Science – Review Article
Med Cannabis Cannabinoids 2019;2:1–13
Developing Robust Standardised Analytical
Procedures for Cannabinoid Quantification:
Laying the Foundations for an Emerging
Cannabis-Based Pharmaceutical Industry
Matthew T. Welling
a Lei Liu
a Arno Hazekamp
b Ashley Dowell
Graham J. King
a Southern Cross Plant Science, Southern Cross University, Lismore, NSW, Australia; b Hazekamp Herbal Consulting
BV, Leiden, The Netherlands
Received: November 14, 2018
Accepted: January 12, 2019
Published online: February 25, 2019
Graham J. King
Southern Cross Plant Science
Southern Cross University, 1 Military Road
Lismore, NSW 2480 (Australia)
E-Mail graham.king @
© 2019 The Author(s)
Published by S. Karger AG, Basel
DOI: 10.1159/000496868
Cannabis sativa · Medicinal cannabis · Cannabidiol ·
Tetrahydrocannabinol · Quantitative analysis · Mass
The plant genus Cannabis is a prolific producer of unique
pharmaceutically relevant metabolites, commonly referred
to as cannabinoids. Robust and standardised methods for
the quantification of cannabinoids within botanical and
drug forms is a critical step forward for an emerging Canna-
bis-based pharmaceutical industry, which is poised for rapid
expansion. Despite a growing body of analytical methods for
the quantification of cannabinoids, few have been validated
using internationally accredited guidelines. Moreover, stan-
dardised methods have yet to be developed for application
at various stages of manufacture as well as for different levels
of processing and refinement. Validation parameters for es-
tablishing robust standardised methods for cannabinoid
quantification within Cannabis-based drug forms are criti-
cally discussed. Determining an appropriate level of specific-
ity (discrimination) among heterogeneous botanical matri-
ces as well as evaluating accuracy (recovery) and inter-labo-
ratory precision (reproducibility) within strict and volatile
regulatory environments are potential obstacles to the es-
tablishment of robust analytical procedures. We argue that
while some of these challenges remain unique to Cannabis,
others are common to botanical-based drug development
and manufacture. In order to address potential barriers to
analytical method standardisation, a collaborative research
initiative inclusive of academic and commercial stakehold-
ers is proposed. © 2019 The Author(s)
Published by S. Karger AG, Basel
Cannabis is a chemically complex [1], pharmaceuti-
cally relevant [2], domesticated [3], monospecific genus
within the angiosperm family Cannabaceae, order Ro-
sales [4]. Plants of this genus produce a group of isopre-
nylated resorcinyl polyketides, also known as cannabi-
noids [5]. Cannabinoids vary structurally in terms of their
isoprenyl residue as well as their resorcinyl alkyl side
chain and moiety (Fig.1). Nine primary isoprenyl topo-
logical arrangements or “types” of cannabinoids occur in
planta [5]. The resorcinyl alkyl residue can also vary by
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Med Cannabis Cannabinoids 2019;2:1–13
DOI: 10.1159/000496868
carbon number [6]. Cannabinoids are synthesised with a
carboxylated resorcinyl moiety that undergoes spontane-
ous decarboxylation via a non-enzymatic reaction, a pro-
cess that can be accelerated at high temperatures [7].
More than 100 cannabinoids have been identified from
Cannabis [1, 8]. While impressive, this catalogue does not
necessarily reflect the metabolic capacity of this genus but
may be a consequence of oxidative instability of plant-
derived cannabinoids, causing a range of degradation
products to occur after synthesis [5].
The current understanding of the bioactivity of can-
nabinoids is based on their interaction with the human
endocannabinoid system, a complex network comprising
of 2 G protein-coupled receptors, a number of thermo-
sensitive transient receptor potential cation channels, at
least 2 endocannabinoids (anandamide and 2-arachi-
donoyl-glycerol), as well as associated biosynthetic path-
ways [9]. The human endocannabinoid system acts as a
versatile broad-spectrum modulator of numerous biolog-
ical systems and is involved in neurological and immuno-
logical pathologies [10, 11]. Delta-9-tetrahydrocannabi-
nol (THC) and cannabidiol (CBD), as well as their C3
alkyl cannabinoid homologues, are considered the most
well studied and clinically relevant plant-derived canna-
binoids (Fig.1), with various combinatory formulations
already scheduled or completed for phase 2 and 3 human
clinical trials [11].
Despite the therapeutic potential of cannabinoids, the
pharmaceutical application of Cannabis has been unten-
able for several decades. This is primarily due to a lack of
high-quality clinical evidence supporting medical use [2],
a situation exacerbated by legislative constraints associ-
ated with the United Nations 1961 Single Convention on
Narcotic Drugs and the 1971 Convention of Psychotropic
Substances, which have severely restricted research into
Cannabis pharmacology and therapeutic use [3, 12].
These constraints have also inhibited clinicians from ex-
ploring therapeutic value and severely limited patient ac-
cess in most legislatures, with clinical application further
blocked by the removal of Cannabis from pharmacopoe-
ias in the 1970s [13, 14]. Until recently, neither of the two
major central regulatory agencies which evaluate drugs,
European Medicines Agency (EMA) and United States
Food and Drug Administration (FDA), approved nor
permitted marketing authorisations for Cannabis or Can-
nabis-related extracts [15]. Indeed, prior to June 2018,
only two synthetic cannabinoid-based pharmaceuticals
had been approved by the FDA, nabilone (Cesamet®)
[16] and dronabinol (Marinol® and Syndros®) [17].
Changes to domestic policy relating to medicinal Can-
nabis have occurred rapidly in a number of countries
[15]. However, there is considerable confusion with re-
gard to the legality and accessibility of these products
both within and between legislatures [15]. Marketed pre-
scription and over-the-counter medicines approved by
regulatory agencies, such as the EMA and FDA, are stan-
dardised and dosage-formulated products that have dem-
onstrated quality, safety and efficacy for their intended
use. These medicines are distinct from Cannabis-based
herbal formulations, artisanal extracts and magistral
preparations (pharmacist-prepared medicines) which
have not been subject to quality assurances associated
Fig. 1. Chemical structures and topological
arrangements of THC- and CBD-type iso-
prenylated resorcinyl polyketides (canna-
binoids). Blue highlights isoprenyl topo-
logical arrangement. Orange highlights
resorcinyl polyketide residue, side-chain
and moiety. CBD, cannabidiol; THC, del-
Robust Standardised Cannabinoid
Analytical Procedures
Med Cannabis Cannabinoids 2019;2:1–13
DOI: 10.1159/000496868
with regular drug marketing authorisation. Many of these
products also require inhalational routes of administra-
tion (vaporisers/e-cigarette style inhalers) or other means
of administration that are poorly characterised in relation
to health outcomes [18].
A number of countries, including Israel, Canada, Ger-
many, and the Netherlands, along with approximately
half of the USA at the state level, have changed their reg-
ulations to allow patient access to medical Cannabis and
related products [15]. At the decentralised national level,
23 European countries have allowed authorisation of the
oromucosal spray nabiximols (Sativex®) for multiple
sclerosis-associated indications [15]. In Australia, medic-
inal Cannabis products were nationally rescheduled to a
Schedule 8 Controlled Drug in November 2016. In order
for medicinal Cannabis products to be lawfully supplied
in Australia, they require entry into the Australian Regis-
ter of Therapeutic Goods (ARTG), an example being
nabiximols (Sativex®) (ARTG ID: 181978). Cannabis
medicinal products which lack sufficient quality assur-
ances to appear in the ARTG can currently be obtained
through special access schemes (
The World Health Organization (WHO)’s Expert
Committee on Drug Dependence met in June 2018 for a
full review on the medicinal use of Cannabis and other
Cannabis-derived substances in response to a series of
landmark studies which provide support for the efficacy
of CBD in treatment-resistant epilepsy (n = 214) [19],
Dravet (n = 120) [20] and Lennox-Gastaut syndromes
(n = 171) [21]. This coincides with the recent approval of
the CBD-based drug Epidiolex® for the treatment of sei-
zures associated with severe forms of epilepsy (Dravet
and Lennox-Gastaut syndromes) [22], with Epidiolex®
being the first Cannabis-derived drug to be approved by
the FDA or any central regulatory agency responsible for
drug evaluation.
Drug development of Cannabis is poised for rapid ex-
pansion, as can be seen by the growing portfolio of chem-
ical phenotypes for therapeutic end-use [23, 24] as well as
the proliferation of patent applications relating to Canna-
bis-based pharmaceutical preparations, delivery technol-
ogies and medical treatments [25]. Despite the promising
global outlook for Cannabis-based drug development, a
critical step forward for this emerging industry is the es-
tablishment of robust standardised methods for the quan-
tification of cannabinoids within a wide and growing
range of Cannabis-based drug forms [26–29]. Refine-
ment of these methodologies and standardisation of ap-
proach will be necessary to establish a foundation for fu-
ture drug development, application and manufacture and
will be required to ensure the identity, efficacy, purity,
quality and potency of impending pharmaceutical-grade
drugs derived from Cannabis.
Cannabis Drug Forms
For a botanical form of Cannabis to be suitable for
marketing as a prescription drug, it must be approved by
an authoritative regulatory agency (FDA, EMA, Austra-
lian Therapeutic Goods Administration, etc.). A Canna-
bis drug product is a finished dosage drug in a form that
consists of the major active ingredient and potentially
other minor active ingredients which may have modula-
tory function (drug substance) together with excipients
(Fig.2). Cannabis drug products can fall into one of two
definitions, depending on the level of purification of the
active ingredient (Fig.2). The official term botanical drug
product was developed by the FDA to describe drug prod-
ucts containing and manufactured from plant materials
and is similar in definition to the term herbal medicinal
product used by EMA [30]. Highly purified or chemical-
ly modified botanical forms are not recognised under the
definition of botanical drug and are, therefore, consid-
ered as conventional drug products (Fig.2). Cannabis-
derived non-prescription over-the-counter botanical
drugs can also be approved by regulatory authorities,
such as the FDA, with the provision that clinical safety
and efficacy has been sufficiently established.
The first stage of Cannabis-derived drug product man-
ufacture involves the harvesting of mature female ra-
cemes (inflorescences) which are cut at the base [31] and
dried in temperature- and humidity-controlled environ-
ments [32]. After removal of stems, dried folium cum flo-
re (leaf with flower) botanical raw material can be further
sorted into low- (foliar) and high- (floral) cannabinoid-
containing tissues. In some legislatures, the latter is dis-
pensed for use as a crude herbal preparation (flos). In ac-
cordance with Good Agricultural Practices and Good
Manufacturing Practice [31], the botanical raw material
is required to be subject to a number of tests relating to
authentication and specification, as well as microbial,
heavy metal, pesticide and aflatoxin contamination
[31, 32].
Additional analyses to consider include tests for adul-
terants, including tobacco and synthetic cannabinoids, as
well as tests for radioactivity if the material is suspected
of being cultivated in a radioactively contaminated envi-
ronment [33]. Production and quality control of Canna-
bis-based biological medicines derived from recombinant
Med Cannabis Cannabinoids 2019;2:1–13
DOI: 10.1159/000496868
DNA technology are also subject to market authorisation
guidelines (e.g., EMA European Guidelines 3AB1a),
which require product purification to remove protein
and nucleic acid contaminants derived from the host cell.
Given the obligate outcrossing nature of Cannabis [34] as
well as heterozygosity within the gene pool [3, 35], seed-
grown Cannabis botanical raw material can be highly
variable in content and composition of target and non-
target active ingredients [31, 36]. Thus, clonal propaga-
tion using callus- [37] or nodal- (axillary buds) [38] me-
diated regeneration is favoured over seed propagation
[22], with explants typically grown under environmen-
tally controlled conditions in an effort to minimise ge-
netic by environment interactions and to maximise prod-
uct uniformity [31].
Cannabis medicine extracts (CME) are synonymous
with botanical drug substances of Cannabis (Fig.2). CME
are derived from Cannabis botanical raw material and
contain the active pharmaceutical ingredient (cannabi-
noids) at various levels of purification and refinement
(Fig.2). Enrichment of active ingredients for manufac-
ture of Cannabis-based drugs can be achieved using aque-
ous (decoction) [39], organic (e.g., ethanolic) [40], super-
critical fluid [41, 42] and edible plant oil-based extraction
[43] (Fig. 2), with aqueous being the least appropriate
given the low solubility of cannabinoids in water [44, 45].
Purification of the botanical drug substance may involve
repeated extraction, chromatographic separation, distil-
lation, as well as winterisation, whereby non-target lipid-
soluble materials, such as waxes, are removed by filtration
at –20°C [46].
The drug product Sativex® is obtained by combining
partially purified THC and CBD with botanical drug sub-
stances [47]. The final botanical drug product incorpo-
rates more than 30 other cannabinoid, terpenoid, fatty
acid and sterol constituents at various concentrations and
is formulated with ethanol (drug vehicle), propylene gly-
col (co-solvent), as well as peppermint (flavour enhancer)
excipients. This is delivered as a 100 µL standardised dose
containing 2.7 mg THC and 2.5 mg CBD. In contrast, the
drug product Epidiolex® uses a highly purified CBD bo-
tanical drug substance and is dose formulated with sesa-
me oil, anhydrous ethanol, sweetener and strawberry fla-
vouring at a concentration of 100 mg/mL.
Fig. 2. Overview of Cannabis-based drug development and manufacture.
Robust Standardised Cannabinoid
Analytical Procedures
Med Cannabis Cannabinoids 2019;2:1–13
DOI: 10.1159/000496868
Botanical drug registration requires the same level of
safety and efficacy evaluation, and similar levels of strin-
gent quality controls, as most conventional drug prod-
ucts which are highly purified, synthetic or chemically
modified [48]. Indeed, for botanical drugs being used in
new and investigational drug applications, the Botanical
Drug Development Guidance for Industry provided by
the FDA recommends a declaration of the quantitative
absolute dry weight description both for the active con-
stituents as well as other chemical constituents that are
known and measurable. Unlike conventional drugs,
which are typically comprised of a single active ingredi-
ent, botanical drugs can contain multiple active ingredi-
ents with specific combinatory formulations being a de-
fining characteristic [22, 31, 47]. Therefore, quantitative
characterisation of cannabinoids and other active con-
stituents present within Cannabis drug products and
substances are required not only for purposes of safety
and efficacy regarding human health [31, 32], but also for
the protection of intellectual property rights. For the lat-
ter, it is important to ensure that the botanical drug is
accurately described upon registration and that the exact
composition is maintained during production. In the
case of drug products derived from Cannabis, accurate
quantification of botanical forms will be critical for qual-
ity control through the manufacturing pipeline from bo-
tanical raw material to the refinement of botanical drug
substances and development of the final drug product
Validation Characteristics for Analytical Procedures
Analytical method validation or method performance
qualification is a measure of how suitable and robust an
assay is over the expected concentration range at which
the analyte will be analysed and provides assurance that
the assay is fit for purpose. Guidance on analytical meth-
od validation for pharmaceutical materials is provided
from a number of sources, such as the International Con-
ference on Harmonisation (ICH) (document Q2 [R1]
[49]), domestically relevant pharmacopeia (United States/
European, etc.) and from local government agencies,
such as the FDA. No one source of validation criteria is
considered superior to another or applicable to every sce-
nario, nor are these guidelines static over time. Multiple
sources of validation criteria require some level of cus-
tomisation [50], which will largely depend upon the in-
tended application of the method as well as on the juris-
diction-specific requirements of a given locality.
Despite the publication of more than 130 methods for
the analysis of cannabinoids between 1990 and 2016 [51],
few examples exist which have been validated using inter-
nationally recognised guidelines, such as those of the ICH
[26]. Whilst a large number of analytical techniques have
been employed for the quantification of Cannabis botan-
ical raw material [28], including the monograph Canna-
bis Flos from the Dutch Office of Medicinal Cannabis,
limited attention has been given to the quantification of
cannabinoids within Cannabis botanical drug substances
or CME [27–29, 52]. This is of significant concern given
the effects different extraction protocols could have on
sample matrices, the chemical composition of active in-
gredients [27], storage requirements [45, 53] and subse-
quent quantitative assessment of cannabinoids. The fol-
lowing sub-sections discuss a validation scheme compli-
ant with the ICH Q2 (R1) guideline and highlight
generalised validation parameters for the quantification
of cannabinoids within CME, with emphasis on potential
challenges for fulfilling these criteria. Evaluation of these
criteria informs the development of all aspects of the ana-
lytical procedure from sampling, extraction and handling
of the starting material through to the optimisation of
parameters associated with the final chemical analysis.
Specificity (Discrimination)
Specificity refers to the capacity to evaluate an analyte
within a sample matrix [49], with the sample matrix refer-
ring to all components of the botanical drug substance
and/or drug product other than the target analyte. Speci-
ficity has consequences for identification and differentia-
tion between compounds having close homology, as well
as for purity testing and for quantitative determination
[49]. Not all analytical procedures across the manufactur-
ing pipeline require a high level of specificity (complete
discrimination) [32, 54], providing they can be compen-
sated by one or more supporting analytical procedure(s)
[49]. For example, thin layer chromatography is suitable
for quantitative and semi-quantitative assessment of can-
nabinoids within botanical raw material [32, 54]. How-
ever, more discriminatory procedures, which include tar-
geted analysis on a larger portfolio of cannabinoids as
well as interfering materials, are typically used during pu-
rification of drug forms in the manufacturing pipeline
[31] (Fig.2).
Chromatographic methods for identification of can-
nabinoids within botanical forms of Cannabis have been
comprehensively evaluated in the literature [28, 55]. The
two leading analytical procedures used for cannabinoid
analysis, liquid chromatography (LC) and gas chroma-
Med Cannabis Cannabinoids 2019;2:1–13
DOI: 10.1159/000496868
tography (GC), have a number of trade-offs with regard
to specificity, sensitivity, accuracy, precision and sample
throughput. The decision to select one methodology over
another is typically informed by a well-defined and clear-
ly understood objective of the procedure. For GC, liquid
samples are heated at high temperatures in the injector
port during vaporisation, resulting in thermal decarbox-
ylation of the cannabinoid resorcinyl moiety [7] (Fig.1).
As a result, GC requires prior sample derivatisation, such
as a silylation, to discriminate between carboxylated can-
nabinoid precursors and decarboxylated cannabinoids
present in the sample. This may not only provide GC with
a higher degree of chromatographic resolution than LC,
but the derivatization process may also allow discrimina-
tion of isomeric cannabinoid species on the basis of silyl
group number [56]. In contrast, LC does not require high
temperatures for chromatographic separation, allowing
for identification of native carboxylated cannabinoids
within botanical forms [26, 57]. LC may also be more ap-
propriate for quantitative cannabinoid determinations as
compared with GC, as the derivatisation process cannot
always be guaranteed to be exhaustive [56, 58, 59].
For identification, ICH guidelines recommend that
analytical tests should have the capacity to discriminate
between structurally similar compounds that are likely to
be present and that the decision to test for false positives
with interfering materials should be established on sound
scientific judgement [49]. A major drawback of common-
ly used LC-diode array detector methods is that ultravio-
let spectra cannot always differentiate between important
cannabinoid structural variants, such as non-aromatic
cyclisation of the isoprenyl residue (e.g., cannabidiolic
acid [CBDA] vs. delta-9-tetrahydrocannabinolic acid
[THCA]) or the length of the resorcinyl alkyl moiety (e.g.,
THCA vs. tetrahydrocannabivarinic acid [THCVA])
(Fig. 1). Baseline separation of cannabinoids also be-
comes increasingly problematic as the portfolio of target
cannabinoids increases, as has frequently been reported
between CBD and cannabigerol (CBG) as well as tetrahy-
drocannabivarin (THCV) [26, 36, 57]. This situation is
likely to be exacerbated with the analysis of complex bo-
tanical drug substances, given the more extensive varia-
tion in cannabinoid concentration within CME [28, 52].
Discrimination between analytes can be more accu-
rately resolved using mass spectrometry (MS) detection
on the basis of mass-to-charge ratio (m/z), with baseline
separation achievable using methodologies such as select-
ed ion monitoring, which allow for simultaneous detec-
tion of cannabinoids and associated ions that coelute
[36]. However, nominal mass measurements from single
mass detectors have limited discriminatory functionality
for isomeric compounds [60, 61], such as delta-8- and
delta-9-THC. LC-MS/MS with triple quadrupole, quad-
rupole-time-of flight (Q-TOF) [27, 61, 62] and Orbitrap
[63] mass analysers can provide sufficient mass resolu-
tion of both parent and fragment ions to allow for dis-
crimination of molecules with identical nominal mass but
different elemental arrangements [60, 61].
Recent non-targeted profiling of CME using high-res-
olution LC-MS/MS has revealed a complex chemical fin-
gerprint from ethanolic and plant oil-based extracts, with
chemical composition and content dependent not only
on the starting material but also on the procedure used
for extraction [27]. Other reports that at least five canna-
binoid species exhibit a m/z of 315.2294 emphasise the
importance of developing analytical procedures with a
high level of inherent selectivity [28]. A high level of dis-
crimination is particularly relevant for targeted medical
applications which rely on structure-activity relation-
ships of a specific cannabinoid species in order to pro-
duce the desired physiological response following phar-
maceutical exposure. For example, S,S stereochemical
isomers of CBD metabolites and their dimethylheptyl an-
alogues have been demonstrated to have potent human G
protein-coupled cannabinoid type-1 receptor binding
compared with R,R isomers which had very weak or no
binding capacity [64]. Isomeric cannabinoids may also
have different stability profiles, as is the case for THCA-A
compared with THCA-B. Here, the positioning of the res-
orcinyl COOH at C-4 of THCA-B affects intramolecular
hydrogen bonding, which may result in improved stabil-
ity and crystallisation behaviours [65, 66] (Fig.1). The
potential for adulteration of botanical raw material with
synthetic cannabinoids [67] is also a concern where anal-
ysis relies on methods with inherent low discrimination,
such as Fourier-transform infrared (FTIR) spectroscopy.
Due to the relatively large molecular size of cannabinoids,
key structural features that appear between 500 and 1,500
cm–1 are difficult to interpret, making the differentiation
of cannabinoid species problematic from FTIR spectra
alone [68].
Accuracy (Recovery)
Accuracy is the measurement of how close the experi-
mental value is to that of the “true” or accepted reference
value of an analyte within a sample matrix. This descrip-
tion of accuracy is also referred to as trueness. Several
methods for assessing accuracy within drug substance
and drug product analytical procedures are acknowl-
edged in the ICH Q2 (R1) [49]. In the case of drug sub-
Robust Standardised Cannabinoid
Analytical Procedures
Med Cannabis Cannabinoids 2019;2:1–13
DOI: 10.1159/000496868
stances, analytical procedures can be compared (1)
against reference material (analytical standards), (2) oth-
er well-characterised independent procedures, and (3)
inferred from the measurement of precision, linearity and
specificity [49]. In addition to methodologies (2) and (3),
drug product analytical procedures can be compared us-
ing synthetic matrices spiked with known quantities of
the excipients or target analyte (standard addition) [49].
Historically, legislative restrictions on the possession,
manufacture and supply of certified cannabinoid refer-
ence standards have contributed to limiting the compara-
tive assessment of accuracy. They have also limited the
availability of sufficient quantities for determining spike
recovery. Potential workarounds which have been ap-
plied to Cannabis include spiking certified reference stan-
dards into blank solvent exhausted botanical matrices at
levels below the upper range of the analytical procedure
[69] or spiking certified reference standards into small
quantities of blank matrices which are potentially non-
representative of the botanical form (i.e., 25 mg) [70]. A
third workaround involves gravimetrically spiking in-
house pentane extracts of botanical raw material [26, 57].
In the latter example, the “true” reference values of the
spiked concentrated Cannabis extracts were assessed us-
ing an empirical procedure (standard addition), with true
values being resolved when determinations of three ex-
tract masses dissolved in solution demonstrated linearity
within the calibration range [26]. Another issue with the
sourcing of certified reference standards for spiked recov-
ery is the authentication of purity. This is a problem most
notable with unstable carboxylated cannabinoids, such as
THCA, which rarely exist without some level of contam-
ination [65] and, moreover, readily degrade under nor-
mal handling conditions [65, 71].
A similar procedure involving spiked recovery for the
evaluation of accuracy (recovery) could be applied to
CME, whereby preparative high-performance LC-based
fractionation of the botanical drug substance could yield
blank sample matrices and purified extracts of cannabi-
noids for spiking botanical drug forms. However, the in-
house development of highly purified reference materials
may also be constrained by the maximum quantities per-
mitted by regulatory authorisations imposed on research
and analysis laboratories. This becomes a practical limita-
tion considering that (1) a minimum of 1 g blank matrix
is considered representative of the botanical form and its
associated putative adsorption sites and cellular interfer-
ences [26], (2) that cannabinoids could yield greater than
73% w/w of the botanical drug substance or CME [52],
and (3) that the assessment of recovery should cover the
range of the analytical procedure [49]. Another consider-
ation is that during the process of cannabinoid fraction-
ation, the CME matrix could be altered to an extent
whereby it is no longer representative. In these circum-
stances, extracts of the related species Humulus lupulus
(hops, family Cannabaceae) [56] or Urtica dioica (family
Urticaceae) [57] could be used as surrogate matrices. The
use of botanically related materials is likely to be a subop-
timal proxy for CME matrices, although this approach
may be necessary given the rarity of Cannabis germplasm
devoid of cannabinoids [72].
Irrespective of physical adsorption and interference
brought about by CME matrices, matrix-dependent ef-
fects on ionisation efficiency when using MS detection
can significantly affect accuracy at the level of detection.
The so-called matrix effect is predominantly associated
with liquid phase- [73] and electrospray ionisation-based
infrastructure [74] and results in a signal non-propor-
tional to the calibrators. Indeed, both the FDA and EMA
guidelines for method validation require assessment and
reduction for suppression of ionisation in MS-based ana-
lytical procedures [75, 76]. Structural analogues can be
used to mitigate ionisation efficiency as well as matrix-
induced ion suppression [53] or enhancement [54]. How-
ever, they may lack sufficient structural similarity to coe-
lute and provide full compensation. This can potentially
amplify ionisation efficiency-associated error in the
method as the non-coeluting analogue provides another
opportunity for ion suppression/enhancement to influ-
ence methodological determinations and quantitative as-
sessment of the target analyte [74, 75].
Isotopically labelled (e.g., deuterated) internal stan-
dards allow for more precise measurement of ionisation
efficiency and recovery of analytes [75, 76]. However, in
some studies, the more commonly available deuterated
forms have been found to have altered physiochemical
properties to those of the target analyte, potentially hav-
ing an impact on extraction recovery [77]. In these cir-
cumstances, 13C and 18O isotopically labelled internal
standards would be more appropriate in LC-MS-based
cannabinoid quantification, although these standards
have limited availability and are typically expensive [75,
76]. Combined with the number of cannabinoids which
could be present within heterogeneous CME [27], this
presents a challenge for laboratories to source a compre-
hensive set of isotopically labelled internal standards for
the purposes of method validation. Universally labelling
biological samples of Cannabis through cultivation under
13C conditions offers one viable option to generate a suf-
ficient quantity and metabolic range of isotopically la-
Med Cannabis Cannabinoids 2019;2:1–13
DOI: 10.1159/000496868
belled reference materials for matrix effect compensa-
tion, as well as for the measurement of accuracy and
spiked recovery [78]. The generation of 13C plants could
be performed in-house or outsourced to companies, such
as IsoLife BV (Wageningen, The Netherlands).
Precision (Reproducibility)
Precision evaluates the degree to which measurements
of a homogeneous sample vary under a given condition
(coefficient of variation) and is generally comprised of
three levels; repeatability (relative standard deviation
(RSD)r), intermediate precision and reproducibility
(RSDR) [49] (Fig.3). Repeatability and intermediate pre-
cision are intra-laboratory evaluations. Repeatability
evaluates the coefficient of variation within a narrow pe-
riod of time (intra-day variation), while intermediate pre-
cision assesses the coefficient of variation against random
events and may include the evaluation of inter-day preci-
sion and/or the assessment of precision between analysts
[49]. Acceptance criteria of 5% RSD have typically been
applied to analytical procedures for quantifying cannabi-
noids [26, 56, 57, 69], although RSDs as high as 15% have
been reported using derivatization and GC-flame ionisa-
tion detector-based analysis [59]. Having developed an
analytical procedure which conforms to the acceptance
criteria, the measurement of precision can be applied to
Cannabis drug forms at key stages in the manufacturing
pipeline in order to establish homogeneity of active con-
stituents. Given the heterogeneous nature of botanical
drugs, the FDA’s Botanical Drug Development Guidance
for Industry recommends that potency testing and quan-
tification of active constituents be conducted on a batch-
to-batch basis.
The legitimacy of cannabinoid determinations among
laboratories is a contentious subject and one which is be-
coming increasingly acknowledged within the literature
[29, 52, 79]. The evaluation of reproducibility involves an
inter-laboratory collaborative trial. This is an assessment
of precision suited to analytical procedure standardisa-
tion which can contribute to the development of method-
ologies for inclusion in pharmacopeia [49, 80]. In a recent
large-scale comparison across six laboratories, which
took into account phenotypic frequency distribution pat-
terns of THC:CBD, the authors found significant and sys-
temic inflation of cannabinoid quantitative determina-
tions between laboratories after controlling for plausible
confounds (i.e. producers, product types etc.) [79]. Anal-
ysis of 200 oil-based CME of Cannabis varieties Bedro-
can®, Bedrobinol® and Bediol® prepared at ten pharma-
cies in Italy showed RSD > 50% among the cannabinoids
tested [29], although variation from the inter-pharmacy
extraction procedures could not be disentangled from
batch-to-batch variation within these registered clonally-
propagated plant strains.
While such assessments may reflect inter-laboratory
variation, the assessment of reproducibility requires the
transfer [81, 82] and testing of a dedicated analytical pro-
cedure with homogeneous materials [49] as opposed to
round-robin audits of non-standardised procedures
among laboratories [79]. The latter provides little infor-
mation on the source of variation or on the condition-
specific measurement of precision required for the evalu-
ation of reproducibility. Reproducibility is a validation
parameter treated as a random variable [83] and quasi-
independent of both the analytical procedure and analyte
under investigation (RSDR [%] = 2C–0.1505, with C = con-
centration as a mass fraction) [84].
The transfer of analytical procedures is required to
meet international analytical technology transfer guide-
lines from the International Society of Pharmaceutical
Engineering and the WHO. Successful transfer of an ana-
lytical procedure is heavily dependent on equipment
equivalence and harmonisation among participating lab-
oratories [82]. As such, collaborative trials for the evalu-
ation of reproducibility are not only subject to financial
Fig. 3. Schematic diagram outlining a proposed validation scheme
for intra- and inter-laboratory analytical procedures.
Robust Standardised Cannabinoid
Analytical Procedures
Med Cannabis Cannabinoids 2019;2:1–13
DOI: 10.1159/000496868
considerations relating to analytical infrastructure [80],
but also require a high level of planning and communica-
tion among sending and receiving laboratories to ensure
transfer activities are sufficient for the measurement of
variability against the acceptance criteria [82] (Fig.3). In
an unlikely scenario where measurements from a number
of independently developed analytical procedures are
congruent with inter-laboratory precision criteria, one
may conclude that a sufficient level of inter-laboratory
robustness has been achieved. However, in such cases,
methodological bias and error would not have been ac-
counted for, with an associated risk that non-standardised
parameters could result in out-of-specification results
over the life cycle of one or more of the analytical proce-
Evaluation of reproducibility is not a requirement for
drug market authorisation [49], and despite acknowl-
edgement for the assessment of reproducibility by gov-
ernment agencies, such as the FDA and pharmacopeia
(United States Pharmacopeia (USP), general chapter
1224, “Transfer of Analytical Procedures” [82]), the as-
sessment of inter-laboratory precision remains largely
unregulated [82]. Although not strictly relevant for the
purposes of drug authorisation, standards of AOAC IN-
TERNATIONAL [83], as well as the International Union
of Pure and Applied Chemistry (IUPAC) [85], recom-
mend that the evaluation of reproducibility be conducted
using a minimum of eight laboratories (Fig.3). A mini-
mum number of laboratories is also identified by the In-
ternational Organization for Standardization (ISO) 5725
(1994). IUPAC will consider concessions for an absolute
minimum of five laboratories in exceptional cases where
the procedure involves expensive and highly specialised
equipment [85]. Having established a cohort of laborato-
ries for the measurement of reproducibility, those with
out-of-specification measurements could be identified
according to “analytical equivalence” which uses a prede-
termined range of acceptable values [50].
Collaborative Research Initiative
Prior to embarking on method performance qualifica-
tion and the assessment of inter-laboratory precision, the
life cycle management of the analytical procedure should
be considered. This includes development of the analyti-
cal target profile which documents the requirements of
the analytical procedure, an acceptable target level of un-
certainty, as well as performance criteria required for val-
idation [81]. Within this framework and as requested by
ICH Q12 guidelines [81], a multivariate analytical quali-
ty-by-design (AQbD) approach could be implemented to
inform development of robust analytical procedures and
quality systems [86]. AQbD approaches can perform in
silico robustness testing of critical method parameters
(flow rate, column length, etc.) early in the development
of an analytical procedure, and this workflow has been
successfully applied to retention modelling for the sepa-
ration of cannabinoids [87]. AQbD approaches establish
a method operable design region or design space to miti-
gate out-of-specification results associated with trial-
and-error, one parameter at a time, optimisation-based
approaches [81, 86]. The former approach reduces the
requirement for large and potentially costly retrospective
changes to the analytical procedure during method per-
formance qualification stages.
Clearly, given the complexities associated with qualifi-
cation of method performance as well as the design, vali-
dation and transfer contributing to life cycle management
of an analytical procedure [81], the establishment of ro-
bust and standardised procedures for cannabinoid deter-
minations in CME requires a collaborative effort. Logisti-
cally, research organisations that may be eligible to par-
ticipate within such an initiative would (1) hold relevant
legal authorisations to conduct research on Cannabis, (2)
have access to germplasm and representative Cannabis
materials as well as analytical reference standards for the
purposes of method development, and (3) be in a position
to receive and supply Cannabis materials from/to par-
ticipating stakeholders nationally and/or internationally.
The success of such a collaboration will be reliant on
an agreed and predefined analytical target profile, inde-
pendent of a specific analytical technique [81, 86, 88]. Im-
portant topics to resolve include: defining what the ana-
lytical procedure is to measure [88], for example the com-
plement of cannabinoids to be included [89] and
expected interfering materials [49], as well as how trans-
ferrable the analytical method is among heterogeneous
CME matrices. The inclusion/exclusion of chemical con-
stituents for quantitative assessment and potency testing
within Cannabis drug forms will be application specific
and dependent on a number of factors, including the ge-
netic uniformity and chemotypic heterogeneity of breed-
ing materials used in biopharmaceutical manufacture.
Quantitative assessment may also need to extend to iso-
meric species (e.g., delta-9-trans-THC, delta-9-cis-THC
and delta-8-THC) which are likely to be present as well as
other chemical constituents potentially capable of modi-
fying pharmacological activity [90]. Given that the pro-
duction of CME may be proprietary and protected by in-
Med Cannabis Cannabinoids 2019;2:1–13
DOI: 10.1159/000496868
tellectual property rights [22, 32, 46], development of an
analytical target profile relevant to industry will require
engagement and collaborative dialogue between academ-
ic and commercial sectors.
A number of public health concerns arise in territo-
ries where legislators permit consumption of CME
which have not been subject to standard quality assur-
ance requirements that are typically associated with
drug marketing authorisation [15, 91]. Reports of inac-
curate labelling and batch-to-batch variation of active
ingredients within magistral CME preparations con-
firm fears that these products lack standardisation [29,
91, 92]. Being able to characterise accurately the com-
plexity of botanical drug forms and to discriminate the
efficacy of their constituents both singularly and in
combination remains challenging [93]. This is high-
lighted by the fact that, so far, only two botanical drug
forms have met the Botanical Guidance definition of a
botanical drug product and received New Drug Appli-
cation approval and prescription drug marketing au-
thorisation by the FDA (Veregen® from Camellia si-
nensis as well as Fulyzaq
] from Croton
lechleri) [30]. Barriers to botanical drug approval have
been associated not only with insufficient clinical evi-
dence, but also inadequate experience in drug develop-
ment programs by some drug sponsors [30].
Regardless of the level of refinement of the finished
dosage form, the establishment of robust, standardised
analytical procedures for CME is an essential step to-
wards adoption of legitimate Cannabis-based botanical
drug substances. This will not only allow for robust dis-
crimination and quantification of active ingredients, but
also inform what interfering materials are likely to be
present within more purified botanical forms. It will fur-
thermore help identify artefacts generated under differ-
ent extraction and purification conditions. Understand-
ing the complexity of CME through well-characterised
and standardised analytical procedures will also facilitate
the development of reliable but less discriminatory pro-
cedures more suited to routine analysis and may help
pave the way forward for gold standard fit-for-purpose
methodologies. This is in the individual and collective in-
terest of all stakeholders, be they policy makers, regula-
tors, drug manufacturers, clinicians, patients or their car-
Statement of Ethics
The authors have no ethical conflicts to disclose.
Disclosure Statement
The authors have no conflicts of interest to declare.
Funding Sources
No funding was received for this study.
Author Contributions
M.T. Welling carried out a detailed literature survey and pre-
pared the manuscript. L. Liu performed a detailed review and revi-
sion of the manuscript. A. Hazekamp performed a detailed review
and revision of the manuscript. A. Dowell contributed to review
and revision of the manuscript. G.J. King conceived the review
topic and performed a detailed review and revision of the manu-
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... In recent years, a great interest in Cannabis sativa L. plants and derived products has been observed, not only due their widespread use for recreational purposes, but also because of their employment in the pharmaceutical, nutraceutical, food, and cosmetic industries [1,2]. Cannabinoids are the most abundant constituents of cannabis plants, with ∆ 9 -tetrahydrocannabidiol (THC) being the main psychoactive compound. ...
... This interest has generated a great expansion of the market for cannabisrelated products. As a result, there is an increasing demand for chemical information about cannabis plants in the forensic, toxicological, and medical fields, as well as in nutraceutical, food, and cosmetics industries, among others [1,2]. In this respect, LC is currently the technique of choice, and several chromatographic assays can be found in the literature that allow the accurate determination of cannabinoids in a variety of samples [6]. ...
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A colorimetric method for the estimation of the total content of cannabinoids in cannabis samples is proposed. The assay is based on the reaction of these compounds with the reagent Fast Blue B (FBB), which has been immobilized into polydimethylsiloxane (PDMS). The reaction and detection conditions have been established according to the results obtained for the individual cannabinoids Δ9-tetrahydrocannabidiol (THC), cannabidiol (CBD), and cannabinol (CBN), as well as for ethanolic extracts obtained from cannabis samples after ultrasonication. In contact with the extract and under basic conditions, the reagent diffuses from the PDMS device, producing a red-brown solution. The absorbances measured at 500 nm after only 1 min of exposure to the FBB/PDMS composites led to responses proportional to the amounts of the cannabinoids in the reaction media. Those absorbances have been then transformed in total cannabinoid content using CBD as a reference compound. The potential utility of the proposed conditions has been tested by analyzing different cannabis samples. The selectivity towards other plants and drugs has been also evaluated. The present method is proposed as a simple and rapid alternative to chromatographic methods for the estimation of the total content of cannabinoids.
... The analysis of cannabinoids in food is especially challenging due to the multitude of product types in the marketplace. Most reported methods have focused on the analysis of inflorescence materials, oils, and resins rather than processed foods (Citti et al., 2018(Citti et al., , 2019Vaclavik et al., 2019;Welling et al., 2019). Recently, a validated HPLC/diode array detector (DAD) method was reported by Ciolino et al. (2018) for the quantitative analysis of 11 cannabinoids in foods, beverages, topical preparations, vapes/e-liquids, and over-the-counter (OTC) pharmaceutical products (Ciolino et al., 2018). ...
... Recognizing the immense public interest around hemp-containing products and the proliferation of methods for the analysis of hemp-containing products, we determined to adopt an established method requiring only minimal matrix extension validation, rather than validating a new in-house method. Although numerous methods are available for the analysis of Cannabis plant material and extracts, few have been extensively validated for complex food matrices such as gummies and edibles (Welling et al., 2019). A notable exception is the method of Ciolino et al. (2018), in which the authors developed and validated an HPLC/DAD method for the analysis of a wide range of cannabinoid-containing products. ...
The 2018 Agricultural Improvement Act removed hemp from Schedule I control, creating a market for hemp products, including cannabidiol-containing products. Due to the market’s rapid growth, little is known about the presence and concentration of cannabinoids in commercial products. Herein, 11 cannabinoids were quantified using liquid chromatography with diode-array detection in a non-representative sampling of 147 products labeled as containing hemp or cannabidiol. A subset of 133 products were analyzed for toxic elements using inductively coupled plasma-mass spectrometry. Cannabinoid content ranged from < LOD – 143 mg/serving, with a median of 16.7 mg/serving. Fewer than half of products surveyed contained cannabidiol concentrations within 20% of their label declarations. The estimated exposure to lead was below the Interim Reference Level of 12.5 µg/day Pb for women of childbearing age, and most products presented concentrations of Δ⁹-tetrahydrocannabinol below LOQ. These findings emphasize the need for further testing and representative investigation of the cannabidiol marketplace.
... The selective analysis of phytocannabinoids represents a hot topic nowadays especially because no standardization has been accomplished notwithstanding all efforts made by private companies and public research institutions [22]. The scenario is rather complex starting from the sampling to the extraction and lastly to the actual analysis and detection. ...
... A long series of analytical methods have been developed, validated, and published in the literature, and many others are under development every day around the world [23]. Although more communication and cross-talk between analytical laboratories have been achieved in the last few years, no agreement has been reached among the scientific community on the validation parameters and criteria for the determination of phytocannabinoids in cannabis samples [22]. ...
The chemical analysis of cannabis potency involves the qualitative and quantitative determination of the main phytocannabinoids: Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD), cannabigerol (CBG), cannabichromene (CBC), etc. Although it might appear as a trivial analysis, it is rather a tricky task. Phytocannabinoids are present mostly as carboxylated species at the aromatic ring of the resorcinyl moiety. Their decarboxylation caused by heat leads to a greater analytical variability due to both reaction kinetics and possible decomposition. Moreover, the instability of cannabinoids and the variability in the sample preparation, extraction, and analysis, as well as the presence of isomeric forms of cannabinoids, complicates the scenario. A critical evaluation of the different analytical methods proposed in the literature points out that each of them has inherent limitations. The present review outlines all the possible pitfalls that can be encountered during the analysis of these compounds and aims to be a valuable help for the analytical chemist. Graphical abstract
... This might be in part due to the regulatory constraints, which necessitate specific licences to handle this scheduled drug and limit the availability of standard compounds and test samples. Due to this, the cannabis testing industry remains poorly established, with only a relatively limited number of laboratories offering analytical services and a lack of standardized protocols for extraction and analysis [5]. ...
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With an increasing appreciation for the unique pharmacological properties associated with distinct, individual cannabinoids of Cannabis sativa, there is demand for accurate and reliable quantification for a growing number of them. Although recent methods are based on highly selective chromatography-mass spectrometry technology, most are limited to a few cannabinoids, while relying on unnecessarily sophisticated and expensive ultra-high performance liquid chromatography and tandem mass spectrometry. Here we report an optimised, simple extraction method followed by a reliable and simple high performance liquid chromatography method for separation. The detection is performed using a time-of-flight mass spectrometer that is available in most natural products research laboratories. Due to the simplicity of instrumentation, and the robustness resulting from a high resolution in the chromatography of isobaric cannabinoids, the method is well suited for routine phytocannabinoid analysis for a range of applications. The method was validated in terms of detection and quantification limits, repeatability, and recoveries for a total of 17 cannabinoids: detection limits were in the range 11–520 pg when using a 1 µL sample injection volume, and the recovery percentages ranged from 85% to 108%. The validated method was subsequently applied to determine cannabinoid composition in the inflorescences of several medicinal Cannabis sativa varieties.
Cannabis sativa L. is undoubtedly the most used recreational drug worldwide because of its desired acute psychotropic effects, like relaxation, euphoria and altered perceptions. In addition, promising medical properties of Cannabis components have gained a lot of attention, resulting in a debate to permit recreational Cannabis use in several countries. In recent years, this controversial plant was increasingly studied and a large number of scientific papers were published. Herbal Cannabis consists of a variable and complex matrix, which makes it challenging to properly seize and prepare the sample for qualitative and quantitative analysis. Moreover, both the adoption of legal cut‐off values in different countries for the Δ9‐tetrahydrocannabinol (THC) content in seizures, and the emergence of cannabidiol (CBD) based products, containing generally small but variable amounts of THC, urged the need for sensitive and reliable analytical techniques to accurately identify and quantify the components of interest. This review presents detailed information on the procedure prior to analysis and covers chromatographic and spectroscopic methods developed for the analysis of cannabinoids in seizures for different forensic purposes, that is, identification/quantification, potency testing, drug‐ and fiber‐type differentiation, age estimation, yield determination and Cannabis profiling. Advantages and drawbacks of existing methods, within a specific forensic context, are discussed. The application of chemometrics, which offers a powerful tool in interpreting complex data, is also explained. This article is categorized under: Toxicology > Cannabis Toxicology > Drug Analysis Forensic Chemistry and Trace Evidence > Presentation and Evaluation of Forensic Science Output Cannabis sativa L. is the most used and seized recreational drug worldwide. For forensic institutes/laboratories, it is important to obtain reliable and reproducible data about seized samples, as it is used in judicial investigations. A thorough consideration about the sampling, sample preparation, instrumental analysis and subsequent data handling is needed, and depends on the forensic purpose. Moreover, chemometrics, which is already applied in certain herbal cannabis studies, will become an important tool in forensics to interpret large and complex data.
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Cannabis has been at the center of scientific attention for some years now. Since its pharmacological potential has been highlighted, cannabis has become a hot topic in research laboratories, leading to the publication of many scientific studies. Focusing on analytical chemistry, an enormous number of analytical methods for cannabinoid (CNB) determination have been published, involving various tech-niques. However, no globally accepted reference method for CNB determination has yet been chosen.This review aims to identify very recent analytical methods developed to analyze phytocannabinoids in cannabis herbal samples. For certain techniques, stagnation in terms of employed operational conditions can be observed. In this context, a reference method of analysis should be proposed and accepted worldwide to standardize CNB determination. In contrast, for other techniques, we are witnessing a scientific ferment, which is resulting in the development of new interesting analytical options. In this regard, particular focus has been given to these niche techniques, which are now emerging in the analytical panorama of cannabis analysis, offering new important perspectives for the future of cannabis testing. Supercritical fluid chromatography and infrared spectroscopy showed tangible advantages when applied to CNB determination in herbal samples.
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Cannabis is a chemically diverse domesticated plant genus which produces a unique class of biologically active secondary metabolites referred to as cannabinoids. The affinity and selectivity of cannabinoids to targets of the human endocannabinoid system depend on alkyl side chain length, and these structural-activity relationships can be utilized for the development of novel therapeutics. Accurate early screening of germplasm has the potential to accelerate selection of chemical phenotypes (chemotypes) for pharmacological exploitation. However, limited attempts have been made to characterize the plasticity of alkyl cannabinoid composition in different plant tissues and throughout development. A chemotypic diversity panel comprised of 99 individuals from 20 Cannabis populations sourced from the Ecofibre Global Germplasm Collection ( and was used to examine alkyl cannabinoid variation across vegetative, flowering and maturation stages. A wide range of di-/tri-cyclic as well as C3-/C5-alkyl cannabinoid composition was observed between plants. Chemotype at the vegetative and flowering stages was found to be predictive of chemotype at maturation, indicating a low level of plasticity in cannabinoid composition. Chemometric cluster analysis based on composition data from all three developmental stages categorized alkyl cannabinoid chemotypes into three classes. Our results suggest that more extensive chemical and genetic characterization of the Cannabis genepool could facilitate the metabolic engineering of alkyl cannabinoid chemotypes.
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In just a few years, cannabidiol (CBD) has become immensely popular around the world. After initially being discovered as an effective self-medication for Dravet syndrome in children, CBD is now sold and used to treat a wide range of medical conditions and lifestyle diseases. The cannabinoid CBD, a non-psychoactive isomer of the more infamous tetrahydrocannabinol (THC), is available in a growing number of administration modes, but the most commonly known is CBD oil. There are currently dozens, if not hundreds, of producers and sellers of CBD oils active in the market, and their number is increasing rapidly. Those involved vary from individuals who prepare oils on a small scale for family and (Facebook) friends to compounding pharmacies, pharmaceutical companies, and licensed cannabis producers. Despite the growing availability of CBD, many uncertainties remain about the legality, quality, and safety of this new “miracle cure.” As a result, CBD is under scrutiny on many levels, ranging from national health organizations and agricultural lobbyists to the WHO and FDA. The central question is whether CBD is simply a food supplement, an investigational new medicine, or even a narcotic. This overview paper looks into the known risks and issues related to the composition of CBD products, and makes recommendations for better regulatory control based on accurate labeling and more scientifically supported health claims. The intention of this paper is to create a better understanding of the benefits versus the risks of the current way CBD products are produced, used, and advertised.
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Cannabidiol (CBD)-based oil preparations are becoming extremely popular, as CBD has been shown to have beneficial effects on human health. CBD-based oil preparations are not unambiguously regulated under the European legislation, as CBD is not considered as a controlled substance. This means that companies can produce and distribute CBD products derived from non-psychoactive hemp varieties, providing an easy access to this extremely advantageous cannabinoid. This leaves consumers with no legal quality guarantees. The objective of this project was to assess the quality of 14 CBD oils commercially available in European countries. An in-depth chemical profiling of cannabinoids, terpenes and oxidation products was conducted by means of GC-MS and HPLC-Q-Exactive-Orbitrap-MS in order to improve knowledge regarding the characteristics of CBD oils. Nine out of the 14 samples studied had concentrations that differed notably from the declared amount, while the remaining five preserved CBD within optimal limits. Our results highlighted a wide variability in cannabinoids profile that justifies the need for strict and standardized regulations. In addition, the terpenes fingerprint may serve as an indicator of the quality of hemp varieties, while the lipid oxidation products profile could contribute in evaluation of the stability of the oil used as milieu for CBD rich extracts.
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The majority of adults in the U.S. now have state-legal access to medical or recreational cannabis products, despite their federal prohibition. Given the wide array of pharmacologically active compounds in these products, it is essential that their biochemical profile is measured and reported to consumers, which requires accurate laboratory testing. However, no universal standards for laboratory testing protocols currently exist, and there is controversy as to whether all reported results are legitimate. To investigate these concerns, we analyzed a publicly available seed-to-sale traceability dataset from Washington state containing measurements of the cannabinoid content of legal cannabis products from state-certified laboratories. Consistent with previous work, we found that commercial Cannabis strains fall into three broad chemotypes defined by the THC:CBD ratio. Moreover, we documented systematic differences in the cannabinoid content reported by different laboratories, relative stability in cannabinoid levels of commercial flower and concentrates over time, and differences between popular commercial strains. Importantly, interlab differences in cannabinoid reporting persisted even after controlling for plausible confounds. Our results underscore the need for standardized laboratory methodologies in the legal cannabis industry and provide a framework for quantitatively assessing laboratory quality.
A generic liquid chromatographic method development workflow was developed and successfully applied to the analysis of phytocannabinoids and Cannabis sativa extracts. Our method development procedure consists in four steps: i) The screening of primary parameters (i.e. stationary phase nature, organic modifier nature and approximate mobile phase pH) was carried out with a generic gradient on a short narrow bore column, using a system able to accommodate numerous solvents/buffers and columns. Instead of complete peak tracking, the number of peaks which can be separated was considered as a response at this level, to save time.ii) The optimization of secondary parameters (i.e. gradient conditions, mobile phase temperature and pH within a narrow range) requires only 12 initial experiments and the use of HPLC modeling software for data treatment. It allows to find out the best retention and selectivity for the selected compounds. Peak tracking was performed with a single quadruple mass detector in single ion recording mode, and UV detection (in a broad wavelength range).iii) The refinement step allows to further adjust column efficiency, by tuning column length and mobile phase flow rate. This can also be done virtually using HPLC modeling software.iv) The robustness testing step was also evaluated from a virtual experimental design. Success rate and regression coefficients were estimated in about 1 min, without the need to perform any real experiment.At the end, this method development workflow was performed in less than 4 days and minimizes the costs of the method development in liquid chromatography.
Introduction Cannabis sativa L. (cannabis) is utilised as a therapeutic and recreational drug. With the legalisation of cannabis in many countries and the anticipated regulation of potency that will accompany legalisation, analytical testing facilities will require a broadly applicable, quantitative, high throughput method to meet increased demand. Current analytical methods for the biologically active components of cannabis (phytocannabinoids) suffer from low throughput and/or an incomplete complement of relevant phytocannabinoids. Objective To develop a rapid, quantitative and broadly applicable liquid chromatography–tandem mass spectrometry analytical method for 11 phytocannabinoids in cannabis with acidic and neutral character. Methodology Bulk diffusion coefficients were calculated using the Taylor–Aris open tubular method, with four reference compounds used to validate the experimental set‐up. Three columns were quantitatively evaluated using van Deemter plots and fit‐to‐purpose performance metrics. Low (1.2 μL²) and standard (3.6 μL²) extra‐column variance ultra‐high pressure liquid chromatography (UPLC) configurations were contrasted. Method performance was demonstrated with methanolic cannabis flower extracts. Results Bulk diffusion coefficients and van Deemter plots for 11 phytocannabinoids are reported. The developed chromatographic method includes the challenging Δ⁸/Δ⁹‐tetrahydrocannabinol isobars and, at 6.5 min, is faster than existing methods targeting similar panels of biologically active phytocannabinoids. Conclusions The bulk diffusion coefficients and van Deemter curves informed the development of a rapid quantitative method and will facilitate potential expansion to include additional compounds, including synthetic cannabinoids. The developed method can be implemented with low or standard extra‐column variance UPLC configurations.
Background: Patients with Lennox-Gastaut syndrome, a rare, severe form of epileptic encephalopathy, are frequently treatment resistant to available medications. No controlled studies have investigated the use of cannabidiol for patients with seizures associated with Lennox-Gastaut syndrome. We therefore assessed the efficacy and safety of cannabidiol as an add-on anticonvulsant therapy in this population of patients. Methods: In this randomised, double-blind, placebo-controlled trial done at 24 clinical sites in the USA, the Netherlands, and Poland, we investigated the efficacy of cannabidiol as add-on therapy for drop seizures in patients with treatment-resistant Lennox-Gastaut syndrome. Eligible patients (aged 2-55 years) had Lennox-Gastaut syndrome, including a history of slow (<3 Hz) spike-and-wave patterns on electroencephalogram, evidence of more than one type of generalised seizure for at least 6 months, at least two drop seizures per week during the 4-week baseline period, and had not responded to treatment with at least two antiepileptic drugs. Patients were randomly assigned (1:1) using an interactive voice response system, stratified by age group, to receive 20 mg/kg oral cannabidiol daily or matched placebo for 14 weeks. All patients, caregivers, investigators, and individuals assessing data were masked to group assignment. The primary endpoint was percentage change from baseline in monthly frequency of drop seizures during the treatment period, analysed in all patients who received at least one dose of study drug and had post-baseline efficacy data. All randomly assigned patients were included in the safety analyses. This study is registered with, number NCT02224690. Findings: Between April 28, 2015, and Oct 15, 2015, we randomly assigned 171 patients to receive cannabidiol (n=86) or placebo (n=85). 14 patients in the cannabidiol group and one in the placebo group discontinued study treatment; all randomly assigned patients received at least one dose of study treatment and had post-baseline efficacy data. The median percentage reduction in monthly drop seizure frequency from baseline was 43·9% (IQR -69·6 to -1·9) in the cannibidiol group and 21·8% (IQR -45·7 to 1·7) in the placebo group. The estimated median difference between the treatment groups was -17·21 (95% CI -30·32 to -4·09; p=0·0135) during the 14-week treatment period. Adverse events occurred in 74 (86%) of 86 patients in the cannabidiol group and 59 (69%) of 85 patients in the placebo group; most were mild or moderate. The most common adverse events were diarrhoea, somnolence, pyrexia, decreased appetite, and vomiting. 12 (14%) patients in the cannabidiol group and one (1%) patient in the placebo group withdrew from the study because of adverse events. One patient (1%) died in the cannabidiol group, but this was considered unrelated to treatment. Interpretation: Add-on cannabidiol is efficacious for the treatment of patients with drop seizures associated with Lennox-Gastaut syndrome and is generally well tolerated. The long-term efficacy and safety of cannabidiol is currently being assessed in the open-label extension of this trial. Funding: GW Pharmaceuticals.
In 1937, the United States of America criminalized the use of cannabis and as a result its use decreased rapidly. In recent decades, there is a growing interest in the wide range of medical uses of cannabis and its constituents; however, the laws and regulations are substantially different between countries. Laws differentiate between raw herbal cannabis, cannabis extracts, and cannabinoid-based medicines. Both the European Medicines Agency (EMA) and the United States Food and Drug Administration (FDA) do not approve the use of herbal cannabis or its extracts. The FDA approved several cannabinoid-based medicines, so did 23 European countries and Canada. However, only four of the reviewed countries have fully authorized the medical use of herbal cannabis - Canada, Germany, Israel and the Netherlands, together with more than 50% of the states in the United States. Most of the regulators allow the physicians to decide what specific indications they will prescribe cannabis for, but some regulators dictate only specific indications. The aim of this article is to review the current (as of November 2017) regulations of medical cannabis use in Europe and North America.
Background: Cannabis use is common, and associated with adverse health outcomes. 'Routes of administration' (ROAs) for cannabis use have increasingly diversified, in part influenced by developments towards legalization. This paper sought to review data on prevalence and health outcomes associated with different ROAs. Methods: This scoping review followed a structured approach. Electronic searches for English-language peer-reviewed publications were conducted in primary databases (i.e., MEDLINE, EMBASE, PsycINFO, Google Scholar) based on pertinent keywords. Studies were included if they contained information on prevalence and/or health outcomes related to cannabis use ROAs. Relevant data were screened, extracted and narratively summarized under distinct ROA categories. Results: Overall, there is a paucity of rigorous and high-quality data on health outcomes from cannabis ROAs, especially in direct and quantifiable comparison. Most data exist on smoking combusted cannabis, which is associated with various adverse respiratory system outcomes (e.g., bronchitis, lung function). Vaporizing natural cannabis and ingesting edibles appear to reduce respiratory system problems, but may come with other risks (e.g., delayed impairment, use 'normalization'). Vaporizing cannabis concentrates can result in distinct acute risks (e.g., excessive impairment, injuries). Other ROAs are uncommon and under-researched. Conclusions: ROAs appear to distinctly influence health outcomes from cannabis use, yet systematic data for comparative assessments are largely lacking; these evidence gaps require filling. Especially in emerging legalization regimes, ROAs should be subject to evidence-based regulation towards improved public health outcomes. Concretely, vaporizers and edibles may offer potential for reduced health risks, especially concerning respiratory problems. Adequate cannabis product regulation (e.g., purity, labeling, THC-restrictions) is required to complement ROA-based effects.
Cannabinoids are a group of terpenophenolic compounds in the medicinal plant Cannabis sativa (Cannabaceae family). Cannabigerolic acid, Δ9-tetrahydrocannabinolic acid A, cannabidiolic acid, Δ9-tetrahydrocannabinol, cannabigerol, cannabidiol, cannabichromene, and tetrahydrocannabivarin are major metabolites in the classification of different strains of C. sativa. Degradation or artifact cannabinoids cannabinol, cannabicyclol, and Δ8-tetrahydrocannabinol are formed under the influence of heat and light during processing and storage of the plant sample. An ultrahigh-performance liquid chromatographic method coupled with photodiode array and single quadruple mass spectrometry detectors was developed and validated for quantitative determination of 11 cannabinoids in different C. sativa samples. Compounds 1 – 11 were baseline separated with an acetonitrile (with 0.05% formic acid) and water (with 0.05% formic acid) gradient at a flow rate of 0.25 mL/min on a Waters Cortec UPLC C18 column (100 mm × 2.1 mm I. D., 1.6 µm). The limits of detection and limits of quantitation of the 11 cannabinoids were below 0.2 and 0.5 µg/mL, respectively. The relative standard deviation for the precision test was below 2.4%. A mixture of acetonitrile and methanol (80 : 20, v/v) was proven to be the best solvent system for the sample preparation. The recovery of all analytes was in the range of 97 – 105%. A total of 32 Cannabis samples including hashish, leaves, and flower buds were analyzed.