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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
a
Graham J. King
a
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 @ scu.edu.au
© 2019 The Author(s)
Published by S. Karger AG, Basel
E-Mail karger@karger.com
www.karger.com/mca
DOI: 10.1159/000496868
Keywords
Cannabis sativa · Medicinal cannabis · Cannabidiol ·
Tetrahydrocannabinol · Quantitative analysis · Mass
spectrometry
Abstract
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
Introduction
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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Welling/Liu/Hazekamp/Dowell/King
Med Cannabis Cannabinoids 2019;2:1–13
2
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-
ta-9-tetrahydrocannabinol.
Robust Standardised Cannabinoid
Analytical Procedures
3
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 (www.tga.gov.au).
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
Welling/Liu/Hazekamp/Dowell/King
Med Cannabis Cannabinoids 2019;2:1–13
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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
5
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
(Fig.2).
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-
Welling/Liu/Hazekamp/Dowell/King
Med Cannabis Cannabinoids 2019;2:1–13
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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
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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-
Welling/Liu/Hazekamp/Dowell/King
Med Cannabis Cannabinoids 2019;2:1–13
8
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
9
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-
dures.
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-
Welling/Liu/Hazekamp/Dowell/King
Med Cannabis Cannabinoids 2019;2:1–13
10
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.
Conclusion
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
TM
[Mytesi
TM
] 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-
ers.
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-
script.
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