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Cannabinoid Preparations (Emerging Issues in Analytical
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The Analytical Chemistry
of Cannabis
Quality Assessment, Assurance, and
Regulation of Medicinal Marijuana
and Cannabinoid Preparations
Brian F. Thomas
Analytical Chemistry and Pharmaceutics, RTI International, Research
Triangle Park, NC, United States

Mahmoud A. ElSohly
National Center for Natural Products Research, Research Institute of
Pharmaceutical Sciences and Department of Pharmaceutics, School of
Pharmacy, University of Mississippi, MS, United States

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DEDICATION

This work is dedicated to my wife Cathy, and my mentors Billy Martin,

Ed Cook, Bob Jeffcoat, and Ken Davis.
LIST OF CONTRIBUTORS

Suman Chandra
National Center for Natural Products Research, Research Institute of Pharmaceutical
Sciences, School of Pharmacy, University of Mississippi, MS, United States
Mahmoud A. ElSohly
National Center for Natural Products Research, Research Institute of Pharmaceutical
Sciences and Department of Pharmaceutics, School of Pharmacy, University of
Mississippi, MS, United States
Michelle Glass
Department of Pharmacology, University of Auckland, Auckland, New Zealand
Hemant Lata
National Center for Natural Products Research, Research Institute of Pharmaceutical
Sciences, School of Pharmacy, University of Mississippi, MS, United States
Raphael Mechoulam
Institute for Drug Research, The Hebrew University of Jerusalem, Jerusalem, Israel
Roger G. Pertwee
Institute of Medical Sciences, University of Aberdeen, Aberdeen, Scotland, United
Kingdom
Brian F. Thomas
Analytical Chemistry and Pharmaceutics, RTI International, Research Triangle Park,
NC, United States
Ryan G. Vandrey
Behavioral Biology Research Unit, Johns Hopkins University School of Medicine,
Baltimore, MD, United States
FOREWORD

Cannabis has been used for thousands of years for recreational,

medicinal, or religious purposes. However, the determination of the
chemical structures of its cannabinoids, terpenes, and many other con-
stituents, and of the pharmacological actions and possible therapeutic
uses of some of these compounds, began less than 100 years ago. This
book begins by describing the cultivation, harvesting, and botanical
classification of cannabis plants, and then goes on to specify how these
plants produce some of their chemical constituents. Subsequent chap-
ters focus on medical formulations of cannabis and cannabis-derived
drugs, on the routes of administration of these formulations, and on
analytical methods that are used in the formulation development and
for the quality control or stability assessment of cannabis constituents.
The penultimate chapter deals with regulatory and additional
formulation-related issues for medical cannabis and cannabinoids,
while the final chapter identifies ways in which analytical chemistry
will most likely contribute to the development of cannabinoid thera-
peutics in the future. This book provides much needed insights into the
important roles that analytical chemistry has already played and is
likely to continue to play in the development of cannabis and its con-
stituents as medicines.

Roger G. Pertwee MA, DPhil, DSc, HonFBPhS

Institute of Medical Sciences
University of Aberdeen
Aberdeen, Scotland, United Kingdom
PREFACE

Pharmacology began with natural products and, over some years on

either side of 1900, evolved into a rigorous scientific discipline domi-
nated, at least in the West, by well-defined chemical entities, either
extracted and processed or synthesized. The two traditions evolved
together, each informing the other, the natural strain by long experi-
ence pointing the way toward how a drug development program might
be structured, the synthetic strain contributing molecular specificity,
with analytical chemistry a common element. The resultant contribu-
tion to modern medicine, with all its caveats and controversies, must
be accounted as one of the great advancements in science.

Natural products pharmacology is very much alive. However, that

“natural” is one cause of the popular misconception that herbs are in
some way better or safer than pills. Though some herbal remedies do
appear to be safe and effective, the opposite is closer to the truth.
Cannabis is a good example. The number of parameters on which can-
nabis products can vary is enormous, from strain, growing conditions,
harvesting methods, and handling to storage and processing of the raw
material to combination with a wide variety of foods and other excipi-
ents in manufacturing to methods of administration (eating, smoking,
“vaping,” applying to mucous membranes). At every step, from plant-
ing through consumption, myriad influences can alter dose, absorption
rate, interactions among constituents, exposure to toxins, and a host of
other factors that can result in underdosing, overdosing, and various
types and levels of acute and chronic poisoning, not excepting an
increase in the probability of lung cancer. Even if quality were well
controlled, which on the whole is very much not the case, this
complexity means that governmental oversight of cannabis products
cannot be as close and complete as that for prescription and over-the-
counter pharmaceuticals. Caveat emptor.
Governments around the world are coming slowly to the conclusion
that, in the absence of draconian enforcement, and to a nontrivial
extent in its presence, people are going to use cannabis for medicine
and recreation. The Internet spreads knowledge of genetic sequencing,
xiv Preface

metabolomics, proteomics, and other disciplines such that people are

going to manipulate cannabis, as they have long done by selective
breeding, to maximize its mental and physical effects and tailor the
quality of those effects. The present legal status in the United States
and elsewhere cannot stop these activities by amateurs, but it does
inhibit research by professionals to investigate the basic science of can-
nabis, and to use this information to better understand neurophysio-
logical function, develop new medicines for people and animals, and
find ways to deal with cannabis addiction. Tight control of marijuana
and inhibition of legal research has arguably led to another paradoxi-
cal effect: driving the chemistry underground, which has resulted in the
proliferation of new and more dangerous synthetic cannabinoids.
There needs to be more involvement by elements of the US Food and
Drug Administration rather than the Drug Enforcement Agency.

Clearly, the policy, regulatory, and research challenges that accom-

pany the study and understanding of cannabis are unique. Despite all
the issues, research continues, understanding of cannabis and its effects
is evolving, policies are in flux, and the literature is ever-changing. The
aim of this book is to provide the reader with a detailed understanding
of the analytical chemistry of cannabis and cannabinoids as the foun-
dation for quality, safety, and utility of cannabis-derived therapeutics,
and offer direction for future advancements.
ACKNOWLEDGMENTS

The authors thank RTI Press and RTI International for their support
of this project, as well as the continued support of RTI research on
cannabis over the years by the National Institute on Drug Abuse.

We appreciate the opportunity to work with the editorial and

production team at Elsevier—Katy Morrissey, Amy Clark, Vijayaraj
Purushothaman, and the many who go unmentioned—in bringing this
first volume in the series “Emerging Issues in Analytical Chemistry”
to fruition.
Chapter 1, “The Botany of Cannabis sativa L.,” was prepared col-
lectively by Dr Suman Chandra, Dr Hemant Lata, and Dr Mahmoud
A. ElSohly at the University of Mississippi, whose work was supported
in part with federal funds from the National Institute on Drug Abuse,
National Institutes of Health, Department of Health and Human
Services, USA, under contract No. N01DA-10-7773.
Thanks to Dayle G. Johnson of RTI International for the cover
design.
We are especially indebted to Dr Gerald T. Pollard for his editorial
assistance. His attention to detail and overall project management are
greatly appreciated.
CHAPTER 1
The Botany of Cannabis sativa L.

Cannabis sativa L. is a widespread species in nature. It is found in

various habitats ranging from sea level to the temperate and alpine
foothills of the Himalayas, from where it was probably spread over the
last 10,000 years.1,2 The age-old cultivation makes its original distribu-
tion difficult to pinpoint.3 Cannabis has a long history of medicinal
use in the Middle East and Asia, with references as far back as the 6th
century BCE, and it was introduced in Western Europe as a medicine
in the early 19th century to treat epilepsy, tetanus, rheumatism,
migraine, asthma, trigeminal neuralgia, fatigue, and insomnia.4,5

As a plant, it is valued for its hallucinogenic and medicinal prop-

erties, more recently being used for pain, glaucoma, nausea, asthma,
depression, insomnia, and neuralgia.6,7 Derivatives are used in
HIV/AIDS8 and multiple sclerosis.9 The pharmacology and
therapeutic efficacy of cannabis preparations and its main active
constituent Δ9-tetrahydrocannabinol (Δ9-THC) have been extensively
reviewed.10 12 The other important cannabinoid constituent of
current interest is cannabidiol (CBD). There has been a significant
interest in CBD over the last few years because of its reported
activity as an antiepileptic agent, particularly its promise for the
treatment of intractable pediatric epilepsy.13,14 Other than Δ9-THC
and CBD, tetrahydrocannabivarin (THCV), cannabinol (CBN), can-
nabigerol (CBG), and cannabichromene (CBC) are major isolates.
Fig. 1.1 shows chemical structures.
Cannabis is also one of the oldest sources of food and textile
fiber.15 17 Hemp grown for fiber was introduced in Western Asia and
Egypt and subsequently in Europe between 1000 and 2000 BCE.
Cultivation of hemp in Europe became widespread after 500 CE.
The crop was first brought to South America (Chile) in 1545, and to
North America (Port Royal, Acadia) in 1606.18 Now its cultivation
is prohibited or highly regulated in the United States.

The Analytical Chemistry of Cannabis. DOI: http://dx.doi.org/10.1016/B978-0-12-804646-3.00001-1

Copyright © 2016 Elsevier Inc. All rights reserved.
2 The Analytical Chemistry of Cannabis

Figure 1.1 Chemical structures of major cannabinoids present in Cannabis sativa. Δ9-THC, Δ9-tetrahydrocan-
nabinol; THCV, tetrahydrocannabivarin; CBN, cannabinol; CBG, cannabigerol; CBC, cannabichromene; CBD,
cannabidiol.

BOTANICAL DESCRIPTION
Table 1.1 describes the botanical nomenclature of C. sativa L. Cannabis
is a highly variable species in terms of botany, genetics, and chemical
constituents. The number of species in the Cannabis genus has long
been controversial. Some reports proposed Cannabis as a polytypic
genus.19 22 However, based on morphological, anatomical, phyto-
chemical, and genetic studies, it is generally treated as having a single,
highly polymorphic species, C. sativa L.23 26 Other reported species
 The Botany of Cannabis sativa L. 3

Table 1.1 Botanical Nomenclature of Cannabis sativa L.

Category Botanical Nomenclature

Kingdom Plantae—Plants
Subkingdom Tracheobionta—Vascular plants
Superdivision Spermatophyta—Seed plants
Division Magnoliophyta—Flowering plants
Class Magnoliopsida—Dicotyledons
Subclass Hamamelididae
Order Urticales
Family Cannabaceae
Genus Cannabis
Species Cannabis sativa L.

are Cannabis indica Lam. and Cannabis ruderalis Janisch, but plants
considered to have belonged to these species are now recognized as
varieties of C. sativa L. (var. indica and var. ruderalis, respectively).
Cannabis sativa and indica are widely cultivated and economically
important; Cannabis ruderalis is hardier and grows in the northern
Himalayas and the southern states of the former Soviet Union but is
rarely cultivated for drug content.

The main morphological difference between indica and sativa is in

their leaves. The leaves of sativa are much smaller and thinner,
whereas those of indica have wide fingers and are deep green, often
tinged with purple; at maturity, they turn dark purple. Indica plants
are shorter and bushier, usually under 6 ft tall and rarely over 8 ft
Indica has short branches laden with thick, dense buds, which mature
early, usually at the beginning of September in the Northern
Hemisphere. Indica buds also vary in color from dark green to purple,
with cooler conditions inducing more intense coloration. Indica flowers
earlier. The natural distribution of indica is Afghanistan, Pakistan,
India, and surrounding areas. The plants of sativa have long branches,
with the lower ones spreading 4 ft or more from the central stalk, as
on a conical Christmas tree. Height varies from 6 ft to more than 20 ft,
with 8 12 ft being the most common. Buds are long and thin and far
less densely populated than in indica, but longer, sometimes 3 ft or
more. Maturation time varies considerably depending on the variety
and environmental conditions. Low Δ9-THC Midwestern sativa varie-
ties (ditchweed) mature in August and September, while equatorial
4 The Analytical Chemistry of Cannabis

varieties mature from October to December. Buds of sativa require

intense light to thicken and swell; indica does not. Sativa tends to be
higher in Δ9-THC and lower in CBD than indica. Sativa is found
all over the world and comprises most of the drug type equatorial
varieties such as Colombian, Mexican, Nigerian, and South African,
where marijuana plants can be very potent. Cannabis has many local
common names, some of which are given in Table 1.2.

Normally, cannabis exhibits a dioecious (male and female flowers

develop on separate plants) and occasionally a monoecious (hermaphro-
dite) phenotype. It flowers in the shorter days (below 12-h photoperiod)
and continues growing vegetatively in the longer photoperiod. Sex is
determined by heteromorphic chromosomes (males being heterogametic
XY, females homogametic XX). Male flowers can be differentiated from
female by their different morphological appearance. At the vegetative
stage, differentiation is difficult because of morphological similarities.
Molecular techniques, however, can differentiate at an early stage.27 32
Cannabis is wind pollinated. For the production of cannabinoids (or
phytocannabinoids), female plants are preferred for several reasons.
First, they produce higher amounts of cannabinoids. Second, once
pollinated, female plants produce seeds at maturity, whereas seed-free

Table 1.2 Common Cannabis Names in Different Languages

Language Common Names

Arabic Bhang, hashish qinnib, hasheesh kenneb, qinnib, tîl

Chinese Xian ma, ye ma
Danish Hemp
Dutch Hennep
English Hemp, marihuana
Finnish Hamppu
French Chanvre, chanvre d’Inde, chanvre indien, chanvrier
German Hanf, haschisch, indischer hanf
Hindi Bhang, charas, ganja
Japanese Mashinin
Nepalese Charas, gajiimaa, gaanjaa
Portuguese Cânhamo, maconha
Russian Kannabis sativa
Spanish Cáñamo, grifa, hachís, mariguana, marijuana
Swedish Porkanchaa
 The Botany of Cannabis sativa L. 5

plants (sinsemilla, a Spanish word) are preferred for their higher yield of
secondary metabolites. Third, if several cannabis varieties are being
grown together, cross-pollination would affect the quality (chemical
profile) of the final product. To avoid this, removing male plants as they
appear, screening female clones for higher metabolite content, and
conservation and multiplication using biotechnological tools ensures the
consistency in chemical profile that is desirable for pharmaceuticals.

CHEMICAL CONSTITUENTS AND PHENOTYPES OF C. SATIVA L.

CBN was the first cannabinoid to be isolated33,34 and identified35 37
from C. sativa. The elucidation of CBN led to speculation that the
psychotropically active constituents of cannabis could be THCs. The
nonpsychotropic compound CBD was subsequently isolated from
Mexican marijuana38 and the structure was determined.39 Gaoni and
Mechoulam, two pioneers of cannabis research, determined the struc-
ture of Δ9-THC after finally succeeding in isolating and purifying this
elusive compound (see Mechoulam Close-up: How to Pamper an
Idea).40 Since then, the number of cannabinoids and other compounds
isolated from cannabis has increased continually, with 545 now
reported. Of these, 104 are phytocannabinoids (Table 1.3). From the
isolation and structural elucidation of Δ9-THC in 1964 until 1980,

Table 1.3 Constituents of Cannabis sativa L.

No. Groups Number of Known Compounds

1 CBG type 17
2 CBC type 8
3 CBD type 8
4 Δ9-THC type 18
5 Δ -THC type
8
2
6 CBL type 3
7 CBE type 5
8 CBN type 10
9 CBND type 2
10 CBT type 9
11 Miscellaneous 22
12 Total cannabinoids 104
13 Total noncannabinoids 441
Total 545
6 The Analytical Chemistry of Cannabis

61 phytocannabinoids were isolated and reported.41 Only nine new

ones were characterized between 1981 and 2005,42 but 31 were
reported between 2006 and 2010. The 13 chemical constituent type
groups shown in Table 1.3 suggests the chemical complexity of the
cannabis plant.42
The concentration of Δ9-THC and CBD, the most abundant canna-
binoids, can be characterized qualitatively and quantitatively.43
Qualitative characterization is based on the Δ9-THC/CBD ratio and
assigning the plant to a discrete chemical phenotype (chemotype). In
1971, cannabis was initially characterized in two phenotypes, drug
type and fiber type, by Fetterman et al.44 A Δ9-THC/CBD ratio .1
was drug type, a lesser ratio was fiber type. In 1973, Small and
Beckstead proposed three categories based on the ratio: drug type if
.1, intermediate if close to 1, and fiber if ,1.45,46 In 1987, Fournier
et al. added a rare chemotype that was characterized by a very low
content of Δ9-THC and CBD with CBG as the predominant
constituent.47

Quantitatively, the plant is characterized by potency through mea-

suring the level of its most abundant cannabinoids, Δ9-THC and CBD,
in its tissues (Fig. 1.2). The levels of cannabinoids are controlled by
the interaction of several genes and also influenced by the growth
environment of the plant.48 52 Numerous biotic and abiotic factors
affect cannabinoid production, including the sex, growth stage, environ-
mental parameters, and fertilization.23,50,53 56

CANNABIS BIOSYNTHESIS
Fig. 1.3 is a schematic of cannabinoid biosynthesis. In the plant,
Δ9-THC, CBD, and CBC are in their acid forms.57 59 Two indepen-
dent pathways, cytosolic mevalonate and plastidial methylerythritol
phosphate (MEP), are responsible for terpenoid biosynthesis. The
MEP pathway is reported to be responsible for the biosynthesis of the
terpenoid moiety.12 Olivetolic acid (OLA) and geranyl diphosphate
(GPP) are derived from the polyketide and the deoxyxylulose phos-
phate (DOXP)/MEP pathways, respectively, followed by condensation
under the influence of the prenylase olivetolate geranyltransferase,
yielding cannabigerolic acid (CBGA). CBGA, in turn, is oxidocyclized
 The Botany of Cannabis sativa L. 7

CBD (7.899)
THCV (6.990)
(A)
80

Internal standard (9.474)

70

60

50
mV

40

30

D-9 THC (8.634)

D-8 THC (8.298)

CBG (9.110)
CBN (9.175)
CBC (7.974)
20

10

0
6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
min
D-9 THC (8.685)
CBD (7.865)

(B)
125

100 Internal standard (9.478)

75
mV

50
D-8 THC (8.433)
THCV (6.983)

CBG (9.109)
CBC (7.973)

CBN (9.182)

25

0
7.0 7.5 8.0 8.5 9.0 9.5 10.0
min

Figure 1.2 Gas chromatography-flame ionization detector (GC-FID) analysis of (A) a high CBD type and
(B) a high Δ9-THC type cannabis plant.

by flavin adenine dinucleotide-dependent oxidases, namely, cannabi-

chromenic acid (CBCA) synthase, cannabidiolic acid (CBDA)
synthase, and Δ9-THCA synthase, producing CBCA, CBDA, and Δ9-
THCA, respectively.60,61
8 The Analytical Chemistry of Cannabis

Geranyl pyrophosphate Olivetolic acid

(GPP) (OLA)

Geranylpyrophosphate:olivetolate
geranyltransferase

Cannabigerolic acid (CBGA)

Tetrahydrocannabinolic acid Cannabidiolic acid Cannabichromenic acid

synthase synthase synthase

Tetrahydrocannabinolic Cannabidiolic Cannabichromenic

acid (THCA) acid (CBDA) acid (CBCA)

Figure 1.3 Biosynthetic pathway of tetrahydrocannabinolic acid, cannabidiolic acid, and cannabichromenic acid.

SELECTION OF ELITE CLONES FOR PLANT PROPAGATION

The quality, safety, and efficacy of starting material are basic prerequi-
sites in the pharmaceutical industry. Cannabis as a feedstock is more
challenging because it is a chemically complex and highly variable
plant due to its allogamous nature. The chemical composition of can-
nabis biomass is affected by a range of factors such as genetics, envi-
ronment, growth conditions, and harvesting stage. Therefore, selection
of elite starting material (female clone) based on chemical composition,
conservation, and mass multiplication using advanced biotechnological
tools is a suitable way to ensure the consistency in chemical profile of
a crop for pharmaceuticals.

In our laboratory, we developed a GC-FID method for screening

and selection of elite biomass based on major cannabinoid content.
 The Botany of Cannabis sativa L. 9

Briefly, quantitative analysis of seven major cannabinoids (THCV,

CBD, CBC, Δ8-THC, Δ9-THC, CBG, and CBN) is done by solvent
extraction followed by analysis using capillary gas chromatography, a
method offering short analysis time and resolution of all cannabinoids
on a single column. Three samples (100 mg each) are used for analysis
from each manicured biomass sample. A 3-mL internal standard (IS)
extraction solvent (100 mg of 4-androstene-3,17-dione 1 10 mL
chloroform 1 90 mL methanol) is added to the sample and allowed to
rest at room temperature for 1 h. The extract is then filtered through
a cotton plug and the clear filtered material is transferred to an
autosampler vial. Samples are placed onto the GC instrument along
with vials of ethanol, internal standard/Δ9-THC mixture (unextracted
standard), and controls. The results are calculated by obtaining an
average percentage of each cannabinoid from the two chromatograms
of each sample. It must be noted that the response factor for the can-
nabinoids relative to IS is 1. Therefore, the area of each cannabinoid
divided by that of the IS multiplied by the amount of IS added (3 mg)
gives the percentage of each cannabinoid in the sample, as 100 mg of
sample is used for analysis. For example, a cannabinoid with the
same peak area as that of the IS represents a 3% concentration in the
sample. The method has been validated to meet FDA-GMP
requirements.
Once a female cannabis plant is screened and selected based on its
cannabinoids profile, it can be used as a mother stalk for future
propagation.

PLANT GROWTH AND CULTIVATION

Cannabis is an annual species and can be grown from seed or vegeta-
tive cuttings under indoor and outdoor conditions. Indoor cultivation
under controlled environmental conditions can generate three or four
crops per year, depending upon required per-plant biomass yield;
outdoor cultivation is limited to one crop per year. Selection of starting
material or variety depends upon the composition of active ingredients
required in the end product.

Propagation Through Seed

Fig. 1.4 shows the typical seeds of a high-Δ9-THC-yielding Mexican
variety of C. sativa L. Cultivation through seed is an ancient and
10 The Analytical Chemistry of Cannabis

Figure 1.4 Cannabis sativa seeds (high-yielding Mexican variety).

traditional method. Seeds can be sown in small biodegradable jiffy

pots containing soil with good aeration and should be kept moist by
watering with a light spray when the upper surface begins to feel dry.
During winter, a heat mat can be used below the pots to increase the
soil temperature and enhance germination.

Normally seeds start sprouting by the fourth day of plantation, and

most of the viable seeds germinate within 15 days. Variation in the rate
of seed germination depends on the variety, seed age, storage conditions,
and soil and water temperatures. Germinated seedlings can be kept under
cool fluorescent light with an 18-h photoperiod until the seedlings are big
enough to transplant to bigger pots. Once transplanted, they can be kept
under full-spectrum grow light (1000 W high-pressure sodium or metal-
halide bulbs) with an 18-h photoperiod for further vegetative growth.

Upon completion of desired vegetative growth, plants may be

exposed to a 12-h photoperiod for flowering. (At this stage, cuttings of
selected healthy plants can be made and maintained at vegetative stage
for screening purposes.) Onset of flowering normally occurs in 2 weeks,
depending upon variety. Being a dioceous species, seed raised plants nor-
mally turn 50% male and 50% female, depending on the variety. Onset
of male flowers normally occurs a week earlier than female flowers.
For the production of useful secondary metabolites, female plants are
preferred as they produce higher cannabinoid content. At this early
flowering stage, male plants can be identified and separated from female
 The Botany of Cannabis sativa L. 11

plants. Once all the male plants are removed, female plants can be
grown to full maturity for the production of sinsemilla (seedless) buds.
Mature buds can be analyzed for cannabinoid content using GC-FID.
Based on this analysis, elite high-yielding clones can be identified and
their vegetative backup cuttings can be used as mother plants for future
propagation.

Vegetative Propagation in Soil

The pharmaceutical industry requires consistency in the active ingredi-
ents of source material. Using cannabis as a source raw material
remains especially challenging. In spite of being grown from seeds
derived from a single cannabis mother plant, a considerable degree of
variation in chemical composition of juvenile plants may be observed.
Therefore, screening of high-yielding female plants and mass propaga-
tion of vegetative clones is the most suitable way to meet the demand
for uniformity of the final product.
Once a best candidate female clone with a specific chemical profile
is screened and selected, a fresh nodal segment about 6 to 10 cm long
containing at least two nodes can be used for vegetative propagation.
A soft apical branch is cut at a 45-degree angle just below a node
and immediately dipped in distilled water. The base of the cutting is
subsequently dipped in rooting hormone and planted in biodegradable
jiffy pots (2 3 2 in) containing coco natural growth medium and a
mixture (1:1) of sterile potting mix and fertilome. At least one node is
covered by soil for efficient rooting. Plants are regularly irrigated and
kept under controlled environmental conditions. Rooting initiates in
2 to 3 weeks, followed by transplantation to bigger pots after 5 to 6
weeks. The cuttings can be maintained at constant vegetative state
under 18-h photoperiod (Fig. 1.5).

Vegetative Propagation in Hydroponics

Vegetative cuttings can also be grown in a hydroponics system.
A small branch consisting of a growing tip with two or three leaves is
cut and immediately dipped in distilled water. The base of the cutting
is dipped in rooting hormone and inserted (B1 in) deep into a rock-
wool cube or a hydrotone clay ball supporting medium. Plants are
supplied with vegetative fertilizer formula and exposed to fluorescent
light under 18-h photoperiod. Rooting initiates in 2 to 3 weeks.
12 The Analytical Chemistry of Cannabis

Figure 1.5 Indoor vegetative propagation of Cannabis sativa. (A) Vegetative cuttings under fluorescent lights,
(B) plant growing under full-spectrum metal-halide lamps.
 The Botany of Cannabis sativa L. 13

In Vitro Plant Regeneration

Tissue culture methods offer an alternative means of vegetative
propagation. Clonal propagation through tissue culture, commonly
called micropropagation, can be achieved in a short time and a
small space. It is possible to produce plants in large numbers
starting from a single clone. However, the process involves several
stages, from initiation and establishment of aseptic cultures to
multiplication, rooting of regenerated shoots, and hardening in soil.
Direct organogenesis is the most reliable method for clonal propa-
gation because it upholds genetic uniformity among progenies.62 64
An efficient micropropagation protocol for mass growing of drug
type varieties using apical nodal segments containing axillary
buds65 67 (Fig. 1.6), as well as the micropropagation of a hemp
variety using shoot tips,68 have been reported. Recently, our group
developed an improved one-step micropropagation protocol using
meta-topolin.69

Plant tissue culture is also considered the most efficient

technology for crop improvement by the production of somaclonal

Figure 1.6 Micropropagation of Cannabis sativa. (A) A representative mother plant, (B and C) fully rooted
cannabis plants, (D) micropropagated plants under the acclimatization condition, and (E G) well-established
micropropagated plants in soil.
14 The Analytical Chemistry of Cannabis

and gametoclonal variants. The callus-mediated cultures have

inheritable characteristics different from those of parent plants due
to the possibility of somaclonal variability,70 which may lead to
the development of commercially important improved varieties.
Micropropagation through callus production has been reported,
including production of roots through cannabis calli,71 occasional
shoot regeneration,63 and high-frequency plant regeneration from
leaf tissue derived calli.72

Quality Control of In Vitro Regenerated Plants

The sustainability of the regeneration systems depends upon the
maintenance of the genetic integrity of micropropagated plants.
Despite its potential, in vitro techniques are known to induce soma-
clonal variations. Further, the frequency of these variations varies
with the source of explants and their regeneration pattern, media
composition, and cultural conditions. Tissue culture induced varia-
tions can be determined at the morphological, cytological, bio-
chemical, and molecular levels with several techniques. At present,
molecular markers are powerful tools used in the analysis of genetic
fidelity of in vitro propagated plantlets. These are not influenced by
environmental factors and generate reliable and reproducible
results.

In our laboratory, DNA-based intersimple sequence repeat (ISSR)

markers have been successfully used to monitor the genetic stability
of the micropropagated plants of C. sativa.73,74 Fully mature in vitro
propagated plants were also analyzed for their chemical profile and
cannabinoid content, and compared with mother plants and vegeta-
tively grown plants from the same mother plant using GC-FID for
quality assurance.75

Our results showed that micropropagated plants were highly

comparable to the mother plant and vegetatively grown plants in terms
of genetics, chemical profile, and cannabinoid content. These results
confirm the clonal fidelity of in vitro propagated plants and suggest
that the biochemical mechanism used to produce the micropropagated
plants does not affect genetics or metabolic content. So these protocols
can be used for mass propagation of true to type plants of C. sativa for
commercial pharmaceutical use.