ACS

Analysis and Extraction of CBD

from Cannabis via RP-HPLC-
MS/DAD, Creating Synthetic
Medicinal Marijuana, and Med-
ical Applications for Epilepsy

Zach Ferris
12-6-2018
Zach Ferris
zferris01@aurora.edu
734-680-7571
Aurora University
Aurora, IL 60506
Wednesday, October 10, 2018

To Dr. Kyle McElhoney:

Analysis and Extraction of CBD from Cannabis via RP-HPLC, Creating Synthetic Medicinal
Marijuana, and Medical Applications for Epilepsy
Medicinal marijuana is currently labeled as a schedule one drug by the Federal Drug
Administration (FDA), which states that it has no medical value and has the potential to be high-
ly addictive. Unfortunately, lowering this classification by proving the previously mentioned
claims as incorrect is rather difficult as it is nearly impossible to study. Fortunately, very limited
organizations have been granted licenses that allow them to study cannabis. Even with the low
numbers of teams working on cannabis research, they still have managed to scientifically prove
that medicinal marijuana treats a wide range of problems ranging from chronic pain to epilepsy.
In short, these teams have helped prove the medical applications of cannabis by using a common
technique for analyzing marijuana: reverse phase high performance liquid chromatography (RP-
HPLC). RP-HPLC allows for the separation and quantification of all the components of canna-
bis. Thorough understanding of the active chemicals in cannabis serves as a framework for iden-
tifying and studying the chemical processes that occur when humans have the compound in their
system. Over one-hundred different cannabinoids in the cannabis plant have been identified, and
two of the most common ones that are studied are tetrahydrocannabinol (THC: commonly asso-
ciated with psychoactive effects) and cannabidiol (CBD: commonly associated with medical
benefits).
There are roughly three and a half million epilepsy cases in the United States, and medic-
inal marijuana is illegal in nineteen states. This means that the patients that happen to live in a
state where it is illegal have no way of having easy access to the least invasive treatment for their
seizures. There currently are numerous kinds prescription pills for treating epilepsy that have
worked in some patients. The problem with not just these prescriptions, but all prescriptions, is
that they result in inevitable side effects, which then require more prescriptions to treat. Once the
patient is caught in this never-ending cycle, they are destined to die a slow death as the side ef-
fects accumulate throughout their life. Medicinal marijuana is a great alternative, as the only side
effect is being in a state of happiness. Regardless, even if medicinal marijuana skeptics don’t
agree on the possible long-term side effects, CBD can be isolated, and the treatment will occur
with no possible side effects resulting from the psychoactive THC.
Families across our country are left heartbroken when a loved one dies to epilepsy all be-
cause of the ignorance of our government. Legalization of medicinal marijuana will allow fami-
lies with loved ones that are affected by epilepsy to begin to repair and live long happy lives.
With the legalization of this harmless compound, our country can get all our best teams involved
in the study. Even with only a handful of organizations studying medicinal marijuana, we’ve ac-
complished so much. Imagine what we can discover on the potential of medicinal marijuana with
all labs having access to study cannabis—the discoveries would roll in exponentially. It may
even be the cure for cancer, we will never know until further studies are done.
Analysis and Extraction of CBD from Cannabis via RP-HPLC-
MS/DAD, Creating Synthetic Medicinal Marijuana, and Medical
Applications for Epilepsy
Zachary L. Ferris
Aurora University, Aurora, IL, USA

ABSTRACT: The purpose of this paper is to describe in depth how cannabis is quantitatively analyzed, how synthetic medicinal
cannabis is designed, introduce the endocannabinoid system, and exemplify the vast medical benefits this plant has to offer and its
possible medical potential upon further research. Reverse-phase high-performance liquid chromatography (RP-HPLC) with dynam-
ic maceration, coupled with mass spectrophotometry and real-time polymerase chain reactions have allowed scientists to quantify
the exact ratios of cannabinoids in any strain of cannabis and determine if the specific strain is contaminated with any pesticides or
bacteria. After the cannabis is analyzed, the individual cannabinoids are extracted by replacing the MS with a UV-Vis DAD so that
the sample remains intact for creating concentrated synthetic marijuana medicines (different HPLC unit). Epileptic patients have
uncontrolled seizures because of chemical imbalances in their endocannabinoid system. Specifically, the body naturally produces
endocannabinoids that function by binding to cannabinoid receptors, inhibiting/regulating neuronal action potential. Primarily,
CBD has been found to mimic the effects of these endocannabinoids. Knowing this, synthetic medicinal cannabis is made with
relatively high concentrations of CBD by extracting the CBD from cannabis via RP-HPLC-DAD. The medical merit of cannabis
has been scientifically proven, and the FDA must acknowledge this so that all epileptic patients have easy access to medicinal mari-
juana. However, due to the complexity of cannabis, its spectrum of effects in the body are not completely understood—leaving the
FDA skeptical in its decision to legalize the plant. Only the surface has been scratched of the complete understanding of this com-
plex plant. This plant’s chemical composition is highly diverse and variable, and only if marijuana is legalized nationally will the
United States be able to start making large contributions to the nationwide effort in decoding its’ chemical processes in the body
and the potential uses of this plant. In terms of increasing efficiency of CBD extraction, it is proposed that HPLC employ C8 col-
umns rather than C18 columns because C8’s intricate combinations of varying polarities would likely reduce retention times and
increase resolution. In order to convince the FDA to legalize medical marijuana nationally, further studies must be done to build a
more wholistic understanding of the plants’ chemistry. This includes, but is not limited to, psychoactive cannabinoid and peripheral
nervous system cannabinoid chemistry.

1 Introduction cannabinol (THC), cannabidiolic acid (CBDA),

Cannabis has acquired a negative stereotype.
People with a lack of understanding of the plant
may associate it with recreational drug users who
desire its psychoactive effects. Contrary to popu-
lar belief, cannabis in and of itself is not a psy-
choactive plant. Over 100 different cannabinoids
(constituents of cannabis) have been isolated
from cannabis: the most commonly studied being
tetrahydrocannabinolic acid (THCA), tetrahydro-
and cannabidiol (CBD). THC, the cannabinoid
associated with inducing psychoactive effects, is
not synthesized in relatively large quantities by
the plant during its life cycle. Furthermore,
THCA is the cannabinoid that is produced by the
plant itself and must undergo decarboxylation to
create THC (and carbon dioxide). THCA is con-
CBD Extraction from Cannabis via RP-HPLC, Medicinal Marijuana, and Epilepsy
verted into THC as seen in figure 1 below.
Figure 1. Decarboxylation of THCA, producing THC and CO2. THCA
is naturally produced during the life cycle of the cannabis plant,
whereas the psychoactive THC is produced due to combustion.
Found from: https://swcarizona.com/decarboxylation
The word “psychoactive” has also developed a
stigma that directly correlates it with having
harmful effects. This is untrue—in this case, psy-
choactive simply means that the cannabinoids
interact with the central nervous system—
specifically the brain. These interactions are
highly beneficial for synaptic regulatory process-
es in the case of epileptic patients and will be ex-
plained in the review section. Cannabis has a
wide array of medical applications from being a
general pain killer to treating more severe condi-
tions, such as epilepsy. The cannabinoid that is
generally associated with having medical value is
CBD. Epilepsy is a condition where the persons
neurons are not functioning properly (firing too
often), inducing seizures. These seizures vary in
length, severity, and frequency. In an Israeli
study, 74 patients were treated with 1 to 20
𝑚𝑔 𝐶𝐵𝐷
per day, and after an average of 5.5
𝑘𝑔 𝑏𝑜𝑑𝑦𝑤𝑒𝑖𝑔ℎ𝑡
months 89% of the patients reported reduction in
seizure frequency [1]. Acquiring medications
with these necessary potencies of CBD is not as
Page 1 of 7
easy as finding a certain cannabis strain in nature.
One option scientists’ have is to make hybrid
strains that are genetically designed to synthesize
larger amounts of CBD. Hybridization is a com-
plex process; therefore, it isn’t as common as the
simpler alternative method: separation and ex-
traction of CBD via reverse-phase high-
performance liquid chromatography (RP-HPLC).
RP-HPLC is the best method for analyzing can-
nabis because the acids remain in their original
form under the LC analytical conditions, whereas
they decompose due to the hot inlet and oven
conditions of GC methods [2]. This is the reason
why gas chromatography (GC) is not used for
analyzing cannabis, as GC uses an oven-heated
injector port, column, and detector. Combustion
of cannabinoids is important to consider because
if a sample of cannabis were to be run through a
GC column, the original content of THCA that
combusts in the column would result in the detec-
tor picking up an increased amount of THC rela-
tive to what was initially in the plant, skewing
results.
RP-HPLC is an analytical technique that
separates compounds based on their relative po-
larities. Two types of separation are possible:
isocratic and gradient. Isocratic separations are
used for samples that contain few analytes that
CBD Extraction from Cannabis via RP-HPLC, Medicinal Marijuana, and Epilepsy
elute at relatively similar times. However, if the
sample were to contain sections of analytes that
have relatively long periods of elution time in
between each other, this would be inefficient.
Gradient separations can correct this issue by
slowly increasing solvent composition between
peaks so that elution dead times can be reduced,
increasing column efficiency (concentration is
stabilized when the analytes get closer to the end
of the column). Although gradient separations
increase efficiency for more complex samples,
they can be extremely difficult to program the
timing of concentration changes into the comput-
er system.
Each HPLC unit contains the following
main components for an isocratic separation:
pump(s), sample injector, column, detector, and
data output (computer). In a gradient separation,
a mixer is also required in order to provide a uni-
form mobile phase concentration throughout the
column. The solvent and column are termed the
mobile and stationary phase, respectively. The
solvent(s) is (are) chosen based on its (their) po-
tential to maximize solubility of the sample, and
to limit interactions between the mobile and sta-
tionary phase. An injection valve sample-loop
autosampler withdraws a precise amount of each
sample and injects the sample directly into the
column. Each cannabinoid will interact with the
non-polar column to various degrees depending
on its affinity for the column. Compounds with a
Page 2 of 7
higher affinity for the column will elute slower
than compounds that don’t interact with the col-
umn as much. A diode array detector (DAD), a
type of spectrophotometer that detects absorb-
ances within the UV-Vis range, is used to deter-
mine the identity of the compound that is exiting
the column, and the quantity that is present. The
cannabinoids that reach the detector first will
produce a spike in the chromatogram and so on.
The relative area of each peak in the chromato-
gram is directly proportional to its percent abun-
dance in the sample.
RP-HPLC analysis of medicinal marijua-
na is also coupled with a mass spectrophotometer
(MS) to detect for pesticides that may still be pre-
sent in the cannabis from the farm where it origi-
nated. The MS separates compound by their mass
to charge ratio (m/z), which is accomplished by
vaporizing and ionizing the sample via elec-
trospray ionization (ESI) and inducing an electri-
cal field. ESI is used because the eluate is already
in the liquid form. Any m/z ratios that aren’t
equal to the standards set (expected values) for
all cannabinoids means that the sample is con-
taminated with pesticides. Finally, a real time
polymerase chain reaction (PCR) is used to detect
for bacteria contamination. PCR works by plac-
ing the sample in a growth medium and incubat-
ing it. Bacteria give off CO2 when incubating,
thus changing the pH of the solution. An indica-
tor in the solution detects this pH change and
CBD Extraction from Cannabis via RP-HPLC, Medicinal Marijuana, and Epilepsy
changes colors. Optical detectors in the PCR
measure the exact color change, providing a
quantitative value of how much bacteria contam-
inates the sample.
The percent content (typically measured
in mass percent) of various cannabinoids in the
plant is termed as its “cannabinoid profile,” and it
is critical that it be quantified accurately and pre-
cisely, so that each patient can choose a strain
that fits them best. That is, every patient’s physi-
ology prefers certain ratios of cannabinoids to
produce the best results, and cannabinoid profiles
must be reproduceable so that the patient is al-
ways getting their ideal ratios of cannabinoids in
their medication. It’s important to note that the
MS is a destructive detector; therefore, the sam-
ple cannot be extracted and devised into concen-
trated marijuana medicines. So, after the MS pro-
vides quantification data for the specific strain,
the rest of the sample is taken to a separate RP-
HPLC unit with a UV-Vis DAD so that the sam-
ple can be collected and made into medicinal ma-
rijuana.
2 Review
The standard operating procedure for making
medical grade cannabis is highly organized and
quite intricate. SC Labs, based in Santa Cruz,
California, is on the forefront in medicinal can-
nabis production. When unprocessed cannabis
samples are delivered to its facilities, they are
Page 3 of 7
immediately entered into a computer inventory
system before any analysis is done—each sample
receiving a unique identification number [3]. The
samples are stored in a temperature and humidity
controlled safe until they are ready to be tested.
The process begins with weighing the sample uti-
lizing an analytical balance. Next, the sample is
frozen on dry ice and immediately milled into a
fine powder. A precise amount of liquid is added
to the powder and uniformly dissolved using a
vortex [3]. The solution is then placed in a sonic
bath—the sound waves separate the plant matter
from the cannabinoid solution. The last step in
separating the plant matter from the cannabinoid
solution is placing the vial in a centrifuge, which
pushes the plant matter to the bottom of the vial.
The solution is extracted using a pipet [3]. In or-
der to determine the best extraction techniques, a
collaborative study between the University of
Modena and the University of Giessen compared
the following four extraction techniques based on
their respective yields of cannabinoids (from the
same sample) after being run through an RP-
HPLC column: dynamic maceration (DM), ultra-
sound-assisted extraction (UAE), microwave-
assisted extraction (MAE), and supercritical fluid
extraction (SFE) [4]. DM involves dissolving the
cannabis and shaking the solution periodically.
UAE and MAE utilize sound-waves and SFE
works by drastically dropping the pressure of the
supercritical solvent, allowing the cannabinoids
CBD Extraction from Cannabis via RP-HPLC, Medicinal Marijuana, and Epilepsy
to precipitate. Their study concluded that DM
with ethyl alcohol (EtOH) as the extraction sol-
vent at room temperature for 45 min was selected
as the best extraction technique for cannabinoids
from cannabis [4].
Now that the cannabinoid solution has
been isolated, it’s placed in an injection valve
sample loop autosampler, which injects a precise
amount of each sample directly into the column.
Sample loop injections are used because repro-
duceable quantitative analysis is pertinent in
providing the future patient with exact contents
of the relevant cannabinoids in their medication.
However, a downside to injection valves is that
an excess amount of sample is required to ensure
that the loop is completely full. Thus, if a limited
amount of sample is available, another injection
method should be considered. However, this isn’t
the case for cannabis analysis as samples are de-
livered in substantial quantities. The sample is
pumped through a packed C18 column and each
cannabinoid interacts differently with the col-
umn, resulting in cannabinoids with the most po-
lar character to reach the DAD detector first. The
C18 column in RP-HPLC employs a highly non-
polar stationary phase. RP-HPLC is used because
the molecular structure of cannabinoids is pri-
marily non-polar. It is also worth mentioning that
there is another type of column used in general
HPLC analysis called normal phase HPLC—
which employs a highly polar stationary phase
Page 4 of 7
such as silica (SiO2) beads. Normal phase HPLC
isn’t used in the extraction of cannabinoids as the
polar stationary phase would have minimal inter-
actions with the primarily hydrophobic canna-
binoids and therefore would result in an unre-
solved chromatogram and impure eluates. It is
important to match the relative polarities of the
stationary phase and the analyte particles, as this
increases the number of equilibrations between
the stationary phase and the cannabinoids—
resulting in more resolved peaks. The difference
between each theoretical interaction of the can-
nabinoid molecules and the packed column is re-
ferred to as its “plate height.” The UV-Vis DAD
is the most common detector used in RP-HPLC
CBD extraction because it is non-destructive;
therefore, synthetic marijuana can be made from
the intact eluate. The DAD detects the canna-
binoids exiting the column by shooting them with
UV light and measuring each cannabinoid’s ab-
sorption. A variable wavelength selector would
be required to distinguish between the highly
similar cannabinoid structures.
As previously mentioned, the UV-DAD is
the most common detector for LC analysis, espe-
cially for cannabis separation, because certain
isolated cannabinoids are used to make higher
concentrated synthetic medical cannabis. As a
reminder, epilepsy is a neuronal condition where
neurons are firing excessively. The reason why
CBD is the main cannabinoid that is isolated
CBD Extraction from Cannabis via RP-HPLC, Medicinal Marijuana, and Epilepsy
from RP-HPLC separation is because it regulates
neurotransmitters and receptors in the central
nervous system (CNS) [4]. This regulation is re-
turning these abnormally firing neurons to their
normal state.
The most unknown fact about human
physiology in regards to its interaction with can-
nabinoids is that the body produces its own can-
nabinoids naturally, which are referred to as en-
docannabinoids (eCBs), and has the correspond-
ing eCB receptors. The two main eCBs are anan-
damide (AEA) and 2-arachidonoylglycerol (2-
AG) and the two types of eCB receptors are
termed type 1 and type 2 (CB1R and CB2R) [5].
The endocannabinoid system (ECS) encompasses
all eCBs and their interactions with eCB recep-
tors which help regulate the central nervous sys-
tem (CNS) and the peripheral nervous system
(PNS). CB1R is found primarily in the CNS, be-
ing the primary eCB receptor of concern when
dealing with epilepsy [5], whereas CB2R is found
in the PNS and therefore regulates synaptic activ-
ity associated with reducing pain [5]. The Uni-
versity of Rome II conducted a study in 2009 fo-
cusing on the relation between AEA and epilepsy
treatment. They concluded that epileptic patients
had 2.55 ± 1.78 pmol/mL of AEA in their cere-
bral spinal fluid (CSF), whereas a healthy control
group had 11.65 ± 7.53 pmol/mL (n = 9 for both
groups, p < 0.01) [6]. Unfortunately, people with
epilepsy are born with insufficient AEA levels.
Page 5 of 7
It’s important to note that there is a lot of genetic
regulation behind the synthesis of endocanna-
binoids but is not discussed as it is beyond the
scope of this paper. CBD (primarily) and THC
may also play a role in helping epileptic patients
considering that eCBs mimic their pharmacologic
effects [5] and the final-outcome of eCBs binding
to CB1R is the inhibition of neurotransmitter re-
lease [5]; therefore, must regulate (reduce) pre-
synaptic calcium concentrations—this anticon-
vulsant activity prevents seizures [7]. Consider-
ing that eCBs inhibit neurotransmitter release in a
regulatory manner and they have very similar
chemistry as CBD (primarily) and THC, CBD
and THC play a role in synaptic action potential
reduction, therefore reducing seizure frequency.
3 Future Steps
It is proposed that RP-HPLC employ C8 columns
rather than C18 columns because this would bet-
ter match the relative polarity of cannabinoids in
general. C18 columns are significantly more hy-
drophobic than C8 columns and would be better
suited for strictly non-polar compounds. Canna-
binoids, although predominantly non-polar, have
various polar functional groups that would resist
interacting with the more hydrophobic C18 col-
umn. In order to increase column efficiency, the
resolution is increased by increasing the amount
of plates, and therefore reducing plate height. C8
columns would be a better choice because there
CBD Extraction from Cannabis via RP-HPLC, Medicinal Marijuana, and Epilepsy
is a better balance of its reduced non-polar char-
acter (relative to C18 columns) to cannabinoids
mixture of varying polarity regions. In addition,
the non-polar interaction time would likely de-
crease due to the shorter silica-modified
CH3(CH2)6CH3 chain and therefore increase the
amount of polar functional group interactions.
This intricate combination of reduced nonpolar
interaction times and increased polar interactions
suggests that there may be increases in resolution
and decreases in retention time.
The goal is to legalize medical marijuana
nationally. To accomplish this, the scientific
community has considered the fact that the FDA
has labeled marijuana as a schedule 1 drug,
meaning that it exhibits no medical value [8]. So,
scientists have designed many experiments (in-
cluding epilepsy studies) in hopes to discover
what medical problems can be cured via marijua-
na. In doing so, studies have predominantly fo-
cused on the non-psychoactive side of cannabis
(CBDs). This has led to a rather drastic imbal-
ance of knowledge regarding the overall chemi-
cal effects in the human body—a lot less is
known on the psychoactive effects of cannabis,
because the FDA’s label of schedule 1 drug isn’t
directly defined this way. Yet, the FDA still uses
this imbalance of knowledge as an excuse to not
legalize; however, is justified. In addition to dis-
covering medical value in CBDs, further studies
on the chemical processes of THC in the body
Page 6 of 7
must be conducted [5] to create a balance in
knowledge of cannabis to increase the probability
of legalization.
4 Conclusion
Epileptic patients are currently provided with no
efficient medications for their condition. They
experience seizures because their synaptic activi-
ty is unregulated due to low levels of AEA and
therefore, experience excessive action potential.
Primarily, CBD has been found to act as a regula-
tory molecule—mimicking the effects of AEA—
which can be introduced into an epileptic pa-
tients’ bloodstream to reduce neuronal action po-
tential. Considering that CBDs can help treat epi-
leptic patients, cannabis can be run through an
RP-HPLC unit to extract the CBD and make con-
centrated marijuana medicines. Prior to CBD ex-
traction, samples of a specific strain are run
through an RP-HPLC-MS unit so that pertinent
quantitative data can be acquired to form canna-
binoid profiles for the patients, so that they know
exactly what ratios of cannabinoids that will be
extracted into their medications. Finally, CBD
can be extracted via an RP-HPLC-DAD unit that
will produce highly pure eluates that can be used
to make a desired medication with the relevant
cannabinoid concentrations for the specific pa-
tient. However, medical marijuana is still illegal
in the eyes of the federal government and is diffi-
cult to obtain for epileptic patients. The FDA is
CBD Extraction from Cannabis via RP-HPLC, Medicinal Marijuana, and Epilepsy Page 7 of 7

skeptical to legalize the plant because of the gen-

eral lack of and imbalance of knowledge of can-
nabis in terms of its chemical activity in the
body. The imbalance of knowledge arises from
the FDA classification of cannabis that it has no
medical value, so scientists have been focusing
the majority of their time and funds on CBD
(non-psychoactive) research. Therefore, legaliza-
tion can become a reality if further studies are
done regarding psychoactive and PNS canna-
binoid chemistry. Having a balanced and in-depth
knowledge of cannabinoid body chemistry is of
utmost importance for the efficacy and probabil-
ity of legalization of medicinal marijuana.

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