please find the instructions in the attached file.

The PowerPoint slides of your research presentation should include at least five parts: introduction such as toxicology and/or pharmacokinetics (20%), sample preparation (10%), analytical method (10%), results (40%) and discussion (20%).

Authors

Elsayed A. Ibrahim1,2, Waseem Gul1, 4, Shahbaz W. Gul4, Brandon J. Stamper4,5, Ghada M. Hadad2, Randa A. Abdel Salam2,

Amany K. Ibrahim

3

, Safwat A. Ahmed3, Suman Chandra1, Hemant Lata1, Mohamed M. Radwan1, Mahmoud A. ElSohly1,4,6

Affiliations

1

National Center for Natural Products Research, School of

Pharmacy, The University of Mississippi, University,

Mississippi, United States of

America

2 Pharmaceutical Analytical Chemistry Department, Faculty

of Pharmacy,

Suez Canal University, Ismailia, Egypt

3 Department of Pharmacognosy, Faculty of Pharmacy,

Suez Canal University, Ismailia, Egypt

4 ElSohly Laboratories Inc., 5 Industrial Park Drive, Oxford,

Mississippi, United States of America

5 Department of Chemistry and Biochemistry, University

of Mississippi, University, Mississippi, United States of

America

6 Department of Pharmaceutics and Drug Delivery, School

of Pharmacy, The University of Mississippi, University,

Mississippi, United States of America

Key words

Cannabis sativa L., Cannabaceae, cannabinoids, GC‑FID,

TMS derivatization, quantitation

received May 29, 2017

revised November 13, 2017

accepted November 25, 2017

Bibliography

DOI https://doi.org/10.1055/s-0043-12408

8

Published online December 13, 2017 | Planta Med 2018; 84:

250

–

259

© Georg Thieme Verlag KG Stuttgart · New York |

ISSN 0032‑0943

Correspondence

Prof. Dr. Mahmoud A. ElSohly

National Center for Natural Products Research, School of

Pharmacy, University of Mississippi

806 Hathorn Road, 135 Coy Waller Complex, 38677 Univer-

sity, Mississippi, USA

Phone: + 6629155928, Fax: + 6629155587

melsohly@olemiss.edu

Supporting information available online at

http://www.thieme-connect.de/products

ABSTRACT

Cannabis (Cannabis sativa L.) is an annual herbaceous plant

that belongs to the family Cannabaceae. Trans-Δ9-tetrahydro-
cannabinol (Δ9-THC) and cannabidiol (CBD) are the two major
phytocannabinoids accounting for over 40% of the cannabis

plant extracts, depending on the variety. At the University of

Mississippi, different strains of C. sativa, with different concen-

tration ratios of CBD and Δ9-THC, have been tissue cultured
via micropropagation and cultivated. A GC‑FID method has

been developed and validated for the qualitative and quanti-

tative analysis of acid and neutral cannabinoids in C. sativa ex-

tracts. The method involves trimethyl silyl derivatization of

the extracts. These cannabinoids include tetrahydrocannabi-

varian, CBD, cannabichromene, trans-Δ8-tetrahydrocannabi-
nol, Δ9-THC, cannabigerol, cannabinol, cannabidiolic acid,
cannabigerolic acid, and Δ9-tetrahydrocannabinolic acid-A

.

The concentration-response relationship of the method indi-

cated a linear relationship between the concentration and

peak area ratio with R2 > 0.999 for all 10 cannabinoids. The

precision and accuracy of the method were found to be

≤ 15% and ± 5%, respectively. The limit of detection range

was 0.11–0.19 µg/mL, and the limit of quantitation was

0.34–0.56 µg/mL for all 10 cannabinoids. The developed

method is simple, sensitive, reproducible, and suitable for

the detection and quantitation of acidic and neutral cannabi-

noids in different extracts of cannabis varieties. The method

was applied to the analysis of these cannabinoids in different

parts of the micropropagated cannabis plants (buds, leaves,

roots, and stems).

Determination of Acid and Neutral Cannabinoids in Extracts
of Different Strains of Cannabis sativa Using GC‑FID

Original Papers

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Introduction
For centuries, Cannabis sativa L., Cannabaceae, has been has been
recognized as an important medicinal plant. Much research has

2

50

been carried out on the medical applications of cannabis, and sev-
eral countries have regarded the plant as an important medicine
[1–4]. In addition, cannabis preparations are consumed by mil-
lions of people all over the world, both for medicinal and recrea-

Ibrahim EA et al. Determination of Acid… Planta Med 2018; 84: 250–259

ABBREVIATION

S

BSTFA N,O‑Bis (trimethylsilyl)-trifluoroacetamide

CBC cannabichromene

CBD cannabidiol

CBDA cannabidiolic acid

CBDV cannabidivarine

CBDVA cannabidivarinic acid

CBG cannabigerol

CBGA cannabigerolic acid

CBL cannabicyclol

CBN cannabinol

DMAP dimethylaminopyridine

Δ8-THC trans-Δ8-tetrahydrocannabinol
Δ9-THC trans-Δ9-tetrahydrocannabinol
THCAA Δ9-tetrahydrocannabinolic acid-A
THCV tetrahydrocannabivarian

TMS trimethylsilyl

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tional purposes. More than 500 secondary metabolites have been
identified in cannabis, of which more than 100 constituents are
cannabinoids [5–7]. Cannabinoids are a class of terpenophenolic
compounds with a C-21 skeleton found exclusively in cannabis [3].

Pharmacological activities of the cannabinoids are very di-
verse, ranging from analgesic and antiemetic to the treatment of
glaucoma and multiple sclerosis [8,9]. Δ9-THC has been proven to
be the most psychoactive ingredient in cannabis. It has many di-
verse pharmacological effects with possible therapeutic value in
the treatment of different medical conditions. These include relief
of nausea and vomiting associated with chemotherapy [10], appe-
tite stimulation [11], reduction of symptoms of multiple sclerosis
[12], and treatment of glaucoma [9], as well as spasticity from spi-
nal cord injury [13]. Other non-psychotropic cannabinoids, mainly
CBD and CBG, are increasingly investigated, showing partially dis-
tinctive effects [14–16]. Scientists all over the world are trying to
explore other possible medical uses for cannabinoids.

Cannabis analysis and understanding the chemistry and chem-
ical profile of cannabis preparations is important in light of the
fact that several states in the United States have legalized the
medical use of cannabis and cannabis preparations.

Several methods have been reported in the literature for the
analysis of cannabinoids in cannabis biomass, extracts, and prep-
arations. These include high-performance thin-layer chromatog-
raphy and TLC [17,18]. Different separation techniques (GC,
HPLC, and UHPLC) have been mainly used in most studies in the
field of cannabis analysis [19–25].

Various LC techniques (HPLC, UHPLC, and LC‑MS) have been
widely used in cannabis analysis. Complete chemical profiling of
multiple compounds, including naturally occurring neutral and
acid cannabinoids, can be obtained by HPLC, which requires no
sample derivatization or decarboxylation [22–29]. The major
drawback with LC is its inadequate resolution for the chromato-
graphic separation of the cannabinoids in a complex plant and
preparation matrices. In addition, it requires multiple sample pu-
rification steps and a relatively longer analysis time.

Ibrahim EA et al. Determination of Acid… Planta Med 2018; 84: 250–25

9

Supercritical fluid chromatography with photodiode array de-
tection is popular due to instrument advancement; however, it is
less sensitive than either GC or HPLC, although the analysis time is
much shorter [30–36].

GC is the most commonly employed technique for the quanti-
tative analysis of cannabinoids, which has been in use for a long
time [37,38]. The method has been used regularly for the analysis
of the cannabinoid content of confiscated cannabis products, in-
cluding marijuana, hashish, and hash oil [39–41]. It is generally
considered faster and simpler than HPLC, so it is often favored.
Since the cannabis plant mainly contains the acidic forms of can-
nabinoids [42], GC cannot differentiate between cannabinoids
and their corresponding carboxylic acids without prior derivatiza-
tion. The high temperature of the injection port transforms the
acid cannabinoids into the neutral cannabinoids: THCAA, CBGA,
and CBDA (native forms of THC, CBG, and CBD, respectively, in
plant material) decarboxylate in the injection port of the GC to
produce THC, CBG, and CBD, respectively.

In this study, an analytical method was developed and validat-
ed using GC‑FID for the analysis of both acid and neutral cannabi-
noids through TMS derivatization. The GC‑FID developed and val-
idated method, according to International Conference on Harmo-
nization (ICH) guidelines [43], has been shown to be very accurate
and highly reproducible, with the analysis of all major cannabi-
noids accomplished within a short time. The method was applied
to the quantitative analysis of 10 different acidic and neutral can-
nabinoids in diverse cannabis plant tissues (leaves, stems, roots,
and buds) produced with the micropropagation technique [44].
The primary application of this method was to differentiate be-
tween cannabis varieties (high THC variety, high CBD variety, or
mixed THC/CBD variety) and to show the genetic stability going
from mother plants to micropropagated plants. Since the canna-
bis plant mainly contains acid forms of cannabinoids, the useful-
ness of this method was further revealed by positive identification
and quantification of the cannabinoid acids like THCAA, CBDA,
and CBGA, along with their neutral species in different types of
cannabis plant materials.

Results and Discussion
A GC‑FID method was developed and validated for the determina-
tion of 10 acid and neutral cannabinoids. Silylation with BSTFA was
used as the derivatization reagent to silylate all cannabinoids by
reacting with the carboxylic and/or hydroxyl groups to form the
TMS derivatives. It has a high reaction rate, which results in a more
complete sample derivatization. BSTFA and its reaction by-prod-
ucts tend to elute early in the GC chromatogram due to their
highly volatile nature, which results in reduced levels of interfer-
ence, high peak resolution, and low noise. Prior to derivatization,
cannabinoid solutions, or plant extracts, are dried under N2 gas
steam at 50°C. BSTFA and TMS derivatives are reactive toward
water and tend to hydrolyze in the presence of moisture or protic
solvents. The solvents must, therefore, be dry to yield the highest
conversion rate.

The silylation process was done in the presence of 2% DMAP as
a catalyst for the silylation process, which is needed to avoid deg-
radation of cannabinoid acids into their corresponding neutral

251

▶ Fig. 1 Chemical structures of the target silylated cannabinoids in
C. sativa.

pA

10987654 min

20

18

16

14

12

3
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▶ Fig. 2 Representative chromatogram of silylated cannabinoids
standards.

Original Papers
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cannabinoids under the high temperature of the injection port.
The chemical structures of the cannabinoids analyzed by this
GC‑FID method are shown in ▶ Fig. 1 as the TMS derivatives.

The optimization of the conditions, particularly the tempera-
ture program and flow rate, was critical for achieving high sensi-
tivity. The optimized GC‑FID separation and chromatogram of the
target cannabinoid standards are shown in ▶ Fig. 2. The chro-

252

matogram shows the 10 cannabinoids quantitated in the plant
material (THCV, CBD, CBC, Δ8-THC, Δ9-THC, CBG, CBN, CBDA,
CBGA, and THCAA). In addition, the chromatogram (▶ Fig. 2)
shows three extra cannabinoids, namely CBDV, CBL, and CBDVA,
for identification by retention time only and for which the method
was not validated. The optimized extraction procedure previously
reported by our group [29] was used for the extraction of all sam-
ples in this study.

The developed method was validated according to ICH guide-
lines [43]. In order to confirm that the method is appropriate for
the analysis of the target cannabinoids in C. sativa plant material
and extracts, several plant tissues were analyzed. The standard
curves of all cannabinoids produced during method development
and validation were linear with correlation coefficient (R2) values
of 0.9997–0.9999 that were achieved within the dynamic range
of 0.5–100 µg/mL of nine target cannabinoids and 1–100 µg/mL
for CBGA. All regression parameters are represented in ▶ Table
1. Calibration curves for all cannabinoids are provided in Fig. 1S,
Supporting Information.

The limits of detection (LOD) ranged from 0.11 µg/mL to
0.19 µg/mL and the limits of quantitation (LOQ) ranged from
0.34 µg/mL to 0.56 µg/mL for the target cannabinoids. The reso-
lution between any two cannabinoids was found to be ≥ 2.0. The
inter-day retention times were measured to determine the repro-
ducibility of the chromatographic method. The coefficients of
variation were below 1% for all cannabinoids, indicating the high
reproducibility of the chromatographic separation. The LOD, LOQ,
and retention times of all the cannabinoids are shown in
▶ Table 2.

The method precision was evaluated by measuring the quanti-
tation of the individual cannabinoids in six replicates on three sep-
arate days. The intra- and inter-day precision and accuracy were
determined in terms of percentage relative standard deviation
(RSD%) and percentage relative error (RE%), respectively, as
shown in Tables 1S and 2S, Supporting Information. The inter-
day RSD% was higher than intra-day. The intra- and inter-day pre-
cision was found to be less than 15%, and the accuracy was within
± 15%. As shown in ▶ Table 3, the measured precision was 6.9–
14.7% for intra-day and 0.53–14.10% for inter-day (Table 2S, Sup-
porting Information). Through comparison with the reference
standard, the accuracy was evaluated and was − 7.10 to 0.62%
for intra-day and − 2.3 to 1.52% for inter-day (Tables 1S and 2S,
Supporting Information).

Tables 1S and 2S (Supporting Information) containing the data
for the inter- and intra-day precision and accuracy and chromato-
grams of the three major varieties of C. sativa leaves, in addition to
the calibration curves for all 13 cannabinoids, are available as Sup-
porting Information

CBDA had the lowest overall intra-day precision, while THCAA
had the highest overall. The intra-day RE% ranged from − 4.33 to
0.62 and the inter-day RE% ranged from − 2.3 to 1.52, which indi-
cates the high accuracy of the developed method. All the values of
precision and accuracy are within the acceptable range, and the
method is accurate, reliable, and precise. The procedure was sta-
ble after minor changes were made, followed by the reinjection of
the same sample preparation.

Ibrahim EA et al. Determination of Acid… Planta Med 2018; 84: 250–259

▶
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25Ibrahim EA et al. Determination of Acid… Planta Med 2018; 84: 250–259

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▶ Table 2 Retention times, LOD, and LOQ levels for all target cannabinoids.

Compound THCV CBD CBC Δ8-THC Δ9-THC CBG CBN CBDA THCAA CBGA CBDV CBL CBDVA

LOD µg/mL 0.12 0.12 0.15 0.14 0.15 0.15 0.13 0.14 0.19 0.11 0.10 0.05 0.10

LOQ µg/mL 0.38 0.35 0.46 0.43 0.45 0.47 0.41 0.43 0.56 0.34 0.50 0.10 0.50

Retention time 4.27 4.87 5.41 5.57 5.73 6.32 6.55 7.39 8.74 9.49 3.73 5.10 6.03

▶ Table 3 Cannabinoids quantities in buds during flowering stage (%w/w).

Buds %w/w (mean)a

Sample CBD CBDA Δ9-THC THCAA CBG CBGA CBC CBN Δ8-THC

V1–19 MP F BD 0.39 2.11 0.03 0.03 0.07 b 0.04 b 0.03

V1–19 P1 F BD 0.34 1.83 0.03 0.02 0.07 b 0.04 b 0.02

V1–19 P2 F BD 0.39 2.00 0.03 0.02 0.07 b 0.04 b 0.02

V1–19 P3 F BD 0.32 1.81 0.03 0.02 0.06 b 0.04 b 0.02

MX MP F BD b b 0.65 6.08 b 0.13 b b b

MX P1 F BD b b 0.64 4.65 b 0.11 b b b

MX P2 F BD b b 0.61 5.73 b 0.12 b b b

MX P3 F BD b b 0.63 7.50 b 0.13 b b b

B4 MP F BD 0.17 1.31 0.46 0.47 0.06 b 0.02 b b

B4 P1 F BD 0.19 1.41 0.52 0.51 0.05 b 0.02 b b

B4 P2 F BD 0.18 1.48 0.52 0.63 0.06 b 0.02 b b

B4 P3 F BD 0.10 1.45 0.37 0.59 0.06 b 0.02 b b

a Mean of n = 3; b Not detected; MX: high THC variety; V1–19: high CBD variety; B4: intermediate variety; MP: mother plant; P1: plant 1; P2: plant 2;
P3: plant 3; F: flowering stage; BD: bud

▶ Table 4 Cannabinoids quantities in leaves during flowering stage (%w/w).

Leaves %w/w (mean)a

Sample CBD CBDA Δ9-THC THCAA CBG CBGA CBC Δ8-THC

V1–19 MP F LF 1.86 0.91 0.020 0.02 0.003 b b b

V1–19 P1 F LF 0.29 0.91 0.020 0.02 0.001 b 0.01 b

V1–19 P2 F LF 0.26 0.82 0.018 0.02 0.005 b 0.01 b

V1–19 P3 F LF 0.28 0.80 0.021 0.02 0.005 b 0.01 b

B4 MP F LF 0.21 0.84 0.199 0.22 0.006 b 0.03 b

B4 P1 F LF 0.15 0.57 0.131 0.15 0.008 b 0.03 b

B4 P2 F LF 0.18 0.65 0.163 0.16 0.002 b 0.03 b

B4 P3 F LF 0.20 0.67 0.178 0.17 0.003 b 0.03 b

MX MP F LF 0.07 1.97 0.074 0.72 b 0.030 0.07 b

MX P1 F LF 0.08 2.30 0.074 0.83 b 0.043 0.08 b

MX P2 F LF 0.07 2.07 0.071 0.92 b 0.029 0.07 b

MX P3 F LF 0.07 2.05 0.223 0.83 b 0.036 0.08 b

a Mean of n = 3; b Not detected; MX: high THC variety; V1–19: high CBD variety; B4: intermediate variety; MP: mother plant; P1: plant 1; P2: plant 2;
P3: plant 3; F: flowering stage; LF: leaf

254 Ibrahim EA et al. Determination of Acid… Planta Med 2018; 84: 250–259

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▶ Table 5 Cannabinoids quantities in leaves during vegetative stage (%w/w).

Leaves %w/w (mean)a
Sample CBD CBDA Δ9-THC THCAA CBG CBGA CBC Δ8-THC

MX MP V LF b b 0.507 1.73 b 0.054 0.12 b

MX P1 V LF b b 0.618 1.66 b 0.052 0.14 b

MX P2 V LF b b 0.559 1.71 b 0.056 0.12 b

MX P3 V LF b b 0.660 1.27 b 0.054 0.13 b

B4 MP V LF 0.077 2.30 0.089 0.96 b 0.039 0.07 b

B4 P1 V LF 0.093 2.75 0.104 1.17 b 0.053 0.09 b

B4 P2 V LF 0.062 1.95 0.070 0.83 b 0.030 0.07 b

B4 P3 V LF 0.065 2.15 0.073 0.92 b 0.031 0.07 b

V1–19 MP V LF 0.298 1.76 0.039 0.03 0.002 0.014 0.01 0.007

V1–19 P1 V LF 0.337 1.78 0.039 0.03 0.001 0.012 0.01 0.007

V1–19 P2 V LF 0.435 1.95 0.040 0.03 0.006 b 0.02 b

V1–19 V P3 LF 0.271 1.47 0.039 0.02 0.002 0.013 b 0.007

a Mean of n = 3; b Not detected; MX: high THC variety; V1–19: high CBD variety; B4: intermediate variety; MP: mother plant; P1: plant 1; P2: plant 2;
P3: plant 3; V: vegetative stage; LF: leaf

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The method was applied to the analysis of the cannabinoids con-
tent in different parts (buds, leaves, stems, and roots) of the tis-
sue cultured micropropagated plants of C. sativa and the results
compared with the values obtained from the same organ of the
mother plant. The compounds were identified in the samples by
matching their retention times with those of the standards. A mix-
ture of acetonitrile:MeOH (80:20, v/v) was used as the optimum
extraction solvent as reported previously [29]. The samples were
obtained from the three major cannabis varieties–drug type (high
Δ9-THC/low CBD), intermediate type (mixed Δ9-THC/CBD), and
fiber type (high CBD/low Δ9-THC) and their analysis showed the
profiles of the target cannabinoids for each variety. The highest
level of THCAA and Δ9-THC was detected in the drug type plants,
and the highest content of CBDA and CBD was clear in the fiber
type. The intermediate variety of buds and leaves showed nearly
equal amounts of overall THC and CBD, which is consistent with
the plant varieties as expected (▶ Tables 3–5). ▶ Figs. 3–5 show
the chromatograms of the TMS derivatives of the extracts of the
buds from the three varieties, while Figs. 2S–5S (Supporting Infor-
mation) show chromatograms of the leaves extracts. In general,
the cannabinoids contents were highest during the flowering
stage in the buds followed by the leaves, with minimal amounts
detected in the stems regardless of the variety (▶ Tables 6,7).

It is noted that the cannabinoid content of the different organs
of the mother plant were consistent with those in the daughter
plants from the micropropagated process. This indicates the ge-
netic stability associated with the micropropagated technique de-
veloped by our group.

The quantities of CBD, CBDA, Δ9-THC, CBG, CBN, and CBC were
very small in the roots but could be clearly observed, and their
identities were confirmed with LC‑MS/MS (data not presented).
To the best of our knowledge, this is the first time to report the
detection of cannabinoids in the roots of C. sativa. Work is in prog-

Ibrahim EA et al. Determination of Acid… Planta Med 2018; 84: 250–259

ress to follow up on this observation and to confirm and quanti-
tate these cannabinoids in the roots using LC‑MS/MS.

This is the first report of a validated GC‑FID method for the si-
multaneous determination and quantification of 10 major sily-
lated acid and neutral cannabinoids in different organ tissues of
micropropagated C. sativa. The method is shown to be accurate,
simple, sensitive, and reproducible; therefore, it is suitable for
the routine analysis of cannabinoids in cannabis plant materials
and extracts.

Materials and Methods

Instrumentation and GC conditions

GC‑FID analysis was performed on an Agilent 6890 N Network GC.
Separation was performed using an Agilent Technologies DB-1MS
0.25 mm × 15 m (0.25 µm film thickness) column. Helium was
used as a carrier gas (at a flow rate of 0.8 mL/min) and for the
FID make-up gas. The inlet was configured in split mode with a
20:1 split ratio and a temperature of 275°C. The oven time pro-
gram began at 190°C for 1 min before ramping at a rate of 30°C/
min to 230°C. The oven was kept at 230°C for 2 min before ramp-
ing at a rate of 5°C/min until reaching 250°C. After holding for
1 min, the oven temperature increased at 20°C/min to 300°C,
where it was held for 2.75 min. Afterward, the oven cooled back
down to 190°C. The total run time was approximately 17.5 min.
The detector temperature was 300°C, and the hydrogen, air, and
make-up flow rates were 40, 500, and 27 mL/min, respectively.
Data analysis was performed using Agilent ChemStation software
(rev. B.04.02).

Statistical analyses

The ChemStation Software, composed of data acquisition, quali-
tative, and quantitative analysis software, was used for method

255

pA
10987654 min

40

35

30

25

20

15

Δ
9
-T
H
C
-T
M
S
IS
T
H
C
A
A
-T
M
S
C
B
G
A
-T
M
S

▶ Fig. 3 Descriptive chromatogram of high THC variety in buds
during flowering stage.

pA
10987654 min

100

80

60

40
20
C
B

D
-T

M
S
C
B

C
-T

M
S
Δ
8
-T
H
C
-T
M
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Δ
9
-T
H
C
-T
M
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C
B

G
-T

M
S
C
B
D
A
-T
M
S
IS
T
H
C
A
A
-T
M
S

▶ Fig. 4 Descriptive chromatogram of high CBD variety in buds
during flowering stage.

pA
10987654 min

70

60
50
40
30
20
C
B
D
-T
M
S
C
B
C
-T
M
S
Δ
9
-T
H
C
-T
M
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C
B
G
-T
M
S
C
B
D
A
-T
M
S
IS
T
H
C
A
A
-T
M
S

▶ Fig. 5 Descriptive chromatogram of intermediate variety in buds
during flowering stage.

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development and data acquisition. Descriptive statistics were cal-
culated with the Microsoft Excel 2010 software.

Standards and reagents

Ten cannabinoids were used as reference standards in this study:
THCV, CBD, CBC, Δ8-THC, Δ9-THC, CBG, CBN, CBDA, THCAA, and
CBGA. They were purchased from Cerilliant as 1.0 mg/mL solu-
tions in MeOH. The purities of all the standards were above 99%,
determined by HPLC and GC‑FID analyses. The compound 4-An-
drostene-3, 17-dione (purity was above 99% confirmed by
GC‑FID) was used as the internal standard (I.S.). It was purchased
from Sigma-Aldrich. BSTFA was purchased from Sigma-Aldrich
(purity was ≥ 99% determined with GC‑MS). DMAP was purchased
from Sigma-Aldrich (purity ≥ 99% determined with GC‑MS). All
solvents (MeOH, acetonitrile, and chloroform) were of HPLC ana-
lytical grade and were purchased from Sigma-Aldrich.

Internal standard and DMAP preparation

A solution of 50 µg/mL of 4-Androstene-3, 17-dione (I.S.) was pre-
pared in a mixture of MeOH and chloroform (9:1, v/v). DMAP solu-
tion (2%) prepared by dissolving 2 grams in 100 mL of MeOH was
used to catalyze the silylation process of the target cannabinoids.

Cannabis plant material

In vitro micropropagated cannabis plants were grown in a climate-
controlled indoor cultivation conditions (light, 700 µmol/m2/s
with 16-h photoperiod, temperature 25–30°C, and relative hu-
midity 60%) at Coy Waller laboratory, School of Pharmacy, Univer-
sity of Mississippi, were used in this study [44]. The plant was
taxonomically identified by Dr. Suman Chandra, and a voucher
specimen (S1310V1) was kept at Coy Waller Laboratory, School
of Pharmacy, University of Mississippi.

Standard solutions preparation

From newly opened vials containing individual cannabinoids with
a concentration of 1.0 mg/mL of each cannabinoid, 400 µL were
mixed together in a four-dram vial, evaporated to dryness under
a gentle flow of N2 gas, and added to 4 mL of MeOH to make a
stock standard solution of 100 µg/mL of each cannabinoid (A). Se-
rial dilutions were made from (A) to make 10 µg/mL (B) and 1 µg/
mL (C) stock standard solutions. These solutions were used to pre-
pare the individual points of the calibration curves.

Calibration curves and quality control samples

Seven concentrations ranging from 0.5–100 µg/mL were pre-
pared from the above stock standard solutions while CBGA was
prepared in the range of 1.0–100 µg/mL. Then 10 µL of a 2%
DMAP solution and 50 µL of I.S. were added to each concentration
of the cannabinoid mixture in GC vials. Samples were evaporated
to dryness under nitrogen at 50°C, and the residue obtained was
then silylated with 100 µL BSTFA for 30 min at 70°C in an oven.
The vials were cooled to room temperature, and the contents
transferred to inserts and injected into the GC‑FID. These solu-
tions were used to construct the calibration curves. The calibra-
tion curves were obtained in triplicate on separate days and con-
structed by plotting the concentrations versus the ratios of the
peak areas (peak area of the analyte to that of the internal stan-

256 Ibrahim EA et al. Determination of Acid… Planta Med 2018; 84: 250–259

▶ Table 6 Cannabinoids quantities in stems during flowering stage (%w/w).

Stems %w/w (mean)a

Sample CBD CBDA Δ9-THC THCAA CBG CBN CBC

V1–19 MP F ST 0.0094 0.0356 0.0008 c 0.0046 c 0.0007

V1–19 P1 F ST 0.0041 0.0159 c c 0.0027 c c

V1–19 P2 F ST 0.0064 0.0253 0.0007 c 0.0033 c b

V1–19 P3 F ST 0.0055 0.0191 0.0011 c 0.0036 c c

MX MP F ST 0.0006 0.0009 0.0082 0.0394 0.0011 0.0009 c

MX P1 F ST 0.0010 0.0015 0.0158 0.0616 0.0017 0.0014 c

MX P2 F ST 0.0007 0.0011 0.0069 0.0335 0.0013 0.0011 c

MX P3 F ST c 0.0007 0.0057 0.0219 0.0011 c c

B4 MP F ST 0.0039 0.0083 0.0019 0.0019 0.0023 c b

B4 P1 F ST 0.0094 0.0222 0.0058 0.0033 0.0061 b b

B4 P2 F ST 0.0066 0.0298 0.0046 0.0049 0.0039 c 0.0007

B4 P3 F ST 0.0048 0.0136 0.0031 0.0024 0.0038 c 0.0009

a Mean of n = 3; b Under the LOQ; c Not detected; MX: high THC variety; V1–19: high CBD variety; B4: intermediate variety; MP: mother plant; P1: plant 1;
P2: plant 2; P3: plant 3; F: flowering stage; ST: stem

▶ Table 7 Cannabinoids quantities in stems during vegetative stage (%w/w).

Stems %w/w (mean)a
Sample CBD CBDA Δ9-THC THCAA CBG CBN CBC

V1–19 MP V ST 0.0074 0.0476 0.0013 0.0018 b 0.0005 0.0007

V1–19 P1 V ST 0.0017 0.0126 0.0008 0.0013 b c c

V1–19 P2 V ST 0.0020 0.0189 0.0009 0.0017 b c c

V1–19 V P3 ST 0.0017 0.0083 0.0010 0.0021 b c c

MX MP V ST c c 0.0043 0.0193 b c c

MX P1 V ST c 0.0006 0.0058 0.0237 0.0009 c c

MX P2 V ST c c 0.0088 0.0210 0.0014 c 0.0007

MX P3 V ST c 0.0006 0.0081 0.0242 0.0015 0.0015 c

B4 MP V ST 0.0179 0.0889 0.0112 0.0318 0.0053 c 0.0013

B4 P1 V ST 0.0151 0.0718 0.0083 0.0256 0.0035 c 0.0009

B4 P2 V ST 0.0075 0.0253 0.0044 0.0041 0.0035 b 0.0007

B4 P3 V ST 0.0074 0.0399 0.0078 0.0230 0.0028 b 0.0011

a Mean of n = 3; b Under the LOQ; c Not detected; MX: high THC variety; V1–19: high CBD variety; B4: intermediate variety; MP: mother plant; P1: plant 1; P2:
plant 2; P3: plant 3; V: vegetative stage; ST: stem

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dard). The regression equation parameters are shown in ▶ Table 1
for all cannabinoids.

Quality control (QC) samples at three different concentrations
(low, medium, and high) for each cannabinoid were independent-
ly prepared in the same way every day (n = 6) and on three consec-
utive days (n = 18) as shown in Table 1S and 2S, Supporting Infor-
mation. All cannabinoids standards, QC samples, and stock solu-
tions were stored at − 20°C.

Ibrahim EA et al. Determination of Acid… Planta Med 2018; 84: 250–259

Preparation of extracts of cannabis plant materials

Cannabis samples from roots, stems, buds, and leaves were dried
separately for 24 h in a 40°C ventilated oven and then powdered.
Triplicates (100 mg each) of the samples (powder) from each or-
gan were weighed into a centrifuge tube and each extracted with
2.5 mL of the extraction solution composed of an acetonitrile/
MeOH mixture (8:2) by sonication for 20 min. The mixture was
centrifuged for 5 min at 1252 ×g. The extraction was repeated
four times. All the supernatants were combined, and the volume
was brought to 10 mL using the extraction solvent. For the roots
and stems, 1 mL of each sample extract was taken into a separate

257

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GC vial, 50 µL of I.S. and 10 µL of 2% DMAP were added, and the
mixture was vortexed and evaporated to dryness under nitrogen
at 50°C. The residue obtained was then silylated with 100 µL
BSTFA for 30 min at 70°C. A 3-µL aliquot of the derivatized sam-
ples were injected for GC‑FID analysis.

For bud samples, the cannabinoids that were expected to be in
high concentrations, 10 µL of the sample extract was taken; for
the cannabinoids that were expected to be in low concentrations,
100 µL of each sample extract was taken and treated in the same
way as mentioned before. For samples of leaves extracts, 50 µL
was taken and processed as mentioned before. Quantitation was
based on the ratio of the peak areas of each analyte to the area of
the internal standard. All experiments were performed in tripli-
cate.

Method validation

The method validation (LOD, LOQ, accuracy, and precision) was
performed according to the ICH Tripartite Guideline for Validation
of Analytical Procedures [43]. The intra-day and inter-day preci-
sion were assessed using a series of measurements.

Sufficient resolution between the different cannabinoids in the
chromatogram resulted in the absence of any interference or
overlap between the analytes tested.

Seven-point standard calibration curves were used to evaluate
linearity. Calibration curves were determined by plotting the peak
area ratio (y) of analytes to the I.S. versus the analyte concentration
(x). Linearity was considered satisfactory if the correlation coeffi-
cient (R2) of calibration was higher than 0.99.

LOD and LOQ were determined as LOD = 3.3σ/S and LOQ =
10σ/S, where σ is the standard deviation of the response of each
cannabinoid and S is slope of the calibration curve of each canna-
binoid.

Accuracy was calculated as RE% and precision was stated as
RSD%. Low, medium, and high QC samples were prepared from
standard stock solutions as a single batch on the same day at each
concentration and then divided into aliquots that were stored at
− 20°C until further analysis. They were analyzed during the same
day to evaluate the intra-day precision (n = 6) and three consecu-
tive days to determine the inter-day precision (n = 18). The intra-
and inter-day precision was required to be ≤ 15%, and the accura-
cy to be within ± 15%.

Extraction and chromatography parameters were optimized.
For extraction, different solvents, extraction methods, and tem-
peratures were tried. It was determined that acetonitrile:MeOH
(80:20, v/v) was the best extraction solvent and ratio. Sonication
in an ultrasonic bath for 20 min then centrifugation for 5 min at
1252 ×g was found to be ideal for the complete extraction pro-
cess. For chromatographic conditions, various parameters such
as different column types, dimensions, and program tempera-
tures were examined.

To determine the degree of carryover, one blank n-hexane
sample was injected immediately after each sample of the calibra-
tion set. The blank sample must not have the analyte and internal
standard peaks at signal-to-noise ratio of ≥ 3.

258

Supporting Information

Tables 1S and 2S containing the data for the inter- and intra-day
precision and accuracy and chromatograms of the three major
varieties of C. sativa leaves, in addition to the calibration curves
for all 13 cannabinoids, are available as supporting information.

Acknowledgements

This work was partially funded by the National Institute on Drug Abuse
(NIDA) contract #N01DA‑15-7793 and partially funded by the Egyptian
and Culture Bureau, Washington D.C.

Conflict of Interest

The authors declare no conflict of interest.

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