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Gul et al.: Journal of AOAC International Vol. 98, No.

6, 2015  1523

DRUG FORMULATIONS AND CLINICAL METHODS

Determination of 11 Cannabinoids in Biomass and Extracts


of Different Varieties of Cannabis Using High-Performance
Liquid Chromatography
Waseem Gul and Shahbaz W. Gul
ElSohly Laboratories, Inc., 5 Industrial Park Dr, Oxford, MS 38655
Mohamed M. Radwan
National Center for Natural Products Research, School of Pharmacy, the University of Mississippi, University, MS 38677, and
Department of Pharmacognosy, Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt
Amira S. Wanas
National Center for Natural Products Research, School of Pharmacy, the University of Mississippi, University, MS 38677, and
Department of Pharmacognosy, Faculty of Pharmacy, Minia University, Minia, Egypt
Zlatko Mehmedic
National Center for Natural Products Research, School of Pharmacy, the University of Mississippi, University, MS 38677
Ikhlas I. Khan
National Center for Natural Products Research, School of Pharmacy, the University of Mississippi, University, MS 38677, and
Department of Bimolecular Sciences, School of Pharmacy, the University of Mississippi, University, MS 38677
Maged H.M. Sharaf
American Herbal Products Association, 8630 Fenton St, Suite 918, Silver Spring, MD 20910
Mahmoud A. ElSohly1
ElSohly Laboratories, Inc., 5 Industrial Park Dr, Oxford, MS 38655, and National Center for Natural Products Research and
Department of Pharmaceutics and Drug Delivery, School of Pharmacy, the University of Mississippi, University, MS 38677

An HPLC single-laboratory validation was performed detection and quantification of these cannabinoids in
for the detection and quantification of the 11 major different types of cannabis plant materials.
cannabinoids in most cannabis varieties, namely,
cannabidiolic acid (CBDA), cannabigerolic acid

T
(CBGA), cannabigerol (CBG), cannabidiol (CBD), he cannabis plant (Cannabis sativa) is an annual
tetrahydrocannabivarin (THCV), cannabinol (CBN), herbaceous plant belonging to the family Cannabaceae.
Δ9-trans-tetrahydrocannabinol (Δ9-THC), Δ8-trans- It is one of the oldest plant sources for food and
tetrahydrocannabinol (Δ8-THC), cannabicyclol
textile fiber (1). The plant is found in a variety of habitats and
(CBL), cannabichromene (CBC), and Δ9-
altitudes (2). The cultivation of hemp started in North America
tetrahydrocannabinolic acid-A (THCAA). The analysis
in 1606 (3). It was legal in the United States for many years,
was carried out on the biomass and extracts of
these varieties. Methanol–chloroform (9:1, v/v) was ending in 1937. Current federal laws prohibit cultivation of
used for extraction, 4-androstene-3,17-dione was cannabis in the United States.
used as the internal standard, and separation was Most recently, however, with the passage of the Farm Bill, it
achieved in 22.2 min on a C18 column using a two- is legal to grow hemp plants in states that enacted laws allowing
step gradient elution. The method was validated for hemp production. There are limits on acceptable levels of
the 11 cannabinoids. The concentration-response Δ9-trans-tetrahydrocannabinol (Δ9-THC) in the produced
relationship of the method indicated a linear plants  (<0.3%). The medicinal use of cannabis is legal in a
relationship between the concentration and peak number of countries, including Canada, the Czech Republic,
area with r2 values of >0.99 for all 11 cannabinoids. and Israel. While federal law in the United States bans all
Method accuracy was determined through a spike sale and possession of cannabis, the legal status of cannabis
study, and recovery ranged from 89.7 to 105.5% with products is in a transitional phase in many states and the District
an RSD of 0.19 to 6.32% for CBDA, CBD, THCV, CBN,
of Columbia in the United States, where use of cannabis for
Δ9-THC, CBL, CBC, and THCAA; recovery was 84.7,
medicinal purposes is allowed. Furthermore, the states of
84.2, and 67.7% for the minor constituents, CBGA,
Colorado, Washington State, and Alaska allow recreational use
CBG, and Δ8-THC, respectively, with an RSD of 2.58
to 4.96%. The validated method is simple, sensitive, of cannabis.
and reproducible and is therefore suitable for the More than 540 different secondary metabolites have been
identified in cannabis. These constituents belong to diverse
phytochemical classes, mainly, cannabinoids, terpenoids, and
Received April 21, 2015. Accepted by AP July 24, 2015
1
Corresponding author’s e-mail: elsohly@elsohly.com noncannabinoid phenols (4). Cannabinoids are the most studied
DOI: 10.5740/jaoacint.15-095 and well known cannabis compounds, exhibiting the typical
1524  Gul et al.: Journal of AOAC International Vol. 98, No. 6, 2015

Figure 1.  Chemical structures of the major cannabinoids of cannabis.

C-21 terpenophenolic skeleton, and their derivatives and Additional plant samples with different cannabinoid profiles
transformation products. To date, a total of 104 cannabinoids were obtained from seizures made by the Drug Enforcement
have been isolated from cannabis (4, 5) and have been the focus Administration (DEA), submitted to our laboratory at the
of extensive chemical and biological research for almost half a
University of Mississippi for potency monitoring.
century since the discovery of the chemical structure of its major
active constituent, Δ9-THC. The plant has been reported for the
treatment of a variety of ailments including, pain, glaucoma, Instruments and Reagents
nausea, depression, and neuralgia (5–9).
In this paper, we report the development and validation of All solvents were of HPLC analytical grade and were
a simple HPLC method for the simultaneous identification purchased from Sigma-Aldrich (St. Louis, MO). A Shimadzu
and quantification of the main 11 cannabinoids in biomass
(Columbia, MD) Ultra Fast LC Prominence system equipped
and extracts of different varieties of cannabis. The method is
currently used for routine analysis of cannabis samples in our with an autosampler, degasser, communications bus module,
laboratories. photodiode array detector, and column heater was used.

Experimental Chromatographic Conditions

Standards
HPLC analysis was performed on a Luna C18(2) column
All cannabinoid standards, namely cannabidiolic acid (150 × 4.60 mm id, 3 µm particle size; Phenomenex, Torrance,
(CBDA), cannabigerolic acid (CBGA), cannabigerol (CBG), CA). The column was equipped with a C18 guard column
cannabidiol (CBD), tetrahydrocannabivarin (THCV), cartridge (Phenomenex). The temperature of the column was
cannabinol (CBN), Δ9-THC, Δ8-trans-tetrahydrocannabinol
maintained at 28°C. The mobile phase consisted of (A) 0.1%
(Δ8-THC), cannabicyclol (CBL), cannabichromene (CBC),
and Δ9-tetrahydrocannabinolic acid-A (THCAA) were isolated (v/v) formic acid in water and (B) 0.1% (v/v) formic acid in
and identified by our group with a purity of more than 95% acetonitrile with the following gradient elution program: started
(Figures 1 and 2). and kept at 30% A and 70% B from 0 to 6 min; then to 23% A
and 77% B in 6 min; kept at 23% A and 77% B for 10 min; after
Cannabis Plant Material
that, the system was restored to the initial conditions in 0.2 min.
Three different varieties of cannabis were grown at the The flow rate was maintained at 1.2 mL/min. Injection volume
medicinal plant garden of the University of Mississippi. was 10.0 µL. UV spectra were recorded from 210 to 400 nm,
Gul et al.: Journal of AOAC International Vol. 98, No. 6, 2015  1525

Figure 2.  Representative chromatogram of the standard cannabinoids mixture.

Figure 3.  Average calibration curves for standard cannabinoids.


1526  Gul et al.: Journal of AOAC International Vol. 98, No. 6, 2015

Figure 4.  Representative chromatogram of an extract of a high Δ9-THC/low CBD cannabis variety.

Figure 5.  Representative chromatogram of an extract of a variety of cannabis rich in both CBD and Δ9-THC.

Figure 6.  Representative chromatogram of an extract of a fiber-type variety of cannabis (high CBD/low THC).
Gul et al.: Journal of AOAC International Vol. 98, No. 6, 2015  1527

Table 1.  LOD and LOQ of various cannabinoids


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

LOD, µg/mL 6.1 1.3 2.5 1.6 0.7 0.3 2.3 1.4 1.1 1.1 1.1
LOQ, µg/mL 18.4 3.9 7.7 4.9 2.1 1.0 6.9 4.2 3.4 3.4 3.4

and the quantification wavelength was set at 220 nm. Run time Sample Solution Preparation
was 22.2 min.
Cannabis samples were dried for 24 h in a 40°C ventilated
Internal Standard Preparation oven and then powdered. A 100 mg amount of the powder was
extracted with 3 mL of internal standard solution by sonication
A solution of 1 mg/mL 4-androstene-3,17-dione was prepared for 15 min. The extract was filtered through a cellulose membrane
in methanol–chloroform (9:1, v/v). filter of 0.2 µm pore size and then diluted 1:1 with methanol.

Stock Standard Solutions Validation

For cannabinoids known to be present at high concentrations The method was validated according to the International
in cannabis, nine concentrations ranging from 0.005 to 5 mg/mL Conference on Harmonization (ICH) Tripartite Guideline for
were prepared in methanol. These were Δ9-THC, THCAA, CBD, Validation of Analytical Procedures (10). Accuracy was measured
and CBDA. Each solution was further diluted with internal by the method of standard addition. The developed method was used
standard solution (1:1) to make a final concentration range of to determine the cannabinoid content before and after spiking. The
0.0025–2.5 mg/mL. For cannabinoids known to exist at low ratio of measured to added amount was used to calculate recovery.
levels in cannabis, seven concentrations ranging from 0.005 The intraday, interday, and intermediate precisions were assessed
to 1.0 mg/mL were prepared in methanol. These were CBG, using a series of measurements. LOD and LOQ were determined as:
CBGA, Δ8-THC, THCV, CBC, CBN, and CBL. Each solution LOD = 3.3σ /S
was further diluted with internal standard solution (1:1) to make a
LOQ = 10σ /S
final concentration range of 0.0025 to 0.5 mg/mL. These solutions
were used to construct individual cannabinoid calibration curves. where σ = SD of response of each cannabinoid and S = slope of
the calibration curve of each cannabinoid.
Calibration Curves
Results and Discussion
Five concentrations, 5.0, 12.5, 25, 50, and 100 µg/mL,
were prepared by mixing the 11 cannabinoids stock standard Several methods have been published for the analysis of
solutions and internal standard solution. These solutions were cannabinoids in cannabis, including TLC (11), HPTLC (12), GC
used to construct the calibration curves (Figure 3). with and without derivatization (5), and HPLC (13). GC is the

Table 2.  Average concentration of the 11 cannabinoids in 13 different samples

Avg. concn, % (w/w)


9
Sample CBG CBGA CBD CBDA Δ -THC THCAA Δ8-THC THCV CBC CBN CBL
a b
Equal — bLOQ 1.28 3.72 0.64 2.16 — — bLOQ bLOQ —
THC-CBD
High THC — bLOQ — — 2.04 12.53 — — — bLOQ —
Low CBD
Fiber type — — 0.06 0.92 — — — — — bLOQ —
1 — bLOQ 0.71 1.22 1.84 5.71 bLOQ bLOQ bLOQ 0.23 —
2 — bLOQ — — 2.47 — bLOQ — 11.2 bLOQ bLOQ
3 — — — — 0.79 bLOQ — — 0.92 0.11 —
4 0.18 1.25 0.19 bLOQ 3.30 12.77 0.25 — bLOQ 0.24 —
5 bLOQ bLOQ 0.80 bLOQ 1.28 bLOQ — bLOQ bLOQ bLOQ —
6 — bLOQ 1.49 3.52 0.69 1.82 bLOQ — bLOQ 0.07 —
7 bLOQ 0.62 1.71 2.50 2.28 9.36 bLOQ — bLOQ 0.09 —
8 bLOQ 0.27 1.49 1.55 2.01 3.74 0.34 bLOQ bLOQ 0.56 —
9 bLOQ 0.51 bLOQ bLOQ 3.62 10.23 0.49 0.22 bLOQ 0.62 —
10 bLOQ 0.59 0.51 1.35 2.84 11.62 0.29 bLOQ bLOQ 0.26 —
a
  — = Not detected.
b
  bLOQ = Below limit of quantitation.
1528  Gul et al.: Journal of AOAC International Vol. 98, No. 6, 2015

most commonly used technique (5); however, GC will not detect This method was used to analyze plant samples submitted to
cannabinoid acids, which will undergo decarboxylation at the our laboratory for potency monitoring by the DEA. Results are
temperature of the injection port. HPLC is a suitable alternative listed in Table 2.
that allows the analysis of both free cannabinoids and their acids
in the same run without the need of derivatization. The developed
Conclusions
method is used for the analysis of cannabinoids of the high Δ9-
THC/low CBD variety, variety rich in both Δ9-THC and CBD,
and the fiber-type variety (high CBD/low Δ9-THC; Figures 4–6). An accurate and reliable analytical method was developed and
validated for the simultaneous identification and quantification
Optimization Studies of the 11 main cannabinoids in biomass and extracts of different
varieties of cannabis. HPLC coupled with a photodiode array
Extraction and chromatography parameters were optimized. detector was used. The method is currently being used for
For extraction, different solvents, extraction methods, and routine analysis of cannabis in our laboratories.
temperature were tried. For chromatographic conditions, various
parameters such as analytical column type and dimensions and Acknowledgments
mobile phase composition were examined. Good separation of
the 11 cannabinoids was observed, and the following approximate The project described was supported in part by the National
retention times (min) were obtained: CBDA (7.77), CBGA Institute on Drug Abuse, Contract No. N01DA-10-7773. The
(8.68), CBG (9.07), CBD (9.41), THCV (10.16), CBN (13.38), authors acknowledge the technical assistance of Candice
Δ9-THC (15.84), Δ8-THC (16.34), CBL (18.63), CBC (19.18),
Tolbert.
and THCAA (19.69; Figure 2). Representative chromatograms
of extracts of the high Δ9-THC/low CBD, approximately equal
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