Order: 800-325-3010 (U.S.) 814-359-3441 (Global) 7
Analysis of Active Cannabis Compounds in Edible
Food Products: Gummy Bears and Brownies
Olga Shimelis,1 Principle R&D Scientist; Kathy Stenerson,1 Principle R&D Scientist;
Margaret Wesley,2 2016 R&D Summer Intern
1MilliporeSigma, Bellefonte, PA
2Pennsylvania State University, University Park, State College, PA
olga.shimelis@sial.com
Introduction
Potency testing in marijuana-infused edibles is an important
problem that analytical labs are facing due to the complexity of the
involved matrices. Among the most popular matrices are gummy
bear candies and brownies. According to one laboratory site, the
concentration of active ingredients in the edibles can range from a
few parts per million to 3.5 parts per thousand.1 In this application, a
procedure was developed to extract active cannabinoid compounds
from gummy bears and brownies. The procedure included a simple
and fast extraction of the active compounds from the studied
foods, and analysis by HPLC-UV using a biphenyl stationary phase
chemistry.
Experimental
Cannabinoid standards from Cerilliant®, available as 1 mg/mL
solutions in either methanol or acetonitrile, were used for this
experiment. The concentration of cannabinoids allowed for the
spiking of both gummy extract and brownies at about 40 ppm
with all compounds. The following compounds were included in
this study: cannabidivarinic acid (CBDVA), cannabidivarin (CBDV),
cannabigerolic acid (CBGA), cannabigerol (CBG), cannabidiolic
acid (CBDA), cannabidiol (CBD), tetrahydrocannabivarin (THCV),
cannabinol (CBN), (-)-Δ9
-Tetrahydrocanabinol (Δ9
-THC),
(-)-Δ8
-Tetrahydrocanabinol (Δ8
-THC), and (-)-Δ9
-Tetrahydrocanabinolic
acid (THCAA). This list of 11 different cannabinoids includes several
acidic forms; thus HPLC analysis was used in order to quantitate these
in their native forms.
The HPLC column used was Ascentis® Express Biphenyl, 2.7 μm
particle size, which gave the best separation of all 11 compounds in
under 13 minutes. The use of this column with superficially porous
particle architecture resulted in low back-pressure, thus, a standard
pressure HPLC system could be used during this experiment.
Sample Preparation
One gummy bear candy, non-spiked, (2.3 g) was dissolved in 20 mL
of warm water. This solution was then spiked with cannabinoids and
extracted using a QuEChERS procedure. The average spiking level
in each gummy bear was 45 ppm for each compound. Bears of four
different colors were tested – orange, yellow, red, and green. After
spiking, the water/candy solution was transferred to 50-mL plastic
QuEChERS extraction tube (55248-U). Acetonitrile (10 mL) was
added, and the tube was shaken for one minute by hand. SupelTM
QuE non-buffered salts (55295-U) were added, and the samples were
shaken for 5 min on an automated QuEChERS shaker. Post-shaking,
the samples were centrifuged for 5 minutes at 5000 rpm. The top
layer was collected and injected directly into the HPLC.
For brownies, a 2.5 g sample of a non-spiked brownie with frosting
was weighted into the QuEChERS extraction tube. This sample
was spiked with cannabinoids and allowed to sit for 30 min prior
to extraction. The average spiking level for the brownies was
40 ppm. The QuEChERS extraction was performed as described
above for gummy bears. Post-extraction, the top acetonitrile layer
was collected into a vial and kept under refrigeration for a minimum
of 3 hours prior to HPLC analysis.
A calibration curve was constructed in acetonitrile bracketing
the expected concentration of 10 μg/mL in the final extracts.
The following calibration points were included: 2 μg/mL, 5 μg/mL,
10 μg/mL, 20 μg/mL and 25 μg/mL.
Results and Discussion
For the gummy bear samples, it was found that neither the red,
yellow, nor green color interfered with detection of cannabinoids
at 220 nm. The red color was partially extracted into acetonitrile,
while the green and yellow colors stayed in the aqueous layer
upon extraction. However, the orange color from the gummy bear,
when extracted into acetonitrile, was found to have an interfering
peak that co-eluted with CBDVA. Thus, for the orange gummy bear,
quantitation of CBDVA was done at 280 nm, where CBDVA has
significant absorbance free of interference. Quantitation was done at
220 nm for the rest of compounds in this study.
While no cleanup was required for gummy bear samples
post-extraction, the co-extractives in the brownie were found to
decrease the recoveries of the analytes if the brownie extract was
injected into HPLC without further processing. The brownie extract
was cleaned by refrigeration to precipitate the co-extracted fats.
The ruggedness of the method for brownies was tested by injecting
the brownie extract multiple times followed by the injection of the
10 μg/mL standard. After 7 injections of the brownie extract, it was
found that the peak retention times were not affected, indicating
that the column was being thoroughly cleaned between injections.
The peak areas for the standards showed a slight decrease of 4%.
Excellent recovery values of above 90% for gummies and above 80%
for brownies were achieved with good accuracies (Table 1).
(continued on next page)

8 Reporter 34.4 |
sigma-aldrich.com/express
Peak
No. Compound
Yellow
Gummy
Orange
Gummy
Red
Gummy
Average
Gummy
and RSD
Average
Brownie
and RSD
 1 CBDVA 90% 92% * 92% 91% (2%) 91% (1%)
 2 CBDV 93% 100% 100% 98% (3%) 93% (5%)
 3 THCV 87% 93% 90% 90% (3%) 87% (1%)
 4 CBDA 94% 90% 95% 94% (3%) 95% (1%)
 5 CBGA 87% 91% 89% 91% (4%) 90% (2%)
 6 CBD 95% 100% 98% 97% (3%) 89% (5%)
 7 CBG 93% 99% 98% 96% (4%) 91% (5%)
 8 CBN 88% 95% 97% 95% (6%) 84% (4%)
 9 Delta-9-THC 93% 99% 100% 97% (3%) 82% (4%)
10 Delta-8-THC 91% 97% 98% 95%(3%) 80% (4%)
11 THCAA 89% 89% 89% 92% (7%) 91% (2%)
Table 1. Recoveries From Spiked Gummy Bears and Brownies
*The orange gummy was done at 280 nm due to the interfering background peak quantitation.
Figure 1. HPLC Chromatogram of Orange Gummy Bear Extract at
(a) 220 nm and (b) 280 nm
The peak elution order is listed in Table 1.
column: Ascentis Express Biphenyl, 10 cm × 2.1 mm I. D., 2.7 μm
particles (64065-U)
mobile phase: (A) 0.1% TFA in water; (B) 0.1% TFA in acetonitrile
gradient: at 47% B, to 50% B in 13 minutes, to 100% B in 0.1 min,
100% B for 3 minutes, to 47% B in 0.1 min, at 47% B for
2.5 minutes
flow rate: 0.70 mL/min
column temp.: 35 °C
detector: UV, 220 nm and 280 nm
injection: 5 μL
pressure: 340 bar
instrument: Agilent 1200, with UV detector
0 2
1
2
3 4
5
6
7
8 9
10
11
1
2
3 4
5
6
7 8 9
10 11
1
2 3
4 5
6 7
8
9 10
11
4 6 8 10 12
Min
0 2 4 6 8 10 12
Min
0 2 4 6 8 10 12
Min
(a)
(b)
Figure 2. HPLC of a Brownie Extract at 220 nm
The peak elution order is listed in Table 1.
Conditions same as Figure 1.
0 2
1
2
3 4
5
6
7
8 9
10
11
1
2
3 4
5
6
7 8 9
10 11
1
2 3
4 5
6 7
8
9 10
11
4 6 8 10 12
Min
0 2 4 6 8 10 12
Min
0 2 4 6 8 10 12
Min
(a)
(b)
Reference
1. Analytical 360, Test Results, Sour Gummy Bears. http://analytical360.com/m/
archived/216628, (accessed July 2016).
Description Cat. No.
SupelTM QuE QuEChERS Products
Non-buffered Extraction Tube 2, 12 mL, pk of 50 55295-U
Empty Centrifuge Tube, 50 mL, pk of 50 55248-U
Ascentis® Express Biphenyl HPLC Column
10 cm × 2.1 mm I.D., 2.7 μm particle size 64065-U
Analytical Standards and Reagents
Cannabidivarinic acid (CBDVA), 1 mg/mL in acetonitrile, CRM C-152
Cannabidivarin (CBDV), 1 mg/mL in methanol C-140
Cannabigerolic acid (CBGA), 1mg/mL in acetonitrile C-142
Cannabigerol (CBG), 1 mg/mL in methanol C141
Cannabidiolic acid (CBDA), 1 mg/mL in acetonitrile C-144
Cannabidiol (CBD), 1 mg/mL in methanol C-045
Tetrahydrocannabivarin (THCV), 1 mg/mL in methanol T-094
Cannabinol (CBN), 1 mg/mL in methanol C-046
(-)-Δ9
-Tetrahydrocanabinol (Δ9
-THC), 1 mg/mL in methanol T-005
(-)-Δ8
-Tetrahydrocanabinol (Δ8
-THC), 1 mg/mL in methanol T-032
(-)-Δ9
-Tetrahydrocanabinolicacid (THCAA), 1 mg/mL in acetonitrile D3415
Accessories
QuEChERS Shaker and Rack Starter Kit, USA compatible plug 55278-U
QuEChERS Shaker and Rack Starter Kit, EU Schuko plug 55438-U
Certified Vial Kit, Low Adsorption (LA), 2 mL, pk of 100 29653-U
 Featured Products
 Related Information
To learn more about our phases incorporating
Fused-Core® technology, visit
sigma-aldrich.com/express
Conclusion
A method was presented for analysis of active cannabinoid
compounds in both brownies and gummy bears. The extraction
procedure involved a salting out step into acetonitrile, and did not
require intensive cleanup. The separation of eleven compounds
was achieved on a biphenyl stationary phase, and was completed
in 13 minutes. The active compound CRM standards are available
through sigmaaldrich.com from Cerilliant.
Food and Beverage Analysis