Extraction of Cannabinoids in Marijuana and Edibles by
QuEChERS
UCT Part Numbers:
ECQUEU750CT-MP - QuEChERS, Mylar packs containing 4 g magnesium
sulfate, 1 g sodium chloride, 0.5 g sodium citrate dibasic sesquihydrate,
and 1 g sodium citrate tribasic; includes 50-mL centrifuge tubes
CSTHC206 - Clean Screen® THC extraction column, 200mg/6mL
SPHPHO7001-5 – Select pH Buffer Pouch, pH 7.0 100 mM phosphate
buffer
GCLGN4MM-5 - GC liner, 4mm splitless gooseneck, 4mm ID x 6.5mm OD
x 78.5mm
January 2015
Summary:
Medical marijuana has been legalized in multiple states across the USA [1]. As a result,
many testing labs are seeking fast and reliable analytical methods to determine the
cannabinoid potency in marijuana and cannabis infused foods (more informally known
as edibles) [2]. This application utilizes the advantages of the QuEChERS technique to
extract cannabinoids in marijuana and cannabis containing foods. This is followed by
either a dilution for marijuana samples or a solid phase extraction (SPE) cleanup for
various complex food samples. This method results in clean extracts for instrumental
detection.
1 gram homogenized food or 100 mg marijuana sample is hydrated with 10 mL reagent
water for 30 min. The cannabinoids are then extracted into 10 mL of acetonitrile (MeCN)
with the aid of EN15662 QuEChERS extraction salts for analyte partitioning and phase
separation. After centrifugation the supernatant is either diluted or undergoes a
secondary SPE cleanup before analysis.
Three representative cannabinoids, tetrahydrocannabinol (THC), cannabidiol (CBD),
and cannabinol (CBN) were selected for analysis in this study. The accuracy and
precision obtained from spiked negative bread samples were excellent. The method
was applied to 6 seized marijuana samples, and the cannabinoid contents and
phenotypic indexes (THC/CBD and (THC+CBN)/CBD ratios) were reported.
Procedure:
1. Extraction
a) Weigh 1 g of homogenized food sample (or 100 mg marijuana) into a 50-mL
centrifuge tube.
b) Add 10 mL of reagent water, let soak and shake occasionally for 30 min.
c) Add 10 mL of MeCN, shake or vortex for 1 min (10 min for marijuana).
d) Add the EN15662 extraction salts in pouch (ECQUEU750CT-MP), and shake
for 1 min manually or use a Spex 2010 Geno-Grinder at 1000 strokes/min.
e) Centrifuge at 3000 g for 5 min. Target analytes were extracted into the upper
MeCN layer (phase separation shown below).
Figure 1. Bread sample after Figure 2. Marijuana sample
extraction after extraction
2. Dilution or Cleanup
OPTION 1: For cannabinoids in marijuana:
Mix 5 μL of the supernatant with 1 mL n-hexane for GC, GC/MS or GC/MS/MS
analysis; or 1 mL mobile phase (50% organic) for HPLC, LC/MS or LC/MS/MS
analysis.
Note: For detection using GC or HPLC detectors which are less sensitive or
selective than MS detectors either dilute the extract less (i.e. take more
supernatant) or use an additional SPE cleanup to remove any remaining
interfering peaks affecting analyte integration.
OPTION 2: For cannabinoids in food samples:
a) Transfer 0.1 to 1 mL supernatant (volume depends on the cannabinoid
contents in the sample and the instrument sensitivity) to a clean test tube.
b) Dilute 10 times with pH 7 phosphate buffer*.
c) Place the SPE columns (UCT p/n CSTHC206) into a glass block or positive
pressure manifold, condition the SPE columns with 3 mL 1:1 n-hexane:ethyl
acetate, soak 1 min, then drain to waste and allow to dry for 1 min. This is
followed by the addition of 3 mL methanol and 3 mL reagent water. Allow
these to draw through the column by gravity.
d) Load the sample by gravity.
e) Rinse the test tube with 3 mL reagent water, apply the rinsate to the SPE
columns, and drain to waste.
f) Repeat step e) with 3 mL n-hexane**, then dry the SPE columns under full
vacuum or pressure for 5 min.
g) Insert a collection rack with test tubes into the manifold, and elute the SPE
columns with 3 x 1.5 mL of 1:1 n-hexane:ethyl acetate by gravity.
h) Evaporate the eluates to dryness under a gentle stream of nitrogen at 40°C.
i) Reconstitute in 1 mL n-hexane for GC, GC/MS or GC/MS/MS analysis, or 1 mL
mobile phase (50% organic) for HPLC, LC/MS or LC/MS/MS analysis.
* pH 7 phosphate buffer is prepared by dissolving the salts in pouch (UCT p/n
SPHPHO7001-5) with 1 L of reagent water.
** Insignificant analyte loss (< 2%) was observed with 3 mL n-hexane wash in
this study, analysts may need to optimize the n-hexane volume for different
matrices and cannabinoid contents.
GC/MS method:
GC/MS Agilent 6890N GC coupled to a 5975C MSD
Injection 2 μL splitless injection at 250 ºC
GC liner 4 mm splitless gooseneck (UCT p/n GCLGN4MM-5), packed
with deactivated glass wool
GC column Restek Rxi®-5sil MS 30m x 0.25mm, 0.25µm with 10m
integrated guard column
Carrier gas Ultra high purity Helium at a constant flow of 1.2 mL/min
Oven temp. Initial temperature at 60 ºC, hold for 1 min; ramp at 30 ºC/min
program to 310 ºC, hold for 2.67 min. Acquire data from 8 to 10 min.
Temperatures Transfer line 280 ºC
Source 250 ºC
Quadrupole 150 ºC
Retention Linearity
Analyte SIM ions (50 ms)
(min) (R2)
CBD 9.078 231.1 246.1 314.2 0.9998
THC 9.357 299.1 231.1 314.2 0.9997
CBN 9.539 295.1 238.1 310.2 0.9998
Results:
Matrix effect was determined by comparing the calibration curve slopes of the matrix
matched standards (bread samples) against those of the calibration standards prepared
in n-hexane, which were found to be insignificant (≤ 20% for all of the 3 analytes), thus
solvent standards (0, 100, 500, 1000 and 2000 ppb) were used to generate calibration
curves using the external standard calibration technique. The responses were found to
be linear over the analytical range (R2 ≥ 0.9997 for 0 - 2000 ppb).
Calibration Curve of THC
THC
Response
R2 = 0.9997
2.00e+005
1.00e+005
0
0 1.00e+003 2.00e+003
Concentration
Recovery and RSD% from Spiked Bread Sample
Analyte Recovery% RSD% (n=6)
CBD 92.6 3.0
THC 93.1 4.6
CBN 96.3 2.0
Cannabinoid Content and Phenotypic Index of 6 Seized Marijuana Samples
Cannabinoids (% dry weight) Phenotypic index
Marijuana
CBD% THC% CBN% THC/CBD (THC+CBN)/CBD
Sample 1 0.16 2.63 0.80 16.9 22.0
Sample 2 0.15 4.47 1.11 29.1 36.3
Sample 3 0.37 5.59 1.22 15.0 18.3
Sample 4 0.54 5.63 1.51 10.5 13.3
Sample 5 0.13 1.02 1.16 7.7 16.5
Sample 6 0.14 2.09 0.60 15.0 19.3
Chromatogram of a Bread Sample Spiked with 10 μg/g Cannabinoids
Abundance
TIC: MS500_6.D\ datasim.ms
450000
400000
CBN
350000
CBD
300000
250000 THC
200000
150000
100000
50000
0
8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60 9.80 10.00 10.20 10.40
Time-->
Chromatogram of a Seized Marijuana Sample
Abundance
TIC:MJ_sample3.D\ datasim.ms
THC
1400000
1300000
1200000
1100000
1000000
900000
800000
700000
600000
500000
CBN
400000
300000
200000
CBD
100000
0
8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60 9.80 10.00 10.20 10.40
Time-->
References:
[1] http://medicalmarijuana.procon.org/view.resource.php?resourceID=000881
[2] http://en.wikipedia.org/wiki/Cannabis_foods
5102-02-01
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