chemical assessment of cannabis by gc-fid
TRANSCRIPT
Chemical Assessment of Cannabis by GC-FID
By:
Rebecca Plessel
Dorman Lab
Penn State University
Estimated over 8% of the US population uses
cannabis 2014 Census puts the US population at 318.9 million
Hall et al, The Lancet 2014, 374 (9698), 1383-1391
Over 25.5 million
cannabis users
1.5 millionRegistered State-legal Medical Cannabis Patients
https://www.mpp.org/issues/medical-marijuana/state-by-state-medical-marijuana-laws/medical-marijuana-patient-numbers/
What is Medical Cannabis?
As defined by the NIH:
“The term medical marijuana
refers to using the whole,
unprocessed marijuana plant or its
basic extracts to treat a disease or
symptom.”
https://www.drugabuse.gov/publications/drugfacts/marijuana-medicine
Medical Cannabis Helps
Reduce pain
Reduce anxiety
Reduce severity and frequency of seizures
Prevent nausea and vomiting
Increase appetite
Strouse, Cannabis and Cannabinoid Research 2016, 1(1), 38-43
The Cannabinoids
Cannabidiol (CBD) Cannabigerol (CBG)
Cannabichromene (CBC)
Delta-9-tetrahydrocannabinol (THC)
Cannabinol (CBN)
CBD “There is a growing interest in the marijuana chemical
cannabidiol (CBD) to treat certain conditions such as
childhood epilepsy, a disorder that causes a child to
have violent seizures.”
Scientists breed marijuana plants high for CBD, which
coincides with a decrease in the plant’s production of
THC.
These drugs are less intoxicating, if at all
"Is Marijuana Medicine?" NIH, July 2015.
Why isn’t it FDA
approved?
http://www.dea.gov/druginfo/ds.shtml
https://www.drugabuse.gov/publications/drugfacts/marijuana-medicine
Schedule I
https://www.dea.gov/divisions/hq/2016/hq081116.shtml
Other Schedule 1 Drugs
DEA. Drug Scheduling. https://www.dea.gov/druginfo/ds.shtml
Schedule 2 Drugs
DEA. Drug Scheduling. https://www.dea.gov/druginfo/ds.shtml
THC has various symptoms
http://headsup.scholastic.com/students/the-science-of-marijuana
Strouse, Cannabis and Cannabinoid Research 2016, 1(1), 38-43
How much do you take? Depends on the disease/symptom:
Amyotropic lateral sclerosis (ALS)
10mg/day THC for 1mo
Nausea from chemotherapy 5mg THC, 4-6
times/day
Chronic pain 2.5-20mg/day THC for roughly
25 days
All currently self-determined
http://www.mayoclinic.org/drugs-supplements/marijuana/dosing/hrb-20059701
How much your body
receives from smoking
10% - 20% THC in the plant
20% - 70% enters the lungs
5% - 24% reaches the brain
Incredible amount of variance
1g plant 1mg – 33.6mg THC entering your brain
If dose is 10mg 1/10 or 3X amount
Hall et al, The Lancet 2009, 374(9698), 1383-1391,
4 Studies
Method Development Rtx – 5 column
Couldn’t resolve the CBC and CBD compounds
Resolved peaks with Rtx-200
Previous Surrogates
JWH 007
Expensive
Undesirable basic nitrogen
4-hydroxycoumarin
Sky liner, IP liner, Cyclosplitter
Degradation in the liner, high pKa
New surrogate is 1-Naphthol
Marijuana Samples
Seized by University Park
Police
Marked for destruction
Provided by Lt. Stephanie
Brookes and Srgt. Monica
Himes for analysis
Sample Prep and Extraction 100mg plant material
Spike in 1-Naphthol
Solvate in 2ml Methanol
Sonicate for 15 minutes
Syringe Filter (0.45um,
PTFE)
Instrument
Agilent GC-dual FID
Equipped with a
Hydrogen generators,
and Nitrogen and
Helium tanks
Fitted with Gerstel
Rail MPS 2
GC Parameters Oven Temp: 80°C 300°C
Ramp: 12°C/min
Split mode
Total Run Time: 23 min
Inlet:
250°C
19.2 psi
Split ratio: 10:1
Carrier Gas: helium
Front Detector:
350°C
H2 Flow: 40ml/min
Air Flow: 450ml/min
Makeup Flow: 48.4 ml/min
Column:
RTX-200, 30m, 0.25mm ID, 0.5 um df
Cannabinoid Peaks
CBC
(16.680)
CBD
(16.815)
THC
(17.334)
CBG
(17.658)
CBN
(18.041)
Mass Spectrum – THC
60 80 100 120 140 160 180 200 220 240 260 280 300 3200
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
60000
m/z-->
Abundance
Scan 4153 (26.826 min): 02181603.D299
314
231
271
243
258
193174 2179177 107 137121 28555 161149 205
Mass Spectrum – CBD
60 80 100 120 140 160 180 200 220 240 260 280 300 3200
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
110000
120000
130000
140000
150000
m/z-->
Abundance
Scan 4043 (26.197 min): 02181603.D231
246
314193174121
207107 2999177 137 159 27155 258219 286
Reagent (Methanol)
1-Naphthol
CBC CBD THC CBG CBN
Mean:(ng/ul)
20.46 18.33 18.29 17.96 17.06 18.02
%RSD: 2.12% 4.34% 4.07% 10.04% 6.75% 9.78%
1-Naphthol
CBC CBD THC CBG CBN
Mean:(ng/ul)
23.26 21.99 21.76 21.58 21.39 22.09
%RSD: 10.63% 11.34% 11.81% 13.18% 11.73% 11.56%
Samples 1-10
Sample 10 – 10x
ParsleySamples 1-10
Sample 10 – 10x
1-Naphthol
CBC CBD THC CBG CBN
Mean:(ng/ul)
13.61 13.08 12.90 12.42 13.42 13.41
%RSD: 7.88% 7.92% 8.51% 8.19% 7.78% 7.25%
1-Naphthol
CBC CBD THC CBG CBN
Mean:(ng/ul)
13.10 12.40 12.42 11.84 12.61 12.81
%RSD: 1.11% 1.36% 1.12% 1.34% 1.36% 0.96%
Non-Homogenized Cannabis(% by Weight)
Samples 1-5
Sample 5– 5x
CBC CBD THC CBG CBN
Mean(%): 0.21 0.04 8.77 0.78 4.18
%RSD: 9.33 5.92 8.56 9.89 7.44
CBC CBD THC CBG CBN
Mean(%): 0.19 0.04 9.75 0.42 3.60
%RSD: 2.56 1.93 0.50 0.75 0.40
Homogenized Cannabis(% by Weight)
Samples 1-5
Sample 5– 5x
CBC CBD THC CBG CBN
Mean(%): 0.18 0.04 6.66 0.43 4.70
%RSD: 5.99 5.05 6.16 4.29 4.98
CBC CBD THC CBG CBN
Mean(%): 0.18 0.04 6.67 0.44 4.48
%RSD: 3.02 3.25 3.55 3.67 3.59
Conclusions If smoked, homogenized cannabis is the better
method of distribution
Provides a more consistent dosage that makes taking
this drug safer for patients
Future Studies
Use QuEChERS as alternative extraction
method
Exploring terpene and pesticide analysis in
cannabis
Terpenes Terpenes are volatile unsaturated hydrocarbons that often
produce a scent.
Plants (and some animals) use to ward off predators or
attract pollinators.
Elsohly, Mahmoud A., Slade, Desmond. “Chemical Constituents of Marijuana: The Complex Mixture of Natural Cannabinoids”, Life Sciences, 78 (2005), 539-548.
Compounds There are 120 terpene in cannabis and only about 70
cannabinoids.
Terpenes Elsewhere Plants:
β-Myrcene Mangoes, Bay Leaves, and Eucalyptus
Pinene Conifers, Pine woods, Citrus Fruits
Limonene Lemons, Oranges, and Rosemary
Personal Care Products
Limonene aftershave lotions, bath products,
cleansing products, hair products, makeup,
moisturizers, perfumes and colognes, skin care
products, and suntan products
Why are we interested? In general aroma is associated with quality
Forensic perspective:
Link between Terpene and cannabinoid production between
strains
Medical perspective:
Link between Terpenes and THC diffusion through the
blood brain barrier
Increase effective distribution of medication
Problem with lung irritation if smoked
Oier Aizpurua-Olaizola et. al, Jour. Nat Prod. 2016, 79, 324-331
Sidney Cohen, JAMA, 1978, 240(16), 1761-1763
Casano et al, Fundación Canna 2011, 925, 115-121
Medicinal Properties of
Terpenes
β-myrcene is known to prevent peptic ulcer disease
Caryophyllene and limonene combined shows anti-
inflammatory properties
CBD, linalool, limonene, and pinene are used to treat acne
Linalool reduces lung irritation caused by smoking
Jeena et al, Jour. Essential Oil Bearing Plants 2014, 17(1), 1-12
Aizpurua-Olaizola et al, Jour. Nat. Prod. 2016, 79, 324-331
Bonamin et al, Chem-Bio Interactions, 2014, 212, 11-19
Ma et al, International Immunopharmacology, 29, 708-713
Terpene StandardsRestek Medical Cannabis Terpenes Standard #1
(-)-α-Bisabolol
Camphene
Δ-3-Carene
β-Caryophyllene
Geraniol
(-)-Guaiol
α-Humulene
p-Isopropyltoluene
(p-cymene)
(-)-Isopulegol
d-Limonene
Linalool
β-Myrcene
Nerolidol
Ocimene
α-Pinene
(-)-β-Pinene
α-Terpinene
γ-Terpinene
Terpinolene
Restek Medical Cannabis Terpenes Standard #2(-)-Caryophyllene oxide 1,8-Cineole (Eucalyptol)
Goals of Terpene Analysis To develop an analytical method for the qualitative
and quantitative analysis of Terpenes in Cannabis
Headspace (HS)
Liquid injection
Solid Phase Microextraction (SPME)
Thermal Desorption Unit of solid material
HS GC-FID Method
Full Evaporation
Technique
Samples were prepared by spiking 10ul of analytical
standards into a headspace vial
Plant matrix spikes contained 10mg of parsley in
headspace vial + 10ul of analytical standards
Elution of
Terpenes on
Rxi-624SilMS
Compound Retention Time
alpha pinene 8.309
camphene 8.638
beta myrcene 8.926
beta pinene 9.041
delta 3 carene 9.372
alpha tepinene 9.494
ocimene 9.644
limonene 9.685
p cymene 9.727
eucalyptol 9.852
gamma terpinene 9.994
terpinolene 10.401
linalool 10.796
isopulegol 11.579
geraniol 12.769
beta caryophyllene 14.813
alpha humulene 15.196
nerolidol 1 15.726
nerolidol 2 16.03
(-)-guaiol 16.756
caryophyllene oxide 16.927
alpha bisabolol 17.402
Headspace Results Resolved terpenes on Rxi624SilMS
Unable to analyze for cannabinoids on Rxi624SilMS
Observed area differences between reagent spikes and matrix spikes
Not always higher or lower in area counts
Adsorption, volatility, matrix interferences
Area count bias throughout chromatogram
More retained compounds displayed lower area counts
Calibration showed less linearity for later eluting compounds
Liquid Injection GC-MS
Liquid Injection GC-MS
Elution on
Rtx200MS
Retention Time Identification Confirmation
8.03 alpha pineneretention match to
standard
8.58 campheneretention match to
standard
9.15 beta pineneretention match to
standard
9.33 beta myrcene NIST match, no standard
9.42 delta 3 careneretention match to
standard
9.83 alpha terpineneretention match to
standard
9.87 d-limonene NIST match, no standard
10.06 ocimeneisomer, NIST match, no
standard
10.34 gamma terpineneretention match to
standard
10.42 p-cymeneretention match to
standard
10.50 ocimeneisomer, NIST match, no
standard
10.72 tepinolene NIST match, no standard
10.66 eucalyptolretention match to
standard
12.15 linaloolretention match to
standard
13.62 isopulegolretention match to
standard
14.73 geraniolretention match to
standard
16.25 beta-caryophylleneretention match to
standard
16.71 alpha humuleneretention match to
standard
17.64 nerolidol (1 or 2)retention match to
standard
17.99 nerolidol (1 or 2)retention match to
standard
18.84 guaoil NIST match, no standard
19.47 alpha-bisbololretention match to
standard
20.21 cayophyllene oxideretention match to
standard
20.91phthalate- lab contaminant NIST match
Restek Medical Cannabis Terpenes Standard #1
Restek Medical Cannabis Terpenes Standard #2
Cannabinoids on
Rtx200MS
CBC CBD THC CBG CBN
26- 28 minute elution period
Liquid Injection Results
Resolved all terpene standards on Rtx200MS column
No area count bias based on retention in column
Analyzed for Terpenes and Cannabinoids in a single
analysis
Headspace SPME 50/30 µm
divinylbenzene/Carboxen
on polydimethylsiloxane
on a StableFlex fiber
Rxi-624SilMS (30m x
0.25mm x 1.4um)
1mm SPME liner
Parameter Optimization Desorption/ Inlet Temperature
230, 250, 270 C
Extraction/ Fiber Exposure Time
5, 10, 20, 40, 60 min
Extraction/ Fiber Exposure Temperature
30, 40, 60, 90, 115, 140, 180 C
Vial Incubation Time
5, 15, 30, 45 min
Optimized Parameters Desorption/ Inlet Temperature: 230 C
Extraction/ Fiber Expose Time: 10 minutes
Extraction/Fiber Extraction Time: 60 C
Vial Incubation Time: 30 minutes
Used same GC parameters as headspace method
SPME Results
SPME Results Matrix interference observed in parsley spiked
replicates
Later eluting compounds absorbed to fiber more
Overloaded fiber in high cal points reducing linearity in
several well retained compounds
HS vs. SPME
Headspace more selective
for more volatile
compounds
Solid Phase Microextraction
more sensitive for larger
molecules
Overall SPME is more
sensitive for terpene analysis
Retention Time HS 2500 ng in vial SPME 1250 ng in vial
8.32 125 43
8.65 124 48
8.94 122 102
9.05 125 61
9.38 132 94
9.50 155 137
9.67 131 124
9.67 141 139
9.78 88 124
9.87 111 77
10.00 127 135
10.40 122 161
11.70 105 205
12.78 86 459
14.83 135 723
15.21 131 804
15.74 30 274
16.05 43 400
16.78 68 679
16.95 81 736
17.42 60 538
Area Counts
HS Analysis of Marijuana
Homogenized
Marijuana
analyzed by HS
GC-FID
Average of 5
replicates display
the Terpene
Signature
Below limit of
quantification ND= non detection
5mg of Marijuana
Compound ng/mg %RSD
alpha pinene 28 10
camphene 10 9
beta myrcene 36 8
beta pinene 26 11
delta 3 carene ND -
alpha terpinene ND -
o cimene 89 10
limonene ND -
p cymene ND -
eucalyptol 22 8
gamma terpinene ND -
terpinolene ND -
linalool 119 9
isopulegol ND -
geraniol ND -
beta caryophyllene 159 8
alpha humulene 58 7
nerolidol 1 ND -
nerolidol 2 65 11
(-)- guaiol ND -
caryophyllene oxide ND -
alpha bisabolol ND -
Future Plans Utilize thermal desorption unit as a direct sampling
technique
Rtx200MS column to analyze for terpenes and
cannabinoids
Split mode to remove water from inlet and to avoid
overloading the mass spectrometer
Thanks to…
Dorman Lab
Questions?
Rebecca Plessel - [email protected]